Article pubs.acs.org/JAFC

Cinnamtannin D‑1 Protects Pancreatic β‑Cells from Palmitic AcidInduced Apoptosis by Attenuating Oxidative Stress Ting Wang,†,∥ Peng Sun,§,†,∥ Liang Chen,§ Qi Huang,† Kaixian Chen,§,† Qi Jia,§ Yiming Li,*,§ and Heyao Wang*,† †

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

§

S Supporting Information *

ABSTRACT: In previous studies, A-type procyanidin oligomers isolated from Cinnamomum tamala were proved to possess antidiabetic effect and protect pancreatic β-cells in vivo. The aim of this study was to unveil the mechanisms of protecting pancreatic β-cells from palmitic acid-induced apoptosis by cinnamtannin D-1 (CD1), one of the main A-type procyanidin oligomers in C. tamala. CD1 was discovered to dose-dependently reduce palmitic acid- or H2O2-induced apoptosis and oxidative stress in INS-1 cells, MIN6 cells, and primary cultured murine islets. Moreover, CD1 could reverse palmitic acid-induced dysfunction of glucose-stimulated insulin secretion in primary cultured islets. These results indicate that reduction of apoptosis and oxidative stress might account for the protection effect of CD1, which provided a better understanding of the mechanisms of the antidiabetic effects of procyanidin oligomers. KEYWORDS: cinnamtannin D-1, pancreatic β-cells, apoptosis, oxidative stress

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INTRODUCTION

definitely useful in improving insulin sensitivity and restoring glucose homeostasis. As a commonly used spice, cinnamon has been investigated as a potential preventive supplement in T2D for over 20 years.11 Procyanidin polymers have been identified to be the main components of cinnamon.12 It can exert antidiabetic effects because of its insulin-like bioactivity13 and the inhibitory effect on α-glucosidase.14 We also found that A- and B-type procyanidin oligomer-rich extracts from different species of cinnamon barks had hypoglycemic effects in streptozotocininduced diabetic rodents.15,16 In a recent study, we isolated welldefined ingredients from cinnamon extracts and found that extracts from Cinnamomum tamala could increase serum insulin levels and protect β-cells in type 2 diabetic db/db mice.17 Strikingly, we found that in C. tamala extracts, the main components were A-type procyanidin trioligomers, including cinnamtannin D-1 (CD1, PubChem CID: 46173958, 43%) and its structural isomer cinnamtannin B-1 (CB1, PubChem CID: 475277, 23.3%).17 We hypothesized that as a potential antioxidant, CD1 might be the main active ingredient in C. tamala to possess antidiabetic activity. This study disclosed that CD1 could protect pancreatic β-cells from palmitic acid (PA)-induced apoptosis and dysfunction. CD1 was found to improve the viability of PA or H2O2 impaired cells and suppress the increased ROS level in INS-1 and MIN6 pancreatic β-cells. In addition, the compound protected β-cells from PA-induced apoptosis and decreased iNOS and NO synthesis in both INS-1 and primary cultured islets. Moreover,

Free fatty acids (FFAs) have multiple physiological effects in pancreatic β-cells, in insulin secretion, energy metabolism, and membrane synthesis.1,2 However, chronic exposure to elevated levels of FFAs could impair β-cell function and induce β-cell apoptosis both in vitro and in vivo,3 which is known as lipotoxicity. Although an elevated circulating FFA level has been recognized as one of the major contributors to type 2 diabetes (T2D), unfortunately, the mechanisms are not yet fully understood. As reported in previous studies, FFAs could induce β-cell apoptosis via various mechanisms, such as endoplasmic reticulum stress (ER stress), elevated production of cytokines, and oxidative stress.2 During the progression of T2D, chronic elevated FFAs damage pancreatic β-cell function and reduce βcell mass.2,4 The failure of β-cells subsequently accelerates the progression of T2D. Therefore, protection of β-cells has been regarded as a new strategy for the treatment of T2D. Pancreatic β-cells are more sensitive to oxidative stress due to its lower expression level of antioxidative enzymes, such as catalase and superoxide dismutase.5 Prolonged exposure to high FFAs causes β-cell mitochondrial dysfunction and the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which subsequently activate stresssensitive intracellular signaling pathways and result in oxidative stress.6,7 Thus, oxidative stress plays an important role in FFAinduced β-cell apoptosis and dysfunction. Research has shown that compounds with antioxidant properties can protect β-cells both in vitro and in vivo.8,9 In addition, some antioxidants, such as α-lipoic acid and RRR-α-tocopherol, have been verified to provide beneficial effects on clinical treatments of T2D.10 Although whether antioxidants are effective therapeutic agents for T2D is still under debate, in fact, many of the compounds are © 2014 American Chemical Society

Received: Revised: Accepted: Published: 5038

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Figure 1. Cinnamtannin D-1 (CD1) protects INS-1 cells from PA-induced decrease in cell viability. (A) Chemical structure of CD1. (B) CD1 protection against PA-induced decreasing of cell viability in INS-1 cells. Cell viability was detected, and the results are shown as inhibition ratio (% of control). All data are expressed as the mean ± SE (n = 6). Asterisks denote significant difference versus the PA-treated alone group (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001). rates were calculated by comparison with the control group (treated with 0.5% BSA alone, 100%). In the H2O2-treated β-cell model, INS-1 or MIN6 cells were preincubated with CD1 for 4 h and exposed to 500 μM H2O2 for 1, 2, and 4 h, respectively. Then the cell viability was measured by MTT assay. Hoechst 33342 staining was performed to evaluate the proportion of apoptotic cells. INS-1 cells were seeded in a 24-well plate on cover glasses. After incubation with different concentrations of CD1 in the presence/absence of 0.4 mM PA for 48 h, the cells were fixed in 4% paraformaldehyde and stained with Hoechst 33342. The photos were taken by fluorescence microscopy (DP70, Olympus, Japan), and the apoptotic ratio in each group was analyzed. Determination of Cellular ROS and NO Production. After the INS-1 cells were preincubated with different concentrations of CD1 for 4 h, 10 μM CM-H2DCFDA was added into cells to load fluorogenic probes at 37 °C for 30 min. Then the cells were incubated with or without 500 μM H2O2 for another 20 min. After removal of H2O2 and multiple washes, the ROS levels were assessed by determination of 2′,7′dichlorofluorescein (DCF), which was converted from CM-H2DCFDA through oxidation. The formation of fluorescent DCF in each well was observed by fluorescence microscopy or quantified at an excitation wavelength 488 nm and emission wavelength of 525 nm by FlexStation 3. To detect the effect of CD1 on PA-induced ROS production, the cells were treated with different concentrations of CD1 in the presence/ absence of 0.4 mM PA for 48 h; after washes, the probe was loaded, and the ROS level in each group was detect by the fluorescence. For further investigation of the effect of CD1 on PA-induced NO generation, INS-1 cells were treated with PA and different concentrations of CD1 for 48 h, and then aliquots of media from each treated group were collected. NO production was then immediately assessed by measuring the amount of nitrite in the supernatant using the Total NO Assay Kit (Beyotime) according to its instructions. Western Blot Analysis and Immunofluorescence Assay. After the INS-1 cells were treated with CD1 in the presence/absence of PA for 48 h, proteins from each group were collected and the expression levels of cleaved caspase-3 and iNOS were detected using Western blot analysis as described previously.19 The following primary antibodies and dilutions were used: rabbit anticleaved caspase-3 (Asp175, 1:1000), rabbit antiphospho-Nuclear Factor Kappa B (NFκB, Ser536, 1:1000), rabbit anti-NFκB (1:1000), rabbit antiphospho-c-Jun N-terminal kinase (JNK, Thr183/Tyr185, 1:1000), rabbit anti-JNK (1:1000), rabbit antiphospho-p38 (Thr180/Tyr182, 1:1000), rabbit anti-p38 (1:1000; all from Cell Signaling Technology, Danvers, MA, USA); rabbit antiNOS2 (iNOS, Santa Cruz Biotechnology, Dallas, TX, USA, 1:500); and mouse anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA, 1:10000). The secondary antibodies were anti-mouse IgG and anti-rabbit IgG (Jackson Laboratory, USA, 1:10000). The expression of protein was measured by optical density using ImageJ (National Institutes of Health, Bethesda, MD, USA). For double-immunofluorescence staining of insulin and iNOS, INS-1 cells or primary cultured islets were grown on coverslips in a six-well

restoration of PA-injured insulin secretion in islets by CD1 was also observed.

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MATERIALS AND METHODS

Materials and Reagents. All general reagents for cell culture were purchased from Hyclone, Shanghai, China; PA, bovine serum albumin (BSA), Hoechst 33342, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) were purchased from Sigma-Aldrich, Shanghai, China. A Total Nitric Oxide Assay Kit was obtained from Beyotime, Shanghai, China. Source of CD1. CD1 (Figure 1A) was isolated from the bark of C. tamala as previously described.17 The structure of CD1 was confirmed by mass spectrometry (MS) and nuclear magnetic resonance (NMR). The compound purity was approved by high-performance liquid chromatography (HPLC: column, Agilent Extend C18 column, 5 μm, 4.6 mm × 250 mm; solvent system, acetonitrile−0.2% acetic acid with gradient elution; flow rate, 1 mL/min; UV detection, 280 nm; Agilent 1200, Agilent Technologies, Santa Clara, CA, USA). CD1 was >95% pure. Cell Culture and Mouse Pancreatic Islets Isolation. The INS-1 rat insulinoma cell line was cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum (FBS) and 50 μM β-mercaptoethanol. The MIN6 mouse insulinoma cell line was maintained in Dulbecco’s modified Eagle medium (DMEM) containing 25 mM glucose, 10% FBS, and 50 μM βmercaptoethanol. All cells at passage 10−15 were kept at 37 °C and 5% CO2 in humidified air. Pancreatic islets were isolated from 6-week-old male C57BL/6J mice (Slac, Shanghai, China). Briefly, the mice were anesthetized by chloral hydrate, and collagenase V (1 mg/mL concentration, Sigma-Aldrich, Shanghai, China) was injected into the pancreas through the common bile duct. The pancreas was quickly removed for digestion at 37 °C for 15 min. The intact islets were handpicked from the digest solution after multiple washes and maintained in RPMI-1640 medium supplemented with 10% FBS, 10000 units/mL penicillin, and 10000 mg/mL streptomycin with 11.1 mM glucose in a sterile cell incubator at 37 °C with 5% CO2 infusion and humidified 95% air. The islets were used in the following experiments after 24 h of culture. All animal care and experiments were permitted by Institutional Animal Care and Use Committees of Shanghai Institute of Materia Medica. Cell Viability Assay and Hoechst 33342 Staining. First, to investigate the effect of CD1 on cell viability in PA-treated pancreatic βcells, INS-1 and MIN6 cells were seeded in 96-well plates. PA/BSA solution was prepared as previously described.18 The cells were treated with different doses of CD1 in the presence or absence of 0.4 mM PA and incubated for 48 h. Cell viability was determined by MTT assay. Briefly, the cells were incubated with MTT regent (0.5 mg/mL) at 37 °C for 4 h, and the absorbency at a wavelength of 492 nm was measured by FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA). The inhibition 5039

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Figure 2. CD1 protects INS-1 β-cells from PA-induced apoptosis. (A) Representative fluorescent images of Hoechst 33342 staining in different treated INS-1 cells. Cells were treated with 0.5% BSA alone, 0.4 mM PA/0.5% BSA with or without 25 μM, and 50 μM CD1 for 48 h, and then Hoechst staining was performed. The brighter nuclei indicated Hoechst 33342-positive apoptotic cells. Scale bar = 50 μm and refers to all panels. (B) Statistical analysis of Hoechst 33342 staining showing the apoptotic rate in differently treated groups. The apoptotic rate was calculated as Hoechst 33342-positive nuclei divided by all nuclei (n = 6). (C) Western analysis of the effect of CD1 on the expression of cleaved caspase-3. (D) Histogram of the statistical results of Western blot analysis data. The optical density of each blot band of cleaved capsase-3 was determined and adjusted by the optical density of β-actin; the blots were performed for at least three independent experiments. All data are expressed as the mean ± SE. Asterisks denote significant difference versus the PA-treated alone group (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001). plate and then incubated with 0.5% BSA alone or PA/BSA or PA/BSA mixed with different concentrations of CD1 for 48 h. The immunofluorescence was performed as previously described.20 Briefly, INS-1 cells or islets were fixed in 4% paraformaldehyde and incubated with primary antibodies (rabbit anti-insulin, Cell Signaling Technology, dilution 1:100; mouse anti-NOS2, Santa Cruz, dilution 1:100) overnight followed with secondary antibodies (Alexafluor 488-conjugated goat anti-rabbit IgG and Alexafluor 546-conjugated goat anti-mouse IgG, Invitrogen, dilution 1:400). Then the nuclei were stained with Hoechst 33342, and the coverslips were mounted and observed by confocal microscope (FV1200, Olympus, Japan). A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was carried out to evaluate the apoptotic cells in islets. After islets were treated with different concentrations of CD1 in the presence/absence of 0.4 mM PA, the apoptotic cells in islets were detected by TUNEL kit (Roche, USA) according to the manufacturer’s instructions. Hoechst 33342 was used for visualization of nuclei. The rate of apoptosis was calculated as the number of TUNEL-positive cells divided by the area of each islet. Determination of Glucose-Stimulated Insulin Secretion (GSIS). Islets of approximately the same size were plated in 24-well plates (10 islets per well) for insulin secretion studies. Islets were incubated with CD1 in the presence/absence of 0.4 mM PA for 48 h. Then the culture medium was removed, and cells or islets were washed and preincubated in Krebs−Ringer buffer (KRB; 135 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 2 mM NaHCO3, 10 mM HEPES, and 0.1% w/v BSA, pH 7.4) without glucose for 1 h at 37 °C. Thereafter, the cells/islets were incubated in KRB containing 5 mM glucose for 1 h at 37 °C and then in 25 mM glucose for another 1 h. The supernatant was collected, and the insulin level was measured by using an insulin ELISA kit (Millipore, Bedford, MA, USA) according to its protocol. Statistical Analysis. All data were expressed as the mean ± SE. The comparison of different groups was assessed by t test or one-way ANOVA analysis followed by Dunnett’s test. Differences were considered statistically significant at p < 0.05.

lines (Supporting Information Supplementary Figure 1A). To evaluate the protective effect of CD1 on PA-induced β-cell apoptosis, we first examined the protective effect of CD1 on cell viability in PA-treated INS-1 cells. It was shown that CD1 could dose-dependently increase cell viability in the presence of 0.4 mM PA in INS-1 cells (inhibitory rates were 95.2 ± 2.2% at 50 μM, 92.1 ± 2.1% at 25 μM, 88.4 ± 2.9% at 12.5 μM, 79.9 ± 3.9% at 6.25 μM, 69.3 ± 1.2% at 3.12 μM, and 67.1 ± 2.1% at 1.56 μM vs 59.7 ± 1.5% for the PA-treated alone group, Figure 1B), which suggested that CD1 could protect pancreatic β-cells from PAinduced decrease in cell viability. To further evaluate whether the protective effect of CD1 on cell viability was associated with apoptosis, Hoechst 33342 staining was then performed to detect the apoptotic rate. In Hoechst 33342 staining, the nuclei of apoptotic cells visualized by fluorescence microscopy displayed a high condensed chromatin compared with normal cells.21 Cells treated with 0.4 mM PA showed brighter nuclei with highly condensed DNA and nuclei shrinkage, whereas an obviously reduced apoptotic rate was observed in CD1 (25/50 μM) and PA cotreated INS-1 cells (Figure 2A,B). The activation of caspase-3 was identified as a key role in the execution of cell apoptosis. In this study, it was found that CD1 significantly reduced cleaved caspase-3 level in PAtreated cells at doses of both 25 and 50 μM compared with PAtreated alone group (Figure 2C,D). All of these data suggested that CD1 could protect β-cells from PA-induced apoptosis. CD1 Attenuates PA-Induced ROS, NO Production, and iNOS Expression in INS-1 β-cells. We then investigated the effects of CD1 on some oxidative stress-associated markers in PA-induced β-cell apoptosis. By detecting DCF fluorescence, ROS generation was significantly decreased in CD1 and PA cotreated cells compared with PA-treated alone INS-1 cells (Figure 3A). Moreover, as shown in Figure 3B, total NO synthesis was obviously inhibited by CD1 in a dose-dependent manner. Increased expression of iNOS can lead to sustained

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RESULTS CD1 Protects INS-1 β-Cells from PA-Induced Apoptosis. There was no cytotoxic effect of CD1 on pancreatic β-cell 5040

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Figure 3. CD1 reduced PA-induced oxidative stress in INS-1 cells. (A) INS-1 cells were treated with different concentrations of CD1 in the presence/ absence of 0.4 mM PA for 48 h. Quantified ROS production in the cells was detected (n = 3). (B) Cells were treated with different concentrations of CD1 in the presence/absence of 0.4 mM PA for 48 h. NO production was then detected (n = 3). (C) INS-1 cells were treated with 25 and 50 μM CD1 in the presence/absence of 0.4 mM PA for 48 h, and the expression levels of iNOS and β-actin were detected by Western blot analysis. (D) The optical density of each blot band of iNOS was determined and modified by the optical density of β-actin; the blots were performed for at least three independent experiments. (E) INS-1 cells were treated as above, and double immunofluorescence of insulin and iNOS was performed. Insulin was marked with green fluorescence, whereas iNOS was red; nuclei were stained by Hoechst 33342 (blue), and figures were merged. Scale bar = 50 μm and refers to all panels. This experiment was repeated at least three times. All data are expressed as the mean ± SE. Asterisks denote significant difference versus the PA-treated alone group (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001).

elevation of NO, which was a major contributor in β-cell dysfunction and apoptosis.22 Therefore, to investigate the expression level of iNOS in pancreatic β-cells, Western blot and immunofluorescence were performed in INS-1 β-cells and primary cultured islets. As expected, CD1 abolished the increasing expression level of iNOS induced by PA (Figure 3C,D). Double-immunofluorescence also showed that there were fewer iNOS-positive cells in CD1 and PA cotreated INS-1 cells than in PA-treated alone cells (Figure 3E), which was consistent with the results of Western blot analysis. Furthermore, to verify the effect of CD1 on iNOS expression in pancreatic βcells, primary mouse islets were isolated and cultured for immunofluorescence staining. Compared with normal cultured islets, PA obviously increased iNOS expression. However, addition of 25 and 50 μM CD1 to culture medium could obviously suppress this deleterious effect (Figure 4A). CD1 Decreases PA-Induced Apoptosis and Protects βCell Function against PA-Induced Impairment of GSIS in Isolated Mouse Islets. To further investigate whether CD1, an A-type proanthocyanidin trimer isolated from C. tamala, could reduce PA-induced apoptosis in primary cultured islets, TUNEL staining was carried out to detect the apoptotic cells by labeling DNA fragmentation.23 After incubation with 0.4 mM PA for 48 h, the number of TUNEL-positive cells increased obviously in

comparison with untreated islets. However, CD1 suppressed this pernicious effect significantly (Figure 4B,C). It is known that chronic exposure to elevated levels of PA can induce β-cell apoptosis as well as impair β-cell insulin secretion. Therefore, the protective effect of CD1 against PA-induced impairment of insulin secretion in primary cultured islets was also investigated. It was found that PA-impaired GSIS function was partly restored by CD1 in a dose-dependent manner (Figure 4D). Together, results suggested that this compound could suppress PA-induced impairment of GSIS as it reduced the apoptosis of pancreatic β-cells. CD1 Suppressed PA-Induced Phosphorylation of NFκB and JNK but Not p38 MAPKs. Multiple mechanisms are involved in PA-induced β-cell apoptosis, such as ER-stress, oxidative stress, and inflammatory effect. CD1 was involved as an antioxidant with the inhibitory activity against cyclooxygenase2.24 Therefore, in addition to iNOS, inhibition of other PAactivated pro-inflammatory gene expression in pancreatic β-cells might be associated with the protective effect of CD1. It was found that PA-induced phosphorylations of NFκB (Figure 5A,D) and JNK (Figure 5B,E) were significantly reduced in the presence of CD1. However, there was no obvious effect of CD1 on PA-activated p38 phosphorylation (Figure 5C,F). 5041

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Figure 4. (A) CD1 affected iNOS expression in primary cultured islets. Islets were isolated and cultured with CD1 and PA for 48 h. Red fluorescence indicated iNOS expression, and Hoechst 33342 showed nuclei. The merged figures are shown in below. Scale bar = 50 μm and refers to all panels. (B) CD1 reduced cell apoptosis in PA-simulated primary cultured islets and restored PA-injured insulin secretion. After islets were treated with CD1 and PA, TUNEL staining was performed to investigate the apoptotic ratio of cells in primary cultured islets. TUNEL-positive nuclei were marked by red fluorescence. Representative TUNEL-positive cells are indicated by white arrows. All nuclei were stained with Hoechst 33342, which showed blue fluorescence. The merged figures are shown below. Scale bar = 50 μm and refers to all panels. (C) Histogram shows the number of TUNEL-positive cells modified by the area of each islet. At least 15 islets were analyzed in each group. (D) After treatment with CD1 and PA, isolated islets were incubated at 5 mM glucose and successive 25 mM. The supernatant was collected and used for insulin determination (n = 3). All data are shown as the mean ± SE. The statistics result showed significant difference versus the PA-treated alone group (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001).

Figure 5. Western blot analysis of NFκB, JNK, and p38 phosphorylation. After the cells were treated with/without different concentrations of CD1 in the presence/absence of PA, the cell lysate was collected and followed phosphorylation proteins were detected. (A) The phosphorylation rate of NFκB was detected; β-actin was used as an internal. (B) The phosphorylation rate of JNK was detected; β-actin was used as an internal. (C) The phosphorylation rate of p38 was detected; β-actin was used as an internal. (D−F) The phosphorylation rate was measured by the expression of phosphorylated protein divided by total protein expression and adjusted by β-actin expression. All blots were repeated at least three times. The statistical data are expressed as the mean ± SE. Asterisks denote significant difference versus the PA-treated alone group (∗, p < 0.05; ∗∗, p < 0.01).

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DISCUSSION

type procyanidin trioligomers, including >43% CD1 and >23% CB1, one structural isomer of CD1.17 A-type polymers were reported to reduce peroxide-induced pancreatic acinar cell injury,27,28 generate insulin-like biological activity,13 and improve insulin sensitivity in vivo.29 However, as effective antioxidants24 and the major components of antidiabetic natural products,17 the effects of A-type polymers in C. tamala have not yet been

Cinnamon exerts antidiabetic properties both in vitro and in vivo, and its antioxidative polyphenol components have been supposed to be the main contributor.25,26 In a previous study, we have verified that C. tamala extracts exert antidiabetic effects and improve pancreas functions in a type 2 diabetic db/db mouse model.17 As reported, the main components of extracts were A5042

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explored on insulin-secreting β-cells. The present work focused on disclosing the mechanisms of the protective effects of CD1, one major A-type polymers in C. tamala. We demonstrated that CD1 could protect pancreatic β-cells from PA-induced apoptosis via attenuating oxidative stress in INS-1, MIN6 pancreatic β-cell line, and primary cultured murine islets. These findings also provide a new understanding of the mechanisms for the antidiabetic effects of procyanidin oligomers. Many antioxidants have been reported to protect β-cells via their antioxidative activity.30,31 Oxidative stress is involved in the pathobiology of T2D and its complications. During the development of T2D, high glucose and FFAs caused mitochondrial damage and overproduction of reactive species, such as ROS and RNS. These free radicals activate several stresssensitive pathways including NF-κB, JNK/SAPK, and PKC, which lead to pancreatic β-cell death, insulin resistance, and impaired insulin secretion.32 Combined with abnormal oxidation, oxidative stress leads to DNA damage, protein oxidationm and lipid peroxidation in various kinds of cells during T2D. We found that CD1 exhibited significant radical scavenging activity in INS-1 and MIN6 cells under oxidative stress condition induced by H2O2. Pretreatment with CD1 dose-dependently attenuated H2O2-induced decrease of cell viability in both INS-1 and MIN6 cells (Supporting Information Supplementary Figure 2A,B). The intracellular ROS level in each group was also detected. H2O2-induced ROS elevation was significantly reduced by pretreatment of CD1 in a dose-dependent manner (Supporting Information Supplementary Figure 2C,D). Therefore, CD1 might also reduce the elevated level of ROS and NO production induced by PA in β-cells. Excessive and sustained generation of NO derived from iNOS plays a pivotal role in FFA-induced β-cell dysfunction and apoptosis.33 Rachek et al. reported that elevated NO production induced by FFA could damage mitochondrial DNA and ultimately lead to apoptosis in INS-1 cells.34 Meanwhile, evoked iNOS activity in pancreatic β-cells could lead to impairment of GSIS both in vivo and in vitro.35,36 Therefore, inhibition of iNOS expression might reduce production of NO and exert a protective effect on β-cells. Hynes et al. demonstrated that inhibition of iNOS by lentiviral vector-based shRNA technology could protect β-cells from cytokine-induced apoptosis.37 In this study, we found that CD1 could significantly suppress PA-induced ROS generation and NO production. PA-induced increase of iNOS expression level was also significantly inhibited by CD1 in both INS-1 cells and primary cultured islets. Similar results had also been confirmed on MIN6 β-cell line (Supporting Information Supplementary Figure 1C,D). Together the results suggested that the antioxidative activity might be one of the main mechanisms of the protective effect of CD1 on PA-injured βcells. Long-term exposure to FFAs results in increased β-oxidation and elevated reactive species levels in both islets38 and β-cell lines.39 Moreover, β-cell failure, including apoptosis and dysfunction, could be mediated obviously by oxidative stress in chronic exposure to elevated levels of FFAs in pancreatic islets.40 Our experiments demonstrated that CD1 could significantly suppress PA-induced oxidative stress and subsequently reduced the apoptotic rate in PA-injured β-cells/islets. Together the results indicated that CD1 could protect pancreatic β-cells from PA-induced apoptosis. As the compound showed great protective effect on cell viability and antiapoptosis in pancreatic β-cells cells, it was inferred that CD1 might have a protective effect on insulin

secretion. In primary cultured islets, CD1 suppressed PAinduced impairment of GSIS. We speculated that the protective effect of CD1 on PA-treated islets could improve insulin secretion. Therefore, CD1 treatment may provide beneficial effects to the function of insulin secretion through its protective effect on pancreatic β-cells. However, the underlying mechanism of the antioxidative effect of CD1 on pancreatic β-cells still needed further investigation. CD1 had been reported to act as an antioxidant with the inhibitory activity against cyclooxygenase-2,24 and cyclooxygenase-2 could be regulated by an inflammatory-associated gene toll-like receptor-4 (TLR4).41 Therefore, the expression of some downstream pro-inflammatory gene was detected in this study. We found that CD1 could significantly reduce PA-induced phosphorylation of NFκB and JNK. However, PA-activated p38 phosphorylation was not attenuated by CD1. Lee et al. had confirmed that JNK activation could be involved with TLR4 signaling pathway in palmitic acid-treated INS-1 cells and decrease inhibitory factor kappa B alpha (IκBα),42 which was in accordance with our results. Therefore, the TLR4 pathway could at least associate with the protective effect of CD1 on pancreatic β-cells. In any case, further investigation on the effect mechanism of CD1 would still be necessary. In conclusion, this study showed that CD1 protects pancreatic β-cells against PA-induced oxidative stress via its antioxidative properties. As the major component of C. tamala, CD1 could be the main active ingredient in C. tamala to possess antidiabetic activity and provide benefit for the protection of pancreatic βcells during the development of T2D.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figures 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(Y.L.) E-mail: [email protected]. *(H.W.) E-mail: [email protected]. Author Contributions ∥

T.W. and P.S. contributed equally to this work.

Funding

This work was supported by a grant from the National Nature Science Foundation of China (81072681), the National High Technology Research and Development Program of China (“863” Program, 2007AA02Z301), the State Key Program of Basic Research of China grant (“973” program, 2009CB918502), and the National Science and Technology Major Project (2012ZX09301001-001). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor S. Seino for his gift of MIN6 cells. REFERENCES

(1) Itoh, Y.; Hinuma, S. GPR40, a free fatty acid receptor on pancreatic β-cells, regulates insulin secretion. Hepatol. Res. 2005, 33, 171−173. (2) Robertson, R. P. β-Cell deterioration during diabetes: what’s in the gun? Trends Endocrinol. Metab. 2009, 20, 388−393. 5043

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