secreting β-cells

In pancreatic β-cells, although H₂O₂ is a metabolic signal for glucose stimulated insulin secretion, it may induce injury in the presence of increased oxidative stress (OS) as in the case of diabetic chronic hyperglycemia. Olea europea L. (olive) leaves contain polyphenolic compounds that may protect insulin-secreting cells against OS. The major polyphenolic compound in ethanolic olive leaf extract (OLE) is oleuropein (about 20%), thus we compared the effects of OLE with the effects of standard oleuropein on INS-1 cells. The cells were incubated with increasing concentrations of OLE or oleuropein for 24 h followed by exposure to H₂O₂ (0.035 mM) for 45 min. H₂O₂ alone resulted in a significantly decreased viability (MTT assay), depressed glucose-stimulated insulin secretion, increased apoptotic and necrotic cell death (AO/EB staining), inhibited glutathione peroxidase activity (GPx) and stimulated catalase activity that were associated with increased intracellular generation of reactive oxygen species (ROS) (fluorescence DCF). OLE and oleuropein partly improved the viability, attenuated necrotic and apoptotic death, inhibited the ROS generation and improved insulin secretion in H₂O₂-exposed cells. The effects of oleuropein on insulin secretion were more pronounced than those of OLE, while OLE exerted a stronger anti-cytotoxic effect than oleuropein. Unlike OLE, oleuropein had no significant preserving effect on GPx; however, both compounds stimulated the activity of catalase in H₂O₂-exposed cells. These findings indicate different modulatory roles of polyphenolic constituents of olive leaves on redox homeostasis that may have a role in the maintenance of β-cell physiology against OS.


IntrOductIOn
Pancreatic β-cells secrete insulin to regulate blood glucose homeostasis.The dysfunction and injury of these cells contribute to the pathogenesis of type 1 and type 2 diabetes (Ihara et al., 1999;Cnop et al., 2005;Del Guerra et al., 2005;Kaneto et al., 2007).Insulin secretion is subject to control by nutrients and by hormonal, neural, and pharmacological factors.Among these, glucose is by far the most important regulator of the machinery of insulin secretion (Gembal et al., 1992).It has been well documented that glycolytic and oxidative events coupled with the generation of reactive oxygen species (ROS) lead to accelerated ATP generation and are key transduction phenomena in β-cell signaling (Pi et al., 2007).However, one of the unique features of β-cells is their relatively low expression of H 2 O 2 -detoxifying antioxidant enzymes such as glutathione peroxidase (GPx) (Kaneto et al., 2007), which could render β-cells susceptible to oxidative damage.Although H 2 O 2 has emerged as a physiological mediator of insulin secretion (Merglen et al., 2004), chronic hyperglycemia has been shown to be involved in β-cell dysfunction, a phenomenon known as glucotoxicity (Robertson et al., 2003;Robertson & Harmon, 2006).The multiple biochemical pathways and mechanisms of the glucose toxicity have in common the formation of ROS, which in excess and over time can cause chronic oxidative stress (OS) (Robertson et al., 2003;Robertson & Harmon, 2006).Oxidative stress plays a central role in cellular injury and cell death, and the cellular responses to OS involve changes in mitochondria and other organelles, notably endoplasmic reticulum, and can lead to a number of cell death modes, which cover a spectrum from apoptosis to necrosis and include autophagy.Apoptosis has been shown to be the main way of β-cell death in the presence of chronic hyperglycemia and OS (Eizirik et al., 2001;Rhodes, 2005).Therefore, the protection of β-cells against OS-induced injury and dysfunction is important in the prevention of diabetes.As in the case of studies on the retardation of diabetes-induced complications by antioxidants (Güneş et al., 2005;Yülek et al., 2007), great attention is now focused on natural antioxidants in the protection of β-cells against OS-induced injury.
Olive (Olea europaea L.) leaves and fruits contain several groups of constituents, including polyphenols, flavonoids, flavones, iridoids and carbohydrates (Gariboldi et al., 1986;Heimler et al., 1992;Romani et al., 1994).These substances have a considerable pharmacological action including antioxidant activity and low toxicity (Soni et al., 2006).Oleuropein, a phenolic secoiridoide, is used as a well known compound of extracts and its concentration is significantly high in leaves and fruit.Oleuropein has a high antioxidant activity in vitro (Speroni et al., 1998), scavenges superoxide anions, hydroxyl radicals, and hypochlorous acid-derived radicals (Visioli et al., 1998).Ol-ive leaves have an ethnomedical usage in the management of diabetes, and the anti-diabetic effect of olive leaves or oleuropein has already been demonstrated in animal models (Gonzalez et al., 1992;Jemai et al., 2009;Eidi et al., 2009).However, the cellular mechanism(s) of their effects responsible for blood glucose control, including the effects on insulin secretion and any relation with cellular redox state have not been studied yet in insulin secreting cells.Thus, we investigated the effects of polyphenolic olive leaf extract (OLE) and oleuropein on intracellular OS, cellular viability, apoptosis, necrosis and insulin secreting function in the INS-1 cell line.

RESEARCH DESIGN AND METHODS
Plant materials and extracts.To obtain oleuropeinrich fraction from olive leaves we used ethanol extraction of leaves previously extracted with petroleum ether.Petroleum ether was used for extraction of pigments and lipophylic material from olive leaves (free fatty/resin acids, sterols, waxes, steryl esters, triglycerides).Olive leaves were collected from the Balıkesir-Edremit region in Turkey in June 2008.Olive leaf samples were handpicked randomly from the trees.They were dried in the shade and crushed.After crushing, 100 g of powdered leaves was extracted by stirring for 12 h at 50 °C in 500 mL petroleum ether.The petroleum extracts were filtered and dried.Then the residue was extracted by stirring 12 h at 50 °C with 500 mL of 95 % ethanol.Following extraction, the ethanol phase was filtered and dried using mini spray dryer (Buchi Laboratory Equipment B-290, Switzerland) to obtain dry olive leaf extract (16.35 g).We used the European Pharmacopeia (EP 6th edn; vol 3, pp 2545-2546) method for quantitative analysis of oleuropein content in the ethanolic extract.We obtained 20.12 ± 0.18 % oleuropein in olive leaf ethanolic extract.
Acridine orange/Ethidium bromide (AO/EB) staining assay.Apoptotic and necrotic changes in the cells were determined morphologically by fluorescence microscopy with AO/EB staining as described by (Ribble et al., 2005).INS-1 cells were seeded in 6-well plates at a density of 5.0 × 10 5 cells per culture well.Cells were preincubated for 24 h with or without oleuropein (0.1 mmol/L) or olive leaf extract (0.1 mg/mL) followed by incubation with H 2 O 2 in Krebs-Ringer buffer for 45 min at 37 °C.Cells were collected and washed three times in phosphate buffered saline (PBS).Cells were centrifuged (1000 r.p.m.) and pellet was gently resuspended in 25 µL of medium containing 3 µL of dye mixture (100 µg/ mL acridine orange and 100 µg/mL ethidium bromide in PBS).Ten microliters of cell-dye mixture was placed on a microscopic slide and images were collected using a fluorescence microscope (Leica Microsystems, Germany) with excitation at 488 nm and emission at 520 nm.For each well, three or more random pictures were obtained.Tests were done in triplicate and minimally 300 cells were counted for each slide.
Intracellular ROS generation.Intracellular ROS generation was evaluated with a modified method previously described by Rosenkranz et al. (1992).The cells were seeded in 96-well plate at a density of 15 × 10 5 cells/ cm 2 and pre-incubated for 24 h with or without different concentrations of oleuropein (10 -1 -10 -4 mmol/L) or olive leaf extract (10 -1 -10 -4 mg/mL) followed by incubation with 15 µmol/L 2΄,7΄-dichlorofluorescin diacetate (DCF-DA, Molecular Probes) in Krebs-Ringer buffer for 1 h at 37 °C.DCF-DA is a membrane permeable probe which is hydrolyzed by cellular esterases resulting in formation of non-fluorescent dichlorofluorescein (DCFH).When oxidized by free radicals, DCFH is converted to fluorescent DCF.After DCF-DA loading, the cells were incubated with 0.035 mmol/L H 2 O 2 for 45 min in Krebs-Ringer buffer at 37 °C.The fluorescence of DCF was quantified at an excitation wavelength of 485 nm and emission wavelength of 528 nm in a microplate fluorometer (Modulus microplate multimode reader, Turner Bio Systems, USA).
Insulin secretion.INS-1 cells were seeded in 24-well plates at a density of 5.0 × 10 5 (in 1 mL medium) cells per culture well.Cells were preincubated for 24 h with or without oleuropein (0.1 mmol/L) or olive leaf extract (0.1 mg/mL) followed by incubation with H 2 O 2 (0.035 mmol/L) in Krebs-Ringer buffer for 45 min at 37 °C.The cells were then washed and preincubated for 30 min in glucose-free buffer supplemented with 0.1 % BSA (bovine serum albumin) and containing (in mmol/L) NaCl 125, KCl 5.9, MgCl 2 1.2, CaCl 2 1.3, Hepes 25 titrated to pH 7.4 with NaOH.The glucose-free buffer was replaced by the same buffer supplemented with 16.7 mM glucose and the cells were incubated for 120 min at 37 °C.The cells were sedimented by centrifugation for 5 min at 2000 r.p.m., and then the supernatants were analyzed for insulin content with an enzyme-linked immunosorbent assay (ELISA) kit (Mercodia, Uppsala, Sweden).
Glutathione peroxidase and catalase activity assay.Glutathione peroxidase (GPx) activity and catalase activity were assayed by using colorimetric assay kits (all from Cayman Chemical Ann Arbor, MI, USA).Cells 2.0 × 10 6 in 6-well plates for each experiment, were preincubated for 24 h with or without oleuropein (0.1 mmol/L) or olive leaf extract (0.1 mg/mL) followed by incubation with H 2 O 2 (0.035 mmol/L) in Krebs-Ringer buffer for 45 min at 37 °C.The cells were scraped and homogenized according to the kit protocols.Absorbance at a specific wavelength for each assay was recorded with a microplate reader.
Chemicals.All cell culture products were obtained from GIBCO ® Invitrogen, USA.H 2 O 2 was purchased Olive polyphenols and H 2 O 2 -induced β-cell toxicity from Merck (Merck & Co., Inc.USA).Oleuropein and all other chemicals, unless otherwise stated, were from Sigma (Sigma-Aldrich Co., USA).
Statistics.Each experiment was performed three times.Data are expressed as mean ± S.D. One-way analysis of variance (ANOVA) was used in multiple group comparisons.Difference with P < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION
Pancreatic β-cells are essential for glucose homoeostasis.The dysfunction of these insulin-secreting cells results in diabetes development.Hydrogen peroxide (H 2 O 2 ) has been demonstrated to be a critical factor in physiological cellular signal transduction for glucosestimulated insulin secretion in β-cells (Pi et al., 2007).However, excessive and/or sustained H 2 O 2 production can directly or indirectly disturb the integrity and the function of these cells.Such disturbances in β-cells contribute to the pathogenesis of diabetes (Del Guerra et al., 2005;Kaneto et al., 2007).
H 2 O 2 is used extensively in cell culture studies for induction of OS instead of the known insulinoma cell specific toxins like alloxan and streptozotocin (Maechler et al., 1999;Pi et al., 2007;Kaneto et al., 2007;Hou et al., 2008).The exposure to H 2 O 2 produces injury in islets via suppressing hyperpolarization of the mitochondrial membrane and it impairs glucose-stimulated insulin secretion in INS-1 cells (Maechler et al., 1999).In our study, starting with the concentration of 25 µmol/L H 2 O 2 , a significant drop in INS-1 cell viability as assessed by MTT (89.1 ± 8.8 % of control) was obtained after 45 min period of treatment (Fig. 1).The concentration of 35 µmol/L H 2 O 2 , which decreases the viability of the cells by about 50 % was used for further experiments (Fig. 1).Oleuropein and OLE were non-toxic at concentrations up to 0.1 mmol/L and 0.1 mg/mL, respectively, as assessed by reduction of MTT (not shown).A 24hour preincubation with the OLE or oleuropein led to a significant improvement in the viability of H 2 O 2 -exposed INS-1 cells, but a complete amelioration was not obtained (Fig. 2).Using AO/EB double staining, four cell stages were identified after exposure to H 2 O 2 -induced stress: living cells uniformly green stained with non-fragmented chromatin, cells with bright green and orange nuclei and condensed or fragmented chromatin (early and late apoptotic cells, respectively), and cells with red intact nuclei (necrotic cells) (Fig. 3).Exposure to H 2 O 2 significantly increased the proportion of apoptotic and necrotic cells (Fig. 4).Preincubation with oleuropein or OLE partly, but significantly increased the proportion of living cells and prevented cell death by necrosis (Fig. 4).Interestingly, neither OLE nor oleuropein did significantly affect the proportion of apoptotic cells after H 2 O 2 exposure (Fig. 4).As expected, increased intracellular ROS production (308.1 ± 56.7 % of control) was found in H 2 O 2 -treated cells (Fig. 5).Although oleuropein and OLE were able partly to decrease ROS production, these effects were only seen at the highest concentrations of these antioxidants (Fig. 5).Our data confirm the ability of OLE to control ROS generation and/or scavenge the intracellular ROS reported in early studies (Visioli et al., 1998;Oliveras-López et al., 2008).Visioli and coworkers (1998) reported the ROS-neutralizing property of oleuropein by both in an in vitro xanthine/xanthine oxidase system and in an ex vivo human polymorphonuclear neutrophil model of superoxide formation.
In our study, the H 2 O 2 -induced OS caused a decrease in insulin secretion to 44.1±6.1 % of that in control cells (Fig. 6A).These data are in agreement with those of Xiong et al. (2006) who reported that 2-hour incubation with H 2 O 2 caused a decrease of glucose-induced insulin secretion in isolated pancreatic islets.When β-cells were exposed to OS inducers, insulin gene expression was shown to be decreased by the activation of the JNK pathway (Tanaka et al., 2002;Sakai et al., 2003;Robertson et al., 2007).The preserving effects of various natural antioxidants on insulin content and insulin mRNA of β-cells have already been demonstrated by others (Kaneto et al., 2007).It is well know that olive polyphenols are powerful antioxidants in vitro and possess other in vivo biological activities that could account for their beneficial health effects (Covas, 2007).Interestingly, in H 2 O 2treated INS-1 cells, the preincubation with oleuropein completely restored insulin secretion to control levels, but the effect of OLE, composed of antioxidant mixture, was only partial (Fig. 6A).
To prevent ROS-induced destruction, cells protect their vital functions by enzymatic or non-enzymatic mechanisms.These include a number of enzymes such as glutathione peroxidase (GPx) which catalyze reactions to get rid of the oxidative effects of ROS.H 2 O 2 is converted into water and molecular oxygen by GPx and catalase.In the present study, while GPx activity was significantly inhibited, catalase activity was increased after H 2 O 2 addition to INS-1 cell (Fig. 6B and 6C, respec-  tively).Interestingly, OLE afforded a complete preservation of the GPx activity, but oleuropein had no significant effect against the H 2 O 2 -induced depression (Fig. 6B and 6C, respectively).The preservation of GPx activity allowed the normal content of GSH to be maintained.Previously, the intensity of insulin secretion has been found to closely correlate with the activity of GPx as demonstrated by using cysteine or synthetic antioxidants against H 2 O 2 -induced toxicity in cell culture (Xiong et al., 2006;Oliveras-López et al., 2008).Our experiments demonstrated that H 2 O 2 activates catalase as a physiological defense response against oxidative attack (Fig. 6C).Interestingly, OLE and oleuropein treatments enhanced this increase even further (Fig. 6C).The exacerbating action of OLE and oleuropein on catalase in the presence of H 2 O 2 is likely related to their pro-oxidant properties.A dual action of polyphenols as both antioxidant and pro-oxidant, has been demonstrated in cell culture systems with the common plant polyphenols, quercetin and epigallocatechin-3-gallate (Raza & John, 2005;Robaszkiewicz et al., 2007).These polyphenols have been shown to decrease production of ROS in the cells but also trigger the production of peroxides in the medium in the presence of H 2 O 2 (Raza & John, 2005;Robaszkiewicz et al., 2007).Thus, the effects of OLE polyphenols on redox systems in our model seem to be complex and might include both antioxidant effects and induction of OS due to formation of ROS in the extracellular medium.Regardless of the localization of catalase, its increased activity reflects an increased catalase-dependent pathway to consume H 2 O 2 and other peroxides.While catalase is primarily located in peroxisomes, it has been reported that extracellular peroxidase activity of catalase is critical for ensuring efficient protection against exogenous H 2 O 2 (Hervé-Grépinet et al., 2008).Generally, oleuropein is believed to be a key active ingredient responsible for the benefits of OLE, and the anti-diabetic properties of olive leaves were until recently attributed only to oleuropein.Oleuropein in olive leaf ethanolic extract obtained in this study was combined with other phytochemicals that naturally occur in live leaves, including other phenolic compounds and flavones such   as tyrosol and hydroxytyrosol (Gordon et al., 2001).The literature indicates that most of the compounds present in olive leaves, have antioxidant properties, preventing or reducing the deleterious effects of OS associated with diabetes (Eidi & Darzi, 2009;Hamden et al., 2009;Cvjetićanin et al., 2010); however, their effects on insulin secretion in experimental cell culture models have not been studied to date.Purified hydroxytyrosol, the major phenolic compound from olive mill waste, has shown hypoglycemic and antioxidant effects in rats (Hamden et al., 2009).Others also isolated and identified another constituent, triterpene oleanolic acid, from olive leaves.It lowered serum glucose and insulin levels in mice fed a high fat diet and enhanced glucose tolerance (Sato et al., 2007).In the case of the present work, it is difficult to explain why oleuropein alone was found to be more effective in normalizing insulin secretion by INS-1 cell than was the antioxidant mixture of OLE.This finding implies different effects of the active ingredients of OLE on the redox-sensitive regulation of insulin secretion.The positive regulatory effect of oleuropein in OLE might be overshadowed by properties of the other constituents of the OLE mixture.It has been demonstrated that the basal physiological effect of H 2 O 2 on glu-cose-stimulated insulin secretion disappears at high H 2 O 2 concentrations, when the antioxidative defense is activated (Pi et al., 2007), as was the case of our model using H 2 O 2 plus OLE.The benefits of olive leaf compounds are supported by recent literature data reporting olive leaf-derived polyphenols as therapeutic agents delaying the progression of advanced glycation end products-mediated inflammatory diseases such as diabetes (Chandler et al., 2010).