Flavonoids protect pancreatic beta-cells from cytokines mediated apoptosis through the activation of PI3-kinase pathway

Cytokine 59 (2012) 65–71

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Flavonoids protect pancreatic beta-cells from cytokines mediated apoptosis through the activation of PI3-kinase pathway Chia-Yu Lin a,⇑, Chih-Chin Ni a, Mei-Chin Yin b, Chong-Kuei Lii b a b

Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan Department of Nutrition, China Medical University, Taichung, Taiwan

a r t i c l e

i n f o

Article history: Received 17 May 2011 Received in revised form 10 January 2012 Accepted 11 April 2012 Available online 9 May 2012 Keywords: Cytokine b-Cell Apoptosis Quercetin Naringenin

a b s t r a c t The preventive effects of four phenolic compounds against cytokines-induced b-cell destruction were assessed in this study. Treatment of INS-1 (832/13) cells with pro-inflammatory cytokine mixtures (interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a) and interferon-c (IFN-c)) resulted in an increased apoptosis. While resveratrol or myricetin failed to prevent cell apoptosis, quercetin or naringenin treatment exhibited an about 40% less in cell death induced by cytokines-mediated damage. This protective effect of quercetin or naringenin might be mediated partially via the activation of the downstream pAkt and pBad pathways, an outcome which was abolished by pretreatment with a specific PI3-kinase inhibitor. Cellular protein levels of p-p38 MAPK and inducible NO synthase (iNOS) were enhanced after cytokines addition; however, the presence of quercetin or naringenin could not suppress their expression. While cytokines induced MnSOD, quercetin or naringnin did not further enhance expression of this protective protein. In addition, the loss of mitochondria membrane potential (MMP) after cytokines treatment might be partially corrected with quercetin or naringenin. However, none of the phenolic compounds tested in this study reversed the blunted glucose-stimulated insulin secretion after cytokines treatment. These results suggest that quercetin or naringenin might possibly be able to protect b-cells from cytokines toxicity by enhancing cell survival through PI3-kinase pathway, independent of p-p38 MAPK or iNOS. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction It is clear that the failure of pancreatic b-cells to adapt to the increased insulin demand due to insulin resistance, and the decreased insulin secretory capacity as well as b-cell mass are the characteristics of type 2 diabetes [1,2]. Cytokines secreted by immune cells that have infiltrated pancreases are thought to be the crucial mediators of b-cells destruction. In addition, there is an inflammatory process in islets of diabetes patients, and many pro-inflammatory cytokines, such as tumor necrosis factor (TNF)a, interleukin (IL)-1b, and interferon (IFN)-c, have been claimed [3]. Exposure of human non-diabetic islets to high glucose results in increased release of IL-1b, followed by nuclear factor kappaB (NF-jB) activation, Fas up-regulation, and impaired b-cell function [3]. Glucose-induced endogenous IL-1b expression also increases

Abbreviations: IL-1b, interleukin-1b; TNF-b, tumor necrosis factor -b; IFN-c, interferon-c; iNOS, inducible NO synthase; MMP, mitochondria membrane potential. ⇑ ce:text>Corresponding author. Address: Department of Health and Nutrition Biotechnology, Asia University, 500, Lioufeng Rd., Wufeng, Taichung 41354, Taiwan. Tel.: +886 4 23323456x20050; fax: +886 4 23321126. E-mail address: [email protected] (C.-Y. Lin). 1043-4666/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2012.04.011

susceptibility of rodent pancreatic b-cell to apoptosis by inhibiting insulin signaling, IRS-2/Akt-mediated signaling [4]. However, conflicted results regarding the deleterious effects of IL-1b production and NF-jB activation after prolonged exposure to high glucose on human islets have also been reported [5]. In addition, IFN-c is known to potentiate IL-1b-induced nitric oxide (NO) production and b-cell death [6]. Treatment with TNF-a increases JNK, reduces Bcl-2 protein content, and induces caspase-3 and cellular apoptosis. Tumor necrosis factor-a also has pronounced inhibitory effects on insulin signaling through the activation of JNK that directly inhibits IRS-1/2 tyrosine phosphorylation and reduces Akt phosphorylation in b-cells [7]. Therefore, many cellular functions of pancreatic b-cells could be compromised by deleterious actions of cytokines through the insulin signaling and its downstream IRS/Akt-mediated pathway. So far, there is increasing evidence of potential benefits of phenolic compounds in the cellular regulations such as redox control and inflammatory responses, and thus may protect against diabetes [8,9]. Quercetin, apigenin, and luteolin treated in RIN cells significantly reduce in IL-1b and IFN-c induced NO production, which is correlated with reduced inducible form of NO synthase (iNOS) mRNA and protein levels through the inhibition of NF-jB activation [10]. These flavonoids also prevent the decrease of

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glucose-stimulated insulin secretion in rat islets by IL-1b and IFN-c [10]. In addition, the in vivo study suggests that quercetin treatment has a protective effect in diabetes by decreasing oxidative stress and preserving pancreatic b-cell integrity, possibly through decreasing lipid peroxidation and NO production, as well as by increasing antioxidant enzyme activities [11]. It also has been found that insulin secretion in the presence of resveratrol is increased in various pancreatic b-cell cultures [12]. All of these studies suggest that the application of phenolic compounds might positively preserve b-cells and their function. Since exposure of b-cells to cytokines may reduce the Akt phosphorylation and therefore possible negatively affect cell survival [13,14], and the activation of PI3 kinase has been described to be protective to b-cells [15], our present study was designed to assess the ameliorative potential of resveratrol, quercetin, myricetin and naringenin against pro-inflammatory cytokines-mediated damage through the activation of PI3 kinase signal transduction in INS-1 (832/13) cells. 2. Materials and methods

were washed and substrate for peroxidase was applied. Absorbance was read spectrophotometrically at 405 nm. 2.4. Measurement of mitochondrial membrane potential (MMP) The mitochondrial membrane potential (MMP) was determined in INS-1 (832/13) cells treated with quercetin, naringenin, quercetin/naringenin in combination with cytokines, or cytokines only (TNF-a 400 U/ml, IL-1b 80 U/ml, and IFN-c 160 U/ml). Cells were harvested after 20 h. Changes in the MMP were measured by flow cytometry using JC-1 10 lg/ml (Molecular Probes, Eugene, OR, USA). JC-1 forms aggregates in cells with a high FL-2 fluorescence during a normal MMP, while loss of membrane potential results in a reduction in FL-2 fluorescence and a shift to monomeric state in FL-1 fluorescence [17]. Briefly, cells were trypsinized, washed in PBS, and resuspended in JC-1 for 15 min at 37 °C. After which, cells were washed with PBS twice and resuspended in PBS. About 10,000 cells were analyzed for fluorescence for each sample in a FL-1 (525 nm, green) versus FL-2 (575 nm, red) on a Cell Lab Quanta SC Flow Cytometer (Beckman Coulter Inc, Brea, CA, USA).

2.1. Materials 2.5. Insulin secretion and content Resveratrol, quercetin, myricetin and naringenin (Sigma– Aldrich, St Louis, MO, USA) were dissolved in dimethyl sulfoxide (DMSO). Stock solutions were prepared at 50 mM for resveratrol, quercetin and naringenin, and at 20 mM for myricetin. PI3 kinase inhibitor, LY294002 (Cell Signaling Technology, Danvers, MA, USA), was also prepared in DMSO at 25 mM. Final concentration of DMSO was between 0.2% and 0.04%. No adverse effect in culture was observed with these concentrations. Cytokines, TNF-a, IL-1b and IFN-c (PeproTech Inc, Rocky Hill, NJ, USA) were dissolved in phosphate-buffered saline (PBS). Stock concentrations of cytokines are 2500 U/ll for TNF-a, 1000 U/ll for IL-1b, and 2000 U/ll for IFN-c. Both phenolics and cytokines were further diluted to the final concentrations in culture medium and were added together for following experiments. PI3 kinase inhibitor was added 1 h before test compounds/cytokines in experiments. 2.2. Cell culture INS-1 (832/13) cells [16] were kindly provided by Dr. Christopher Newgard (Duke University Medical Center, Durham, NC, USA). Cells were cultured in RPMI 1640 culture medium, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 50 lM 2-mercaptoethanol, 100 U/ml penicillin and 100 lg/ml streptomycin in a humidified atmosphere at 37 °C and 5% CO2. Culture medium was changed the day after seeded and subsequently every other day. 2.3. DNA fragmentation measurement DNA fragments were analyzed by the Cell Death Detection ELISA Plus Kit (Roche Applied Science, Mannheim, Germany). Different concentrations of cytokines (Cytokines 1: TNF-a 200 U/ml, IL-1b 40 U/ml, and IFN-c 80 U/ml; Cytokines 2: TNF-a 100 U/ml, IL-1b 20 U/ml, and IFN-c 40 U/ml; or Cytokines 3: TNF-a 50 U/ml, IL-1b 10 U/ml, and IFN-c 20 U/ml) with or without test compounds were treated for 24 h. For blocker study, vehicle or PI3 kinase inhibitor, LY294002 (50 lM) was pretreated 1 h before treatment. At the end of study, cells were incubated with lysis buffer and the resulting cell lysate was then centrifuged at 200g for 10 min. Twenty microliter aliquots of the supernatant were then added to microtiter plate with 80 ll of immunoreagent (mixture of anti-histone-biotin and anti-DNA-peroxidase). The plate was incubated for 2 h at room temperature. After incubation, plates

Acute insulin release in response to glucose stimulation was performed at the end of treatment. First, cells were washed and preincubated in Krebs–Ringer bicarbonate buffer (KRB) containing 2.8 mM glucose and 0.5% BSA. KRB was then replaced with KRB 2.8 mM glucose for 1 h (basal state), followed by an additional 1 h in KRB 16.7 mM glucose (glucose stimulated state). Supernatants from both basal and glucose stimulated states were collected for insulin secretion analysis using insulin ELISA kit (Millipore, Billerica, MA, USA). Cells were extracted with 1 ml acid ethanol (0.18 mM HCl in 70% ethanol) overnight at 4 °C followed by insulin ELISA to measure insulin content. 2.6. Western blot On the day of the experiments, medium was changed, and groups of cells were treated with vehicle or the specific PI3 kinase inhibitor, LY294002 (50 lM), for 1 h before addition of vehicle control or cytokines (TNF-a 400 U/ml, IL-1b 80 U/ml, and IFN-c 160 U/ml) with or without quercetin or naringenin for 24 h. At the end of the incubation, cells were washed in PBS and collected in RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate; Sigma, St Louis, MO) with protease and phosphatase inhibitor cocktail (Sigma). Whole cell lysates were then incubated on ice for 10 min and cleared by centrifugation at 8000g for 10 min at 4 °C to pellet the cell debris. Protein concentrations of sample supernatants were determined by Bio-Rad protein assay. Sample (about 50 lg total protein) were then subjected to SDS–PAGE (4–12% gel) and electrically transferred to PVDF membranes. After transfer, membranes were blocked with 5% nonfat dry milk in TBS with 0.1% Tween 20 (TBS-T) and incubated overnight at 4 °C with rabbit polyclonal Akt, rabbit polyclonal pAkt (Ser473), rabbit polyclonal p-p38 MAPK, rabbit polyclonal b-actin (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal pBad (Ser136) (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal MnSOD (1:5000), goat polyclonal SIRT1 (1:5000), rabbit polyclonal COX2 (1:1000), and rabbit polyclonal iNOS (1:200) (Abcam, Cambridge, MA, USA) antibodies. Membranes were then washed with TBS-T and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000, Millipore, Billerica, MA, USA) or anti-goat IgG (1:5000, Abcam) for 1 h. Blots were developed using chemilu-

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minescent HRP substrate (Millipore). Membranes were visualized by Fujifilm LAS-4000mini and analyzed using Fujifilm multi gauge. 2.7. Statistical analysis Statistical analysis was performed using the nonpaired twotailed t test or determined by a one-way ANOVA when comparing more than two groups. Data are presented as means ± SEM. A p value < 0.05 is considered statistically significant. 3. Results 3.1. Prevention of cytokines-induced apoptosis by phenolic compounds Different concentrations of cytokines (Cytokines 1: TNF-a 200 U/ml, IL-1b 40 U/ml, and IFN-c 80 U/ml; Cytokines 2: TNF-a 100 U/ml, IL-1b 20 U/ml, and IFN-c 40 U/ml; or Cytokines 3: TNF-a 50 U/ml, IL-1b 10 U/ml, and IFN-c 20 U/ml) were used here to analyze for apoptotic cells by cell death ELISA. Cytokines treated at different concentrations significantly induced cell apoptosis dose dependently (Fig. 1, p < 0.05). Naringenin treatment alone has less basal apoptotic cells (Fig. 1, p < 0.01). While resveratrol and myricetin did not protect cells from cytokines-induced toxicity, quercetin and naringenin might significantly lower the apoptotic cells at cytokines 1 treatment (Fig. 1, p < 0.05). Quercetin and naringenin still induced less cell death after cytokines 2 or 3 treatments. 3.2. Recovery of the reduced MMP from cytokines toxicity with phenolics We further examined the influence of MMP by quercetin and naringenin in the presence of cytokines. Apoptotic cells containing depolarized mitochondria after cytokines treatment has indicated a 3-fold more of the loss of potential as compared to the vehicle control (Fig. 2, p < 0.05). The reduced MMP after cytokines could partially be recovered with the presence of quercetin and naringenin (Fig. 2, p < 0.05).

Fig. 2. Cytokines-induced reduction in MMP measured by JC-1 was partially recovered with the treatment of quercetin (20 lM) or naringenin (50 lM) in INS-1 (832/13) cells. After treatment, cells were labeled with JC-1 fluorescent dye which measures the different distribution of the dye in the form of aggregates (normal cells) and monomeric form (apoptotic cells) by flow cytometry analysis. Data are shown as the means ± SEM of 5 individual experiments.

1b 40 U/ml, and IFN-c 80 U/ml) or cytokines alone. While there was a 10-fold increase in GSIS in control group, treatment of cytokines significantly inhibited this effect. However, neither quercetin nor naringenin treatment reverse the decreased GSIS after cytokines toxicity (Fig. 3A). Insulin contents were no different among groups (Fig. 3B). 3.4. Expression of SIRT and MnSOD Expression of SIRT1, which is involved in cell survival, both in mRNA (data not shown) and protein (Fig. 4A) levels might not be influenced by any of the treatments. Antioxidative enzyme MnSOD protein expression was induced in cells treated with quercetin in the presence of cytokines or with cytokines alone. Quercetin/ naringenin did not further modify MnSOD level after cytokines (Fig. 4B). 3.5. Cytokines-induced p-p38 MAPK and iNOS protein expression

3.3. Glucose-stimulated insulin secretion (GSIS) Glucose-stimulated insulin secretion (Fig. 3A) and insulin content (Fig. 3B) were analyzed after 1 day treatment of quercetin 20 lM or naringenin 50 lM with cytokines (TNF-a 200 U/ml, IL-

While protein levels of p-p38 MAPK and iNOS were significantly induced after cytokines treatment (Fig. 5A and B, p < 0.05), neither quercetin nor naringenin could inhibit their expression. The result of iNOS mRNA expression (data not shown) among groups was also similar to that of protein. However, COX2 protein expression (Fig. 5C) was not different among all groups. 3.6. Quercetin and naringenin enhanced the reduced pAkt protein expression after cytokines toxicity

Fig. 1. INS-1(832/13) cells were analyzed for cell apoptosis after treatment of cytokines and phenolic compounds. Quercetin at 20 lM and naringenin at 50 lM significantly prevented Cytokines 1 (TNF-a 200 U/ml, IL-1b 40 U/ml, and IFN-c 80 U/ml) induced cell apoptosis (p < 0.05). Data are means ± SEM of at least 6 individual experiments.

While cytokines treatment might cause a reduction in pAkt expression (Fig. 6A, p < 0.01), a significant elevation of pAkt was observed in the presence of quercetin or naringenin during cytokines treatment (Fig. 6A, p < 0.05). Quercetin or naringenin possibly preserved the loss of pAkt due to cytokines toxicity. Pretreatment of cells with a PI3 kinase-specific inhibitor, LY294002, significantly inhibited quercetin or naringenin-induced Akt phosphorylation in the presence of cytokines (Fig. 6A, p < 0.05). Interestingly, PI3 kinase inhibitor likely abolished the protective effect of quercetin or naringenin from cytokines toxicity as indicated by an increased apoptotic cells (Fig. 7, p < 0.05), suggesting that the activation of the PI3 kinase-Akt pathway might be one of the mechanisms by which quercetin or naringenin protects pancreatic b-cells from cytokines toxicity. Although it did not reach significance, pBad levels were elevated after quercetin or naringenin treatment during

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Fig. 3. (A) Cells were tested for glucose-stimulated insulin secretion (GSIS) by static incubation assay (basal: 2.8 mM glucose and stimulated: 16.7 mM glucose). While control cells significantly increased GSIS, cytokines blunted insulin secretion. However, neither quercetin nor naringenin reversed the reduced GSIS after cytokines. (B) Insulin contents were similar among groups. Data are shown as the means ± SEM of 4–5 individual experiments.

the cytokines toxicity and dropped back to the control levels with the presence of PI3 kinase-specific inhibitor, which possibly imply the anti-apoptotic effects of pBad (Fig. 6B). 4. Discussion

Fig. 4. Expression of SIRT1 protein (A) levels was similar among groups. MnSOD protein (B) was increased in cytokines with quercetin/naringenin treatment or cytokines alone. Data are shown as the means ± SEM of 4–5 individual experiments.

Pro-inflammatory cytokines that infiltrated pancreases may mediate b-cell dysfunction of diabetes [3]. Since inflammatory reactions depend on a cluster of cytokines rather than on a single cytokine only, and pro-inflammatory cytokines (IL-1b, TNF-a, and IFN-c) might work together to potentiate their cellular effects [3,6], we have tested the combined effects of these cytokines. The concentrations chose here were based on concentrations used by others applied both in cell lines and islets [18–21], and our dose-response studies. The activation of PI3 kinase-Akt in its phosphorylated state is known to be responsible for anti-apoptotic activities and cell survival [22,23]. Our data possibly imply the necessity of activation of PI3 kinase signal for the anti-apoptotic effects of quercetin or naringenin during cytokines toxicity. Similar to previous study that suggests the role of PI3 kinase-Akt pathway in cellular protection. The presence of albumin in cell culture increases the reduced Akt phosphorylation after a cytokine mixture (IL-1b, IFN-c, and TNFa) treatment for 2 days. The inhibition of PI3 kinase by LY294002 and wortmannin antagonizes the protective effects of albumin from cytokines-induced b-cell death [14]. Moreover, Bad, a member of Bcl-2 family involves in the regulation of apoptosis, gets phosphorylated by Akt [24], which links upstream with cell survival signals and downstream with anti-apoptotic signals. Therefore, increased levels of pAkt might correspond to phosphorylation of Bad as observed in our study. Hence, this work might reflect a possible role of Akt dependent Bad phosphorylation which leads to b-cell survival, and imply the importance of insulin signal transduction in pancreatic b-cells. Other insulin signaling pathways and/or downstream factors of Akt, such as GSK-3 or FoxO, should be investigated to verify their roles in the future. Moreover, mitochondria might participate in the development of apoptosis by cytokines. Treatment of MIN cells with TNF-a and IFN-c synergistically induces the production of reactive oxygen species (ROS), loss of MMP, and apoptosis [25]. Similar result also has been shown in INS cells which have been treated with IL-1b leading to a significant reduction in the MMP [26]. It is possible that the loss of MMP causes an increase in the permeability of mitochondrial membrane and leads to the

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Fig. 6. Densitometric measurements of pAkt (A) and pBad (B) protein levels among different groups were performed. There was a decreased Akt phosphorylation after cytokines treatment alone compared with control (A, p < 0.01). The presence of quercetin or naringenin significantly increased Akt phosphorylation to that of control levels after cytokines toxicity (A, p < 0.05). The PI3 kinase inhibitor, LY294002, blocked the activation of Akt expression (A, p < 0.05). Phosphorylated Bad levels expressed the similar patterns as for pAkt (B). Data are shown as the means ± SEM of 5–6 individual experiments.

Fig. 5. Densitometric measurements of p-p38 MAPK, iNOS and COX2 protein levels were analyzed among different groups. Cytokines treatment induced higher expression of (A) p-p38 MAPK and (B) iNOS (p < 0.05), but did not affect (C) COX2 levels as compared to control. Quercetin or naringenin did not modify p-p38 MAPK and iNOS expression after cytokines treatment. Data are shown as the means ± SEM of 4–6 individual experiments.

release of small proapoptotic molecules such as cytochrome c and mitochondria-derived activators of caspases, which consequently induce caspase-dependent apoptotic cell death [27]. Therefore, maintenance of MMP could be an important parameter for mitochondrial function and one of the indicators for cellular health.

Quercetin and naringenin might have potential to prevent the loss of MMP due to cytokines toxicity in our study. Many studies have suggested that cytokines-induced b-cells apoptosis is preceded by complex modifications in gene expression. Pro-inflammatory cytokines might induce the formation of ROS [25,28,29], which could trigger an inflammatory response through the activation of transcription factor NF-jB. NF-jB then translocates into the nucleus where it activates a variety of inflammatory genes such as iNOS, COX2, cytokines (IL-6, IL-1b, TNF-a etc.) and chemokines (MCP-1, IL-8, etc.) [30–32]. The pro-inflammatory cytokines IL-1b and TNF-a could activate NF-jB, and their expression is possibly induced in response to NF-jB activation, thus forming an amplifying feed-forward loop and a vicious cycle,

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Fig. 7. Analysis of apoptosis by the Cell Death Detection ELISA. Cells were treated with vehicle control and cytokines (TNF-a 400 U/ml, IL-1b 80 U/ml, and IFN-c 160 U/ml) with or without quercetin or naringenin for one day. Blocker or vehicle was added 1 h before treatment. Protective effect of quercetin or naringenin from cytokines-induced cell apoptosis was abolished by PI3 kinase inhibitor (p < 0.05). Data are means ± SEM and are expressed as a percentage of the control. Individual experiments were repeated for 4–6 times.

eventually lead to b-cell dysfunction and cell death [32]. Moreover, NF-jB regulated gene expression after cytokines treatment includes the up regulation of MnSOD and down regulation of Pdx1 [28,29]. Although the expression of protective proteins, such as mitochondrial MnSOD or heat shock protein 70, is induced by cytokines, up-regulation of the iNOS and the consequent intracellular production of NO, together with changes in the expression of other NF-jB regulated genes, eventually lead to b-cell death [21,28]. Since cytokines-induced b-cell damages could be via the formation of ROS [28,29], the antioxidant enzyme MnSOD might be involved in cellular defenses against deleterious radicals and/or cellular repair during diabetes [33]. Overexpression of MnSOD could reduce cytokines-induced activation of NF-jB in b-cells by 80%. However, catalase (CAT), glutathione peroxidase (GSHPx), and the cytoplasmic Cu/ZnSOD (cytoplasmic isoform of SOD) do not have effect [29]. It seems like that the cellular MnSOD status might not contribute significantly to cell viability and integrity in our study, although quercetin has been shown to increase the reduced antioxidant enzyme activities, such as SOD, GSHPx, and CAT activities, which protects against streptozotocin-induced oxidative stress and b-cell damage in rat pancreas [11]. IL-1b and IFN-c-induced NF-jB activation and subsequent transcriptional regulation of iNOS and COX2 expression have been shown to be responsible for NO production and destruction of b-cells [18,19]. However, the p-38 inhibitor, partially prevents cytokine-induced apoptosis, is not associated with lowered iNOS expression or reduced nitrite production [34]. Several flavonoids have been implied to inhibit the NF-jB dependent genes, iNOS and COX2 expressions. Quercetin, apigenin, and luteolin treated in RIN cells significantly reduce in IL-1b and IFN-c induced NO production, which is correlated with reduced iNOS mRNA and protein levels through the inhibition of NF-jB activation after 48 h. These flavonoids also prevent the decrease of GSIS in rat islets by IL-1b and IFN-c [10]. However, quercetin or naringenin might not prevent cytokines-induced INS-1 cell death through the regulation of iNOS, p-p38 MAPK, or the downstream COX2 expressions in our study. Moreover, dietary phenolic compounds might stimulate the deacetylase activity of SIRT1, which regulates various cellular processes such as cell survival, stress related processes, and life span extension in response to caloric restriction [35,36]. SIRT1 overexpression completely prevents the increased iNOS and NO

production by cytokines, which involves the inhibition of the NFjB signaling pathway [36]. In addition, SIRT1 also functions as a positive regulator of insulin secretion in response to glucose [36,37]. However, the protective effects of quercetin and naringenin might not be associated with SITR1 in our study. Cytokinesmediated signal transduction in b-cells seems to involve pathways leading to the activation of JNK, ERK, and STAT1, besides the NF-jB pathway [7,18–20,34]. In summary, we have demonstrated the inhibitory effects of quercetin and naringenin against toxicity of cytokines in b-cells, an effect that might not be associated with reduced iNOS or p-p38 MAPK expression. Quercetin and naringenin induce pAkt activation which could participate in b-cell protection from cytokines-induced cell apoptosis, possibly by a NO-independent mechanism. Furthermore, MMP reduction after cytokines toxicity might be improved in the presence of quercetin and naringenin treatments, suggesting the maintenance of mitochondrial membrane integrality and cellular protection. Apparently, quercetin and naringenin, contained in many fruits and vegetables, could be potent agents to benefit b-cell mass preservation in diabetes. Acknowledgements The author deeply thanks Dr. Christopher Newgard (Duke University Medical Center, Durham, NC) and Dr. Peter Butler (Larry Hillblom Islet Research Center, University of California, Los Angeles, CA) for assistances, and Dr. Cheng-hong Hsieh from Asia University, Taiwan, for technical assistance. Funding Information: This work was supported by research grants from the National Science Council in Taiwan (NSC 972320-B-468-002-MY3). References [1] Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 2003;52:102–10. [2] Kahn SE. The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of Type 2 diabetes. Diabetologia 2003;46:3–19. [3] Donath MY, Storling J, Berchtold LA, Billestrup N, Mandrup-Poulsen T. Cytokines and beta cell biology: from concept to clinical translation. Endocr Rev 2008;29:334–50. [4] Venieratos PD, Drossopoulou GI, Kapodistria KD, Tsilibary EC, Kitsiou PV. High glucose induces suppression of insulin signalling and apoptosis via upregulation of endogenous IL-1beta and suppressor of cytokine signalling-1 in mouse pancreatic beta cells. Cell Signal 2010;22:791–800. [5] Welsh N, Cnop M, Kharroubi I, Bugliani M, Lupi R, Marchetti P, et al. Is there a role for locally produced interleukin-1 in the deleterious effects of high glucose or the type 2 diabetes milieu to human pancreatic islets? Diabetes 2005;54:3238–44. [6] Heitmeier MR, Scarim AL, Corbett JA. Interferon-g increases the sensitivity of islets of Langerhans for inducible nitric-oxide synthase expression induced by interleukin 1. J Biol Chem 1997;272:13697–704. [7] Natalicchio A, De Stefano F, Orlando MR, Melchiorre M, Leonardini A, Cignarelli A, et al. Exendin-4 prevents c-Jun N-terminal protein kinase activation by tumor necrosis factor-{alpha} (TNF{alpha}) and inhibits TNF{alpha}-induced apoptosis in insulin-secreting cells. Endocrinology 2010;151:2019–29. [8] Crozier A, Jaganath IB, Clifford MN. Dietary phenolics: chemistry, bioavailability and effects on health. Nat Prod Rep 2009;26:1001–43. [9] Dembinska-Kiec A, Mykkänen O, Kiec-Wilk B, Mykkänen H. Antioxidant phytochemicals against type 2 diabetes. Br J Nutr 2008;99(E Suppl. 1):ES109–17. [10] Kim EK, Kwon KB, Song MY, Han MJ, Lee JH, Lee YR, et al. Flavonoids protect against cytokine-induced pancreatic beta-cell damage through suppression of nuclear factor kappaB activation. Pancreas 2007;35:1–9. [11] Coskun O, Kanter M, Korkmaz A, Oter S. Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and beta-cell damage in rat pancreas. Pharmacol Res 2005;51:117–23. [12] Chen WP, Chi TC, Chuang LM, Su MJ. Resveratrol enhances insulin secretion by blocking K(ATP) and K(V) channels of beta cells. Eur J Pharmacol 2007;568:269–77. [13] Jambal P, Masterson S, Nesterova A, Bouchard R, Bergman B, Hutton JC, et al. Cytokine-mediated down-regulation of the transcription factor cAMPresponse element-binding protein in pancreatic beta-cells. J Biol Chem 2003;278:23055–65.

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