Caffeic Acid Phenethyl Ester (Propolis Extract) Ameliorates Insulin

Caffeic acid phenethyl ester (CAPE), extracted from propolis, was evaluated for the ameliorative effects on insulin resistance and the mechanisms were...
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Caffeic Acid Phenethyl Ester (Propolis Extract) Ameliorates Insulin Resistance by Inhibiting JNK and NF-κB Inflammatory Pathways in Diabetic Mice and HepG2 Cell Models Jiarui Nie,† Yaning Chang,*,† Yujia Li,† Yingjun Zhou,† Jiawen Qin,† Zhen Sun,† and Haibin Li‡ †

The State Key Laboratory of Bioreactor Engineering, College of Bioengineering, East China University of Science and Technology, Shanghai 200237, People’s Republic of China ‡ Bee Forest Industry Co., LTD, Shanghai 200030, People’s Republic of China ABSTRACT: Caffeic acid phenethyl ester (CAPE), extracted from propolis, was evaluated for the ameliorative effects on insulin resistance and the mechanisms were identified, using non-insulin-dependent diabetes mellitus (NIDDM) model mice and insulin resistance (IR) model cells. After 5 weeks of CAPE supplementation, insulin sensitivity, hyperlipidemia, and peroxisome proliferator-activated receptor-α (PPAR-α) levels were improved in mice. Proinflammatory cytokines in serum and the expressions of tumor necrosis factor-alpha (TNF-α) mRNA in tissues were markedly downregulated from CAPE-treated mice. In vitro, CAPE supplement significantly improved glucose consumption, glucose uptake, glycogen content, and oxidative stress and decreased expression of glucose-6-phosphatase (G6Pase) mRNA in cells. Both in vivo and in vitro, CAPE enhanced p-Akt (Ser473) and p-insulin receptor substrate (IRS)-1 (Tyr612), but inhibited p-JNK (Thr183/Tyr185), p-NF-κB p65 (Ser536), and nuclear translocation of p-NF-κB p65 (Ser536). In summary, CAPE can ameliorate insulin resistance through modulation of JNK and NF-κB signaling pathway in mice and HepG2 cells. KEYWORDS: caffeic acid phenethyl ester, insulin resistance, inflammation, JNK, NF-κB

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INTRODUCTION The population of non-insulin-dependent diabetes mellitus (NIDDM) is gradually increasing in developed countries.1 NIDDM is also called type 2 diabetes mellitus (T2DM) with characteristics of hyperglycemia, insulin deficiency, and insulin resistance, and is a chronic disease lacking insulin sensitivity.2 Considering the cause of the disease, there is considerable evidence that obesity-induced NIDDM and insulin resistance are associated with metabolic inflammation.3,4 Obesity, one of the diabetic complications, is characterized by the excess of lipid profile such as total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), triglyceride (TG), and adipose tissue mass but little high-density lipoprotein cholesterol (HDL-c).4 In addition, peroxisome proliferator-activated receptors (PPARs), regulating nutrient−gene interaction, play an important role in glucose and lipid metabolic processes, which prevent the metabolic syndrome and become effective targets for treatment.5 The effective therapy for T2DM is controlling blood glucose strictly, and decreasing the risk of diabetic complications. The c-Jun N-terminal kinase (JNK) and the nuclear factor-kappa B (NF-κB) signaling pathways contribute to inflammation and play key roles in obesity and insulin resistance.3,4,6−8 In addition, these signaling pathways regulate the expression of proinflammatory molecules. The proinflammatory cytokine tumor necrosis factor-alpha (TNFα) is a mediator in both of these pathways. TNF-α is able to activate the JNK signaling pathway by binding to its receptors, and the activity of JNK can also regulate the expression of the TNF gene.9 Similarly, monocyte chemotactic protein (MCP)-1 and interleukin (IL)-6 are proinflammatory cytokines that are associated with insulin resistance and T2DM.10,11 IL-6 is able to © 2017 American Chemical Society

inhibit the signal transduction of insulin receptor and reduce insulin sensitivity in mouse and HepG2.10 T2DM, obesity, and hyperinsulinemia are associated with adipocyte dedifferentiation which is induced by increase of MCP-1.11 In addition, oxidative injury also plays a vital role in the pathological mechanisms of diabetes. Under diabetic conditions, oxidative stress mediator reactive oxygen species (ROS) are referred to the progression of β-cell deterioration and insulin resistance.12 In diabetic complications, hyperglycemia can induce proinflammatory cytokine MCP-1 and TNF-α secretion, which is regulated by ROS and NF-κB pathways.13 In JNK and NF-κB pathways of the diabetic condition, proinflammatory cytokines and oxidative stress inhibit tyrosine phosphorylation of insulin receptor substrate (IRS)-1, which can also lead to an increase in the activation of JNK phosphorylation and a decrease in Akt phosphorylation.2,12 In addition, IRS-1, Akt, and JNK are three upstream targets of NF-κB. The activated phosphorylation of JNK and inhibited phosphorylation of Akt can increase the activation of NF-κB.2,7 These diabetic and insulin resistant processes lead to activation of the NF-κB and JNK pathways which inhibit insulin biosynthesis and action.12 In consideration of side effects of drugs, diverse scientific investigations are being incessantly conducted into natural products which have a preventive or therapeutic effect on improving insulin sensitivity.1,14 Specifically, propolis, a resinous substance of natural product, is widely used in medicine Received: Revised: Accepted: Published: 9041

June 22, 2017 August 10, 2017 August 11, 2017 August 11, 2017 DOI: 10.1021/acs.jafc.7b02880 J. Agric. Food Chem. 2017, 65, 9041−9053

Article

Journal of Agricultural and Food Chemistry

Figure 1. High pressure liquid chromatogram of CAPE. Retention time of (A) CAPE standard and (B) CAPE sample. CAPE, caffeic acid phenethyl ester.

and food,15 obtained by bees from plants, exudates, and shoots.14 Some reports demonstrated that propolis is able to improve lipid metabolism and insulin sensitivity in T2DM rats.1,16 Also, propolis exerts effects of antioxidation and antiinflammation.17 At present, it remains largely unknown what compound in propolis supplementation might influence glucose metabolism in insulin resistance and diabetes. Caffeic acid phenethyl ester (CAPE), an active compound in propolis, can suppress oxidative stress in diabetic liver18 and heart.19 Interestingly, CAPE has been proven to inhibit NF-κB and contributes to the anti-inflammatory activity of propolis, while the synergism among CAPE, galangin, and other compounds does not.20,21 Therefore, it is promising and meaningful to investigate the antidiabetic effect and mechanisms of CAPE. In this study, we investigated whether CAPE extracted from propolis has protective effects on insulin resistance and to explore the relevant mechanism. NIDDM model mice in vivo and human hepatoma HepG2 cell in vitro were used in this study to validate if CAPE exerts the ability of diabetes improvement by JNK and NF-κB signaling pathway.

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mol/L HCl was added to pH = 3.5. Acticarbon (w/v = 3%) was added and stirred for 2 min. This procedure was repeated three times. The crude solution was filtered through a 0.45 μm membrane, loaded on a 717# (Shanghai resin factory, Shanghai, China) macroporous resin column (2.6 cm × 30 cm), and washed with deionized water at 1.00 mL/min. The column was then eluted with chloroform−methanol (20:1) mixed solvent at 1.00 mL/min. CAPE eluent was collected at 35−45 min and vacuum concentrated. CAPE extract was freeze-dried to powder and stored at −20 °C until use. The content of CAPE extract was calculated using authentic CAPE standards (purity >97%, purchased from Sigma-Aldrich, Shanghai, China) as the standard. Two milligrams of CAPE extract was dissolved in 100 mL of MeOH (Aladdin, Shanghai, China), and the mixture was filtered through a 0.45 μm membrane. The content of CAPE was detected by a high pressure liquid chromatograph (LC-2010A, Shimadzu, Japan) equipped with a UV detector and a HP-C18 (InertSustain, Tokyo, Japan) capillary column (4.6 mm × 250 mm, 5.0 μm). The HPLC settings were as follows: the mobile phase was acetonitrile (Sigma-Aldrich, Shanghai, China)/water/glacial acetic acid (Aladdin, Shanghai, China) (v/v) = 55:44:1. The flow rate (1.0 mL/ min), wavelength (325 nm), column temperature (30 °C), and injection volume (20 μL) were set. One milligram of authentic CAPE standard was dissolved in 1 mL of MeOH as stock solution. Stock solutions were used to manufacture the calibration of curves of CAPE standard (y = 5498.4x; r2 = 0.9993). As shown in the CAPE chromatogram (Figure 1), the yield of CAPE was 3.79 ± 0.2% (w/w) from propolis and the purity of CAPE extract was 87.3 ± 2.2%. Thus, this CAPE extracted from propolis was used in the present study. Animal and Diets. Generation of nongenetic mouse models of non-insulin-dependent diabetes mellitus (NIDDM) was performed according to the procedure of Mu et al.22 Before model establishment, three-week-old male C57BL/6J mice were purchased from SLAC Laboratory Animal (Shanghai, China). After 3 days of acclimatization, mice were placed on the high fat (HF) diet D12492 (60% of kilocalories from fat, purchased from Research Diets, New Brunswick, NJ, USA). After HFD feeding for 3 weeks, mice were injected once with low-dose streptozotocin (STZ, intraperitoneal at 90 mg/kg, purchased from Sigma-Aldrich, Shanghai, China) to induce partial insulin deficiency. STZ was dissolved in 0.1 mol/L sodium citrate

MATERIALS AND METHODS

Extraction and Purification. The refined propolis (100.0 g, supplied by Bee Forest Industry Co., LTD, Shanghai, China) homogenate with chloroform−methanol (20:1, purchased from Aladdin, Shanghai, China) mixed solvent was put into a 1000 mL volumetric flask. The solution was extracted at 50 °C in an ultrasonic cell disintegrator (JY92-2D, Xinzhi Bio-Science, Ningbo, China) for 30 min. The extract was centrifuged (CENCE, Changsha, China) at 4500 rpm for 15 min, the supernatant was obtained, and then 2 mol/L HCl (Aladdin, Shanghai, China) was added until pH = 3.0. The solution was combined with an equal volume of petroleum ether (Aladdin, Shanghai, China) and centrifuged at 5000 rpm for 15 min. In addition, the supernatant was obtained, vacuum concentrated, and then diluted with ethanol (Aladdin, Shanghai, China) to 200 mL. The solution was extracted at 50 °C in the ultrasonic cell disintegrator for 20 min, and 1 9042

DOI: 10.1021/acs.jafc.7b02880 J. Agric. Food Chem. 2017, 65, 9041−9053

Article

Journal of Agricultural and Food Chemistry Table 1. Changes of Body Weight (BW), Food Intake, and Tissue Weight (TW) after CAPE Treatment in Micea normal

HF + C15

HF + C30

HF CON

12.3 ± 0.9 22.4 ± 1.4 b 24.5 ± 0.8 b 2.8 ± 0.6 b

12.3 ± 0.9 24.9 ± 1.3 a 25.6 ± 1.1 b 2.6 ± 0.5 b

12.4 ± 0.7 24.4 ± 1.8 a 24.9 ± 1.2 b 2.8 ± 0.5 b

12.4 ± 0.9 24.8 ± 1.1 a 26.9 ± 2.0 a 3.2 ± 0.6 a

3.9 ± 0.3 1.6 ± 0.5 1.3 ± 0.1 b

4.2 ± 0.1 1.7 ± 0.2 2.1 ± 0.4 a

4.1 ± 0.5 1.8 ± 0.4 1.9 ± 0.4 a

4.3 ± 0.5 1.9 ± 0.2 2.2 ± 0.4 a

BW before model establishment (g) after model establishment (g) posttreatment (g) food intake (g/day) TW liver wt (% BW) muscle wt (% BW) epididymal fat (% BW) a

Significant differences were considered when p < 0.05 and are indicated by different letters. CON, control; C15, CAPE-15 mg/kg; C30, CAPE-30 mg/kg.

Table 2. Effects of CAPE on HOMA-IR in Micea normal before model establishment serum fasting glucose (mmol/L) after model establishment serum fasting glucose (mmol/L) serum fasting insulin (μIU/mL) HOMA-IR posttreatment serum fasting glucose (mmol/L) serum fasting insulin (μIU/mL) HOMA-IR

HF + C15

HF + C30

HF CON

4.6 ± 0.2

4.5 ± 0.1

4.6 ± 0.3

4.4 ± 0.3

4.5 ± 0.3 b 16.8 ± 0.8 a 3.4 ± 0.3 b

22.0 ± 0.6 a 10.0 ± 0.6 b 9.7 ± 0.5 a

21.8 ± 0.5 a 9.8 ± 0.7 b 9.5 ± 0.4 a

21.9 ± 0.4 a 10.0 ± 0.7 b 9.7 ± 0.4 a

4.7 ± 0.3 d 16.9 ± 0.8 a 3.5 ± 0.2 d

15.5 ± 0.5 b 12.4 ± 0.8 b 8.5 ± 0.6 b

12.2 ± 0.7 c 12.1 ± 1.6 b 6.6 ± 0.9 c

23.6 ± 0.6 a 8.8 ± 0.7 c 9.3 ± 0.8 a

a

Significant differences were considered when p < 0.05 and are indicated by different letters. CON, control; C15, CAPE-15 mg/kg; C30, CAPE-30 mg/kg. Blood and Tissue Collection. Mice were fasted for 12 h and blood was collected from the retro-orbital plexus after treatment for 5 weeks. After blood collection, liver, muscle, and epididymal adipose tissues were weighed (Table 1), collected, and stored at −80 °C for further tests. Biochemical Determinations. TC, TG, HDL-c, and LDL-c concentrations in mouse serum were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Peroxisome proliferator-activated receptor-α (PPAR-α) concentration was measured by mouse ELISA kits (Jining, Shanghai, China). Serum glucose levels were measured by mouse ELISA kits (Sigma-Aldrich, Shanghai, China). Serum insulin levels were measured by commercial ELISA kits (Crystal Chem, Downers Grove, IL, USA). Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated by the formula fasting glucose (mmol/L) × fasting insulin (μU/mL)/ 22.5. All parameters above (TC, TG, HDL-c, LDL-c, PPAR-α, serum glucose, serum insulin) were measured using a ThermoFisher spectrophotometer Go-1510 (ThermoFisher, Shanghai, China). Levels of cytokines (TNF-α, IL-6, MCP-1) in serum were assayed by Milliplex Map multiplex commercial kit (Millipore, USA) in accordance with the manufacturer’s approach and measured by using Luminex 200 Milliplex MAP (Darmstadt, USA). Cell Culture and Treatment. Human hepatoma HepG2 cells (Keygen, Nanjing, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, purchased from Keygen, Nanjing, China) with 10% fetal bovine serum (ThermoFisher, Waltham, MA, USA), 100 IU/mL penicillin (Keygen, Nanjing, China), and 100 μm/mL streptomycin (Keygen, Nanjing, China) at 37 °C in 5% CO2 and passed every 3 days by trysinization. After 70−80% confluence, the cells were washed by phosphate-buffered saline (PBS) twice and incubed overnight in DMEM (serum-free). Then cells were cultured in normal (5 mM glucose) DMEM as a control group, high glucose (25 mM glucose) DMEM plus palmitic acid (PA, 0.25 mM, purchase from Aladdin, Shanghai, China) as an IR group, and high glucose DMEM plus PA in the presence of CAPE with different concentrations for 24 h. Cell viability in serum-free medium incubation of CAPE was

buffer (pH 4.5). Body weight (BW) changes of model establishment are shown in Table 1. Three weeks after STZ injection, as shown in Table 2, hyperglycemia and insulin resistance were observed in the majority of HF diet/STZ-treated mice as previously reported.23 After 6 weeks of HF diet feeding (9 weeks of age), 30 NIDDM model mice with similar body weight and degrees of hyperglycemia were randomly divided into two groups of CAPE and one group of vehicle supplement. For CAPE treatments, the three groups of NIDDM model mice were given by HF diet and gavage with (a) C15 (n = 10), CAPE 15 mg/kg body weight, and (b) C30 (n = 10), CAPE 30 mg/kg body weight (CAPE was dissolved in edible polyethylene glycol (PEG)-400 (Sigma-Aldrich, Shanghai, China), and the dosage was performed according to Bezerra et al. with modification24), and (c) control (n = 10), the same dosage of vehicle. The nondiabetic normal control experimental group (C57BL/6J mice, n = 10) was given normal diet (AIN-93G diet) and gavage with vehicle. All groups were treated for 5 weeks and were provided free fresh food and water. Food intake (per day) and body weight (per week) of mice were tested. Body weight of posttreatment and food intake are shown in Table 1. The mice were individually housed in a control room with temperature (22 ± 3 °C) and humidity (50% ± 10%) and 12-h light (7 am to 7 pm)/dark cycle. The care of the animals was according to the National Institute of Health guidelines for the Care and Use of Laboratory Animals. All studies were approved by the Institute Animal Care and Use Committee (Protocol number: SYXK (Hu) 2014-0016). All the experimental diets were packed in zipper plastic freezer bags (0.5 kg/batch) and stored at −20 °C in the dark and thawed before feeding. Oral Glucose Tolerance Test. Mice were given glucose solutions by gavage (2 g/kg) after fasting overnight. After glucose was given, mouse blood was obtained from a tail prick and tested at 0, 15, 30, 60, 90, and 120 min using a OneTouch Glucose Meter (Johnson & Johnson, New Brunswick, NJ, USA). Oral glucose tolerance test (OGTT) of all groups was measured before and after 5 weeks of intervention. 9043

DOI: 10.1021/acs.jafc.7b02880 J. Agric. Food Chem. 2017, 65, 9041−9053

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Journal of Agricultural and Food Chemistry

rpm for 10 min at 4 °C, and the supernatants were used for analyzing. Nucleoprotein extraction of mouse tissues and HepG2 cells were prepared and performed using a nucleoprotein protein extraction kit (Sangon Biotech, Shanghai, China) in accordance with the manufacturer’s approach. Treated cells or tissues were harvested and washed twice with PBS. Cold hypotonic buffer was added, and samples were homogenized at 4 °C. The solution was centrifuged (Cence, Changsha, China) at 3000 rpm for 5 min, and then the supernatant was immediately discarded. Then cold hypotonic buffer was added, the sample was shaken and washed for 30 s and centrifuged (Cence, Changsha, China) at 5000 rpm for 5 min at 4 °C, and the supernatant was discarded. 0.2 mL of lysis buffer (2 μL PMSF, 1 μL of phosphatase inhibitor, and 0.2 μL DTT) was added to the precipitate, and the sample was shaken on ice for 20 min and then centrifuged (Cence, Changsha, China) at 15000 rpm for 10 min at 4 °C. The supernatant is a nuclear extract and is preserved at −70 °C before analysis. The total protein and nucleoprotein was measure using a BCA protein assay kit (Keygen, Nanjing, China) in accordance with the manufacturer’s approach. The protein of liver, muscle, and HepG2 samples was mixed with 2× Laemmli sample buffer (Sigma-Aldrich, Shanghai, China), and the protein of epididymal adipose samples was mixed with 5× Laemmli sample buffer (Sigma-Aldrich, Shanghai, China) and then boiled at 95 °C for 5 min. Equal amounts of each protein sample were separated by SDS−PAGE (Keygen, Nanjing, China) and transferred to PDVF membranes (Millipore, Billerica, MA, USA) which were blocked with 5% skim milk powder (Sangon Biotech, Shanghai, China) in TBST buffer (50 mM Tris, pH 7.5, 0.05% Tween-20, and 500 mM sodium chloride) for 2 h at room temperature. Incubation of the primary antibodies was carried out at 4 °C overnight for p-IRS-1 (Tyr612) (ThermoFisher, Waltham, MA, USA), p-JNK (Thr183/Tyr185), p-Akt (Ser 473), p-NF-κB p65 (Ser536), α-tubulin, and lamin A (Cell Signaling Technology, Danvers, MA, USA), and samples were rinsed three times with TBST buffer for 5 min each. After washing, the membranes were incubated in anti-rabbit secondary antibody conjugated to horseradish peroxidase (Cell Signaling Technology) at room temperature for 2 h. Chemiluminescence (ECL, PerkinElmer Life Science, Boston, MA, USA) and scanning with a Tannon imaging system (Tannon-4200, Shanghai, China) were used to detect protein bands. Tannon Image 1.0 software was used for densitometry analysis. Statistical Analyses. All the experiments were calculated with mean and standard error values. Statistical comparisons among experimental groups were analyzed by one-way ANOVA and Duncan’s multiple-comparison test using the SPSS software program (Version 21; SPSS). Significant differences were considered when p < 0.05 and are indicated by different letters. Unpaired, two-paired t tests were also used to determine statistical significance.

assessed with an MTT assay kit (Keygen, Nanjing, China) according to the manufacturer’s approach. Five replications of treatments and two repetitions of experiment were required in MTT analysis. Glucose Consumption Analysis. Glucose consumption was determined according to Yan et al.25 with some modification. Briefly, HepG2 cells (4 × 103 cells/well) were cultured in a 96-well plate with five wells for 5 mM glucose DMEM and five wells for 25 mM glucose DMEM plus PA left as blanks. After the treatments for 24 h followed by insulin (100 nM, purchased from Sigma-Aldrich, Shanghai, China) incubation for 30 min, the medium was removed and glucose consumption was calculated by the glucose concentration of blank wells minus glucose concentrations in plated wells by using the SBA90 biosensor (Biology Institute of Shandong Academy of Sciences, Jinan, China). Five repetitions of treatments and two replications of experiment were required. Glucose Uptake Estimation. According to Yan et al.,25 glucose uptake was determined with some modification. HepG2 cells (4 × 104 cells/well) were cultured in a 24-well plate. The 24-h treatments were followed by insulin (100 nM) for 30 min and 0.1 mM 2-NBDG (Keygen, Nanjing, China) for 45 min incubation at 37 °C. Images were obtained using identical acquisition settings on a Nikon ECLIPSE Ti-S fluorescence microscope (Nikon, Tokyo, Japan). Three independent replicates of the experiment were required. Glycogen Content Determination. HepG2 cells (3 × 105 cells/ well) were cultured in a 6-well plate. After 24-h treatments, cells were washed by PBS and incubated insulin (100 nM) for 30 min. Then glycogen content of cells was estimated using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s approach. Three independent replicates of the experiment were required. ROS Level Estimation. ROS levels were analyzed according to Tian et al.26 with some modification. HepG2 cells (4 × 103 cells/well) were cultured in a 96-well plate, and after the treatments for 24 h, the medium was removed and washed by PBS three times. Treated cells were incubated in DMEM with 2′,7′-dichlorofluorescein diacetate (DCFH-DA, 10 μM, purchased from Beyotime, Shanghai, China) for 30 min at 37 °C. ROS levels were examined by a fluorescence plate reader (Molecular Devices Spectra Max M3, Sunnyvale, CA, USA) at 488 nm for excitation and 525 nm for emission. The increase of intracellular ROS was expressed as the increased value compared to the control group. Five repetitions of treatments and two replications of experiment were required. Real-Time PCR Analysis. Total RNA from HepG2 cells or tissues was extracted by RNA extraction kit (Takara, Beijing, China) in accordance with the manufacturer’s protocol. The cDNA was synthesized from RNA by the cDNA kit (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s protocol. SYBR Green Kit (Tiangen, Beijing, China) was used for real-time PCR. Specific primers for genes were used as follows. For glucose-6phosphatase (G6Pase): 5′-CATTGACACCACACCCTTTGC-3′ and 5′-CCCTGTACATGCTGGAGTTGAG-3′; PCR product size was 89 bp. For β-actin of HepG2: 5′-GGACTTCGAGCAAGAGATGG-3′ and 5′-AGCACTGTGTTGGCGTACAG-3′; PCR product size was 234 bp. For TNF-α: 5′-GAGTCCGGGCAGGTCTACTTT-3′ and 5′CAGGTCACTGTCCCAGCATCT-3′; PCR product size was 235 bp. β-Actin of C57BL/6J mice: 5′-TTCCTTCTTGGGTATGGAAT-3′ and 5′-GAGCAATGATCTTGATCTTC-3′; PCR product size was 203 bp. A CFX96 system (Bio-Rad, Hercules, CA, USA) was used for the reactions as follows: 95 °C for 15 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s. Three independent replicates of the experiment were required. Isolation of Total Protein and Nucleoprotein and Western Blot Assay. Total protein of mouse tissues (100 mg) was extracted by homogenization with 1 mL of cold RIPA lysis and extraction buffer (ThermoFisher, Waltham, MA, USA) and phosphatase and protease inhibitors (Roche, Shanghai, China). Total protein of HepG2 was extracted by homogenization with cold NP-40 assay lysis buffer (Keygen, Nanjing, China) containing PMSF (Keygen, Nanjing, China) with phosphatase and protease inhibitors (Roche, Shanghai, China). Total protein was centrifuged (Cence, Changsha, China) at 12000

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RESULTS Body and Tissue Weight and Food Intake. Before the model establishment, body weights were similar in all mice (Table 1). Body weights of NIDDM model mice were significantly higher than those fed normal mouse diet after model establishment (6 weeks of HF diet and single low dose STZ treatment). After 5 weeks of HF or normal diet with or without CAPE treatment (posttreatment), mice fed the HF + 15 and HF + C30 had mean body weights of 25.6 and 24.9 g, which were significantly lower than the 26.9 g of those fed the HF diet, but no significant differences compared with the 24.5 g of normal group mice. The trends of food intake in mice fed HF + C15 and HF + C30 were similar to that of body weight. No marked difference was observed in liver weight and muscle weight among all groups, but epididymal fat of normal mice was 1.3 g, which was significantly lower than that of model mice (HF + C15:, 2.1 g; HF + C30, 1.9 g; HF CON, 2.2 g). Treatments did not cause any toxicity or abnormal daily behaviors in mice. 9044

DOI: 10.1021/acs.jafc.7b02880 J. Agric. Food Chem. 2017, 65, 9041−9053

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Journal of Agricultural and Food Chemistry

Figure 2. Effects of CAPE on OGTT in mice. (A) OGTT, (B) AUC, (C) TC, (D) TG, (E) HDL-c, (F) LDL-c, and (G)PPAR-α in HF-diet-fed or normal-fed mice (mean ± SEM, n = 10 per group, P < 0.05).

Glucose Tolerance and Glucose Homeostasis. HOMAIR was used to analyzed insulin sensitivity. At the end of the study, the highest serum fasting glucose and HOMA-IR indexes and the lowest serum fasting insulin were found in mice fed HF diet and given gavage of vehicle. Mice given a gavage of CAPE 15 mg/kg and 30 mg/kg body weight showed 34.3% and 48.3% lower serum fasting glucose level compared to mice fed HF diet and given gavage of vehicle (Table 2). Low-dose and high-dose CAPE also reduced HOMA-IR indexes compared to the HF group (Table 2). OGTT areas under the curve (AUC) were

20.8% lower for the HF + C30 groups in comparison with HF (Figures 2A and 2B), suggesting that CAPE improved insulin sensitivity, homeostasis, and glucose tolerance. Serum Lipid Level and Inflammatory Markers. TC, TG, HDL-c, and LDL-c concentrations in serum were analyzed to confirm the anti-hyperlipidemic effects of CAPE (Figure 2C− F). The HF group exhibited a dramatic increase in TC, TG, and LDL-c levels and a decrease in HDL-c levels in serum compared with the normal group, which was significantly alleviated by administration of high-dose CAPE. Moreover, HF 9045

DOI: 10.1021/acs.jafc.7b02880 J. Agric. Food Chem. 2017, 65, 9041−9053

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Figure 3. Effects of CAPE on proinflammatory cytokines in HF-diet-fed or normal-fed mouse serum. Proinflammatory cytokine levels of (A) IL-6, (B) TNF-α, and (C) MCP-1 (mean ± SEM, n = 10 per group, P < 0.05).

in the normal group, while low-dose and high-dose CAPE treatments significantly inhibited these increases (Figures 4E, 4F, 6E, and 6F), while only HF + C30 supplementation markedly lowered the phosphorylation of JNK and NF-κB in muscles (Figures 5D and 5E). In addition, the nuclear translocation of NF-κB was assayed with Western blot on pNF-κB p65 (Ser536) in nucleus. The HF + C15 and HF + C30 treatments also significantly decreased the translocation of NFκB into nucleus in muscle and liver, but only high-dose CAPE treatment can markedly reduce the nuclear translocation of NFκB in epididymal adipose tissues (Figures 4G, 5F, and 6G). These data suggest that CAPE treatment stimulated insulin signaling by downregulating inflammatory markers through the NF-κB pathway and the JNK pathway. Effect of CAPE on Cell Viability, Glucose Metabolism, and Oxidative Stress. The doses of CAPE in subsequent experiment were evaluated by cell viability through MTT assays (Figure 7A). CAPE at concentrations exceeding 100 ng/mL reduced cell viability. Thus, CAPE concentrations below 60 ng/ mL were used in subsequent experiments. It is obvious that the glucose consumption and glucose uptake in the IR cell model decreased compared to the control. Besides, the dose of high glucose and PA had little toxicity. The results above indicated that the IR cell model was successfully built using HepG2 cells. CAPE significantly enhanced glucose consumption by 12.6%, 17.1%, and 24.3% at 10, 30, and 60 ng/mL, respectively (Figure 7B). Meantime, CAPE at three concentrations was able to prevent the inhibition of glucose uptake induced by high glucose and PA (Figures 7C and 7D). The expression of G6Pase mRNA was increased markedly in IR model cells

group mice showed significantly lower PPAR-α level in serum when compared with normal group mice (Figure 2G). However, the PPAR-α level was increased by 38.6% in highdose CAPE-treated mice, compared with HF group mice (Figure 2G). Proinflammatory cytokine levels of IL-6, TNF-α, and MCP-1 were attenuated by 36.2%, 32%, and 4.7% in highdose CAPE-treated mice, compared with HF group mice (Figure 3). Both low-dose and high-dose CAPE administration reduced levels of proinflammatory cytokines in serum and exhibited anti-inflammatory effects. The adverse effects on these metabolic and inflammatory parameters were mitigated by CAPE supplement. CAPE Stimulated Insulin Signaling by Downregulating Inflammatory Markers through NF-κB Pathway and JNK Pathway in Mice. In order to determine the metabolic and anti-inflammatory mechanisms of CAPE in mice, we assessed changes in insulin signaling molecules and inflammatory-related genes and protein. CAPE significantly decreased the expression of TNF-α mRNA in livers and epididymal adipose tissues of NIDDM mice (Figures 4A and 6A). The HF + C30 supplementation produced a 1.54-fold, 1.64-fold, and 1.49-fold increase of p-IRS1 (Tyr612) levels in livers, muscles, and epididymal adipose tissues, respectively, as compared to the HF group (Figures 4B, 4C, 5A, 5B, 6B, and 6C). Consistently, HF + C15 and HF + C30 treatments significantly increased the levels of p-Akt (Ser 473) as compared to HF group in muscles and epididymal adipose tissues (Figures 5C and 6D), but there was no significant difference in that of liver (Figure 4D). In the HF group, p-JNK (Thr183/Tyr185) and p-NF-κB p65 (Ser536) in livers and adipose tissues were much higher than 9046

DOI: 10.1021/acs.jafc.7b02880 J. Agric. Food Chem. 2017, 65, 9041−9053

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Figure 4. Effects of CAPE on JNK and NF-κB pathways in livers from NIDDM model or normal-fed mice. (A) TNF-α mRNA expression. (B) Western blotting of IRS-1 phosphorylation at Tyr612, AKT phosphorylation at Ser473, JNK phosphorylation at Thr183/Tyr185, NF-κB p65 phosphorylation at Ser536, α-tubulin (loading control for T: total protein), and lamin A (loading control for N: nucleoprotein). (C) Densitometric analysis of p-IRS1, (D) p-AKT, (E) p-JNK, (F) p-NF-κB p65 in total protein, (G) p-NF-κB p65 in nucleoprotein (mean ± SEM, n = 10 per group, P < 0.05).

JNK pathway. The intracellular ROS levels were measured by DCFH-DA to determine if CAPE has the effect of reducing ROS production. The level of ROS in HepG2 cells was significantly increased by high glucose plus PA treatment, compared with control group (Figure 7G). 30 and 60 ng/mL CAPE markedly attenuated ROS production in IR condition.

compared to the control (Figure 7E). At 60 ng/mL, CAPE suppressed the expression of G6Pase mRNA as much as the control. Additionally, glycogen content was significantly recovered when HepG2 cells were treated with CAPE as comparison with IR model cells (Figure 7F). Intracellular ROS levels may be one of the signaling mediators in regulating the 9047

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Figure 5. Effects of CAPE on JNK and NF-κB pathways in muscles from NIDDM model or normal-fed mice. (A) Western blotting of IRS-1 phosphorylation at Tyr612, AKT phosphorylation at Ser473, JNK phosphorylation at Thr183/Tyr185, NF-κB p65 phosphorylation at Ser536, αtubulin (loading control for T: total protein), and lamin A (loading control for N: nucleoprotein). (B) Densitometric analysis of p-IRS1, (C) p-AKT, (D) p-JNK, (E) p-NF-κB p65 in total protein, (F) p-NF-κB p65 in nucleoprotein (mean ± SEM, n = 10 per group, P < 0.05).

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According to the above results, we selected 30 and 60 ng/mL to investigate further mechanisms of CAPE improving insulin resistance in HepG2 cells for following experiments. CAPE Inhibits Upregulation of JNK and NF-κB Pathways Induced by High Glucose and PA in HepG2 Cells. Protein levels of p-IRS1 (Tyr612) were inhibited but pJNK (Thr183/tyr185) was activated in IR cells, however these adverse effects were significantly prevented by 30 and 60 ng/ mL CAPE treated in cells in a dose-independent way (Figures 8A, 8B, and 8D). Results of Figures 8C and 8E also indicated that CAPE at 30 and 60 ng/mL protected p-Akt (Ser473) and decreased p-NF-κB p65 (Ser536) activation in IR model cells. Treatments of C30 and C60 can also significantly lower the translocation of NF-κB into nucleus in cells (Figure 8F). These results supported the hypothesis that CAPE blocked JNK and NF-κB signaling pathways by activating p-IRS1(Tyr612) and pAkt (Ser473) and inhibited p-JNK (Thr183/tyr185) and p-NFκB p65 (Ser536).

DISCUSSION

Insulin resistance and obesity-induced T2DM are connected with metabolic syndrome and inflammation.3,4 An active lifestyle approach is an essential factor to manage the risk of NIDDM and T2DM development. As a strategy to recover or prevent metabolic syndrome or disease, it is necessary to research certain foods or their bioactive compounds. Although several reports suggested that propolis has an antidiabetic effect without apparent side effects,1,27−30 few have investigated which compound contributes to antidiabetes and what are the underlying mechanisms. CAPE, isolated from propolis, possesses anti-inflammatory31 activity and alleviates type 1 diabetic mellitus (T1DM) effect on mice in vivo.32 Therefore, we investigated whether CAPE is able to improve glucolipid metabolic disorder and insulin resistance related to inflammatory markers and pathways such as the JNK and NF-κB signaling pathways. This study demonstrated that CAPE is an effective compound in propolis for HF/STZ-induced NIDDM in mice and insulin resistance in HepG2 cells. 9048

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Figure 6. Effects of CAPE on JNK and NF-κB pathways in adipose tissues from NIDDM model or normal-fed mice. (A) TNF-α mRNA expression. (B) Western blotting of IRS-1 phosphorylation at Tyr612, AKT phosphorylation at Ser473, JNK phosphorylation at Thr183/Tyr185, NF-κB p65 phosphorylation at Ser536, α-tubulin (loading control for T: total protein), and lamin A (loading control for N: nucleoprotein). (C) Densitometric analysis of p-IRS1, (D) p-AKT, (E) p-JNK, (F) p-NF-κB p65 in total protein, (G) p-NF-κB p65 in nucleoprotein (mean ± SEM, n = 10 per group, P < 0.05).

CAPE extracted from propolis to administer to animals. In the present study, we also found that CAPE showed diminished levels of HOMA-IR and a significant decrease in the AUC of the OGTT, although effects were little less than in the normal group. It demonstrated that CAPE can improve insulin sensitivity in NIDDM mice. For lipid metabolism aspect, a previous study showed that water and ethanol extracts in

Considering relevance to human obesity-induced diabetes, C57BL/6J mice with HFD/STZ are good to evaluate compounds for the therapy of NIDDM in vivo.23 A previous study reported that CAPE exhibits an improvement in glucose sensitivity and amelioration in hepatic steatosis in diet-induced obesity (DIO) mouse model.24 However, in their study, they used pure CAPE to treat animals. In our present study, we used 9049

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Journal of Agricultural and Food Chemistry Figure 7. continued

fluorescence as fold related to the control. Cell viability, glucose uptake, G6Pase mRNA expression, glycogen content, and ROS fluorescence values are represented as fold relative to the HF control. Data are represented as mean ± SD. Significant differences were considered when p < 0.05 and are indicated by different letters. C, control; IR, insulin resistant model; C10, CAPE-10 ng/mL; C30, CAPE-30 ng/mL; C60, CAPE-60 ng/mL.

propolis reduced TC, TG, and LDL-c levels, but induced HDLc level in fasting rat serum.27 Our results showed that the 5week-CAPE treated mice were observed to have not only reduced TC, TG, and LDL-c and increased HDL-c but also reduced food intake and body weight compared to the HF diet group. Additionally, PPAR-α is able to modulate these lipid profiles and inflammatory pathway, as well as maintaining homeostasis.5 Although PPAR-α does not directly affect plasma glucose or insulin levels in diabetic subjects, PPAR-α agonist combined with antidiabetic agent benefited both lipid and glucose metabolism in T2DM.5 Furthermore, PPAR-α can suppress the NF-κB pathway through controlling transcription of IκB-α and IKK genes.5 Our data showed that CAPE intervention increased PPAR-α in mice, which might explain the changes of serum lipid levels in our study. These findings demonstrated that CAPE is able to resist obesity-induced diabetes and insulin resistance in NIDDM mice. IR cell model is better to be used to investigate T2DM in vitro, because it has different glucose metabolism compared with normal cells.25 Thus, HepG2 cells that formed into IR models were used as a cellular model in vitro to analyze the antidiabetic effect of CAPE. At present, we used 25 mM glucose and PA to induce the IR model, and investigate the protective effect of CAPE on IR cells. We found that CAPE was able to decrease glucose consumption and glucose uptake in IR cells, which were in agreement with previous studies.25,33 A gluconeogenic gene, G6Pase, is induced in diabetes.25,30 The high levels of G6Pase mRNA found in IR cells may explain why the glycogen contents in IR cells were low in the present study. CAPE can protect these adverse effects at 60 ng/mL. Our data demonstrated that CAPE can improve insulin resistance caused by high glucose and PA in HepG2 cells. Metabolic inflammation34 in tissues and oxidative stress13 in cells can interfere with insulin action through inhibiting the insulin signaling pathway. The excess of proinflammatory cytokine, IL-6, can disturb physiologic insulin levels through inhibiting tyrosine phosphorylation of IRS-1 and decreasing activation of Akt, which is an important kinase in regulating insulin’s downstream metabolic actions.10 Other proinflammatory cytokines, like TNF-α and MCP-1, can be elevated in obesity and cause insulin resistance in animals and in cells.2,11 Activation of NF-κB causes transcription of TNFα and MCP-1, resulting in an increased expression of TNFα and MCP-1.13 Our collected data had confirmed, similarly to a study reported by Choi et al.,27 that CAPE significantly reduced proinflammatory cytokines (TNF-α, IL-6, and MCP-1) in serum. TNF-α and oxidative stress are also able to activate the JNK pathway in diabetic conditions.12 ROS, which is related to oxidative stress, can increase MCP-1 production and activate the NF-κB pathway in diabetes.13 In our study, CAPE exerted the ability to reduce proinflammatory cytokine TNF-α in the NIDDM model and oxidative stress relative mediator ROS production in the IR cell model. Similarly, CAPE had

Figure 7. Effects of CAPE on cell viability and glycometabolism in HepG2 cells with IR induced by high glucose and PA. HepG2 cells were incubated in low glucose (5 mM) DMEM, high glucose (25 mM) DMEM with or without 10, 30, 60 ng/mL CAPE for 24 h. (A) Cell viability. (B) Glucose consumption assays from HepG2 cells incubated with different medium after 24-h treatment. (C) Glucose uptake of 2-NBDG assays from HepG2 cells incubated with different medium after 24-h treatment: (a) control, HepG2 cells incubated in low glucose DMEM; (b) IR; (c) CAPE-10 ng/mL; (d) CAPE-30 ng/ mL; (e) CAPE-60 ng/mL. (D) The quantitative estimation of Nikon fluorescence microscope. Tannon Image 1.0 software was used for densitometry analysis. (E) G6Pase mRNA expression. (F) Glycogen content represented as fold related to the control. (G) ROS 9050

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Figure 8. Effects of CAPE on JNK and NF-κB pathways in HepG2 cells. (A) Western blotting of IRS-1 phosphorylation at Tyr612, AKT phosphorylation at Ser473, JNK phosphorylation at Thr183/Tyr185, NF-κB p65 phosphorylation at Ser536, α-tubulin (loading control for T: total protein), and lamin A (loading control for N: nucleoprotein). (B) Densitometric analysis of p-IRS1, (C) p-AKT, (D) p-JNK, (E) p-NF-κB p65 in total protein, (F) p-NF-κB p65 in nucleoprotein (mean ± SEM, n = 3 per group, P < 0.05). C, control; IR, insulin resistant model; C30, CAPE-30 ng/mL; C60, CAPE-60 ng/mL.

effects on JNK, NF-κB is activated by obesity and insulin resistance.7 Previous studies indicated that CAPE can inhibit NF-κB.24,37 In vivo, the present study showed that CAPE treatment reduced TNF-α mRNA expression which was related to the increase in tyrosine phosphorylation of IRS-1 in liver and adipose tissues. Both in vivo and in vitro, NIDDM mice and IR cells induced a significant impairment in the phosphorylated levels of Akt and IRS-1 and activation in the phosphorylated levels of JNK and NF-κB p65. CAPE supplement enhanced phosphorylation of Akt and IRS-1 and inhibited JNK and NFκB p65 phosphorylation in HepG2 cells and in mice livers, muscles, and adipose tissues. In addition, CAPE can inhibit nuclear translocation of NF-κB p65 in NIDDM mice and IR cells. These results indicated that CAPE could reverse insulin resistance and inflammation through JNK and NF-κB pathways. Therefore, our study’s findings are valuable and promising for understanding that CAPE is one of the compounds of propolis that has antidiabetic effect. Furthermore, we also identified

antioxidant activity to prevent ROS production in human neutrophils at 10 μM concentration.35 It demonstrated that CAPE can affect both TNF-α and ROS, which were two signaling activated mediators in regulating JNK and NF-κB pathways. These results might show an underlying mechanism of CAPE improving insulin resistance. We used Western blot analysis in order to further investigate the signaling pathway of protective effects of CAPE against T2DM. Studies suggest that JNK3,12 and NF-κB6−8 pathways contribute to insulin resistance and link with inflammation and disrupted glucose homeostasis, and thus could be potential therapeutic targets for diabetes. JNK feedback inhibits signal transduction of insulin receptor through decreasing tyrosine phosphorylation of IRS-1.36 In addition, tyrosine phosphorylation of IRS-1 can activate the PI3K pathway to induce phosphorylation of Akt,2 which is an important mediator related to insulin’s downstream metabolic actions and inflammation.10 Activation of Akt can also lead to inhibition of NF-κB by phosphorylation.34 In a manner similar to the 9051

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Glucose Regulates a Proapoptotic Program in Retinal Pericytes. Diabetes 2002, 51, 2241−2248. (8) Bierhaus, A.; Schiekofer, S.; Schwaninger, M.; Andrassy, M.; Humpert, P. M.; Chen, J.; Hong, M.; Luther, T.; Henle, T.; Klöting, I.; Morcos, M.; Hofmann, M.; Tritschler, H.; Weigle, B.; Kasper, M.; Smith, M.; Perry, G.; Schmidt, A.-M.; Stern, D. M.; Haring, H.-U. Diabetes-Associated Sustained Activation of the Transcription Factor Nuclear Factor-kappaB. Diabetes. Diabetes 2001, 50, 2792. (9) Bennett, B. JNK: a new therapeutic target for diabetes. Curr. Opin. Pharmacol. 2003, 3, 420−425. (10) Senn, J. J.; Klover, P. J.; Nowak, I. A.; Mooney, R. A. Interleukin6 Induces Cellular Insulin Resistance in Hepatocytes. Diabetes 2002, 51, 3391−3399. (11) Sartipy, P.; Loskutoff, D. J. Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 7265−70. (12) Kaneto, H.; Matsuoka, T. A.; Nakatani, Y.; Kawamori, D.; Miyatsuka, T.; Matsuhisa, M.; Yamasaki, Y. Oxidative stress, ER stress, and the JNK pathway in type 2 diabetes. J. Mol. Med. (Heidelberg, Ger.) 2005, 83, 429−39. (13) Quan, Y.; Jiang, C. T.; Xue, B.; Zhu, S. G.; Wang, X. High glucose stimulates TNFα and MCP-1 expression in rat microglia via ROS and NF-κB pathways. Acta Pharmacol. Sin. 2011, 32, 188−93. (14) Madrigal-Santillan, E.; Madrigal-Bujaidar, E.; Alvarez-Gonzalez, I.; Sumaya-Martinez, M. T.; Gutierrez-Salinas, J.; Bautista, M.; Morales-Gonzalez, A.; Garcia-Luna y Gonzalez-Rubio, M.; AguilarFaisal, J. L.; Morales-Gonzalez, J. A. Review of natural products with hepatoprotective effects. World J. Gastroenterol 2014, 20, 14787−804. (15) Burdock, G. A. Review of the biological properties and toxicity of bee propolis (propolis). Food Chem. Toxicol. 1998, 36, 347−363. (16) Li, Y.; Chen, M.; Xuan, H.; Hu, F. Effects of encapsulated propolis on blood glycemic control, lipid metabolism, and insulin resistance in type 2 diabetes mellitus rats. Evidence-Based Complementary Altern. Med. 2012, 2012, 981896. (17) Bufalo, M. C.; Ferreira, I.; Costa, G.; Francisco, V.; Liberal, J.; Cruz, M. T.; Lopes, M. C.; Batista, M. T.; Sforcin, J. M. Propolis and its constituent caffeic acid suppress LPS-stimulated pro-inflammatory response by blocking NF-kappaB and MAPK activation in macrophages. J. Ethnopharmacol. 2013, 149, 84−92. (18) Yilmaz, H. R.; Uz, E.; Yucel, N.; Altuntas, I.; Ozcelik, N. Protective effect of caffeic acid phenethyl ester (CAPE) on lipid peroxidation and antioxidant enzymes in diabetic rat liver. J. Biochem. Mol. Toxicol. 2004, 18, 234−8. (19) Okutan, H.; Ozcelik, N.; Yilmaz, H. R.; Uz, E. Effects of caffeic acid phenethyl ester on lipid peroxidation and antioxidant enzymes in diabetic rat heart. Clin. Biochem. 2005, 38, 191−6. (20) Borrelli, F.; Maffia, P.; Pinto, L.; Ianaro, A.; Russo, A.; Capasso, F.; Ialenti, A. Phytochemical compounds involved in the antiinflammatory effect of propolis extract. Fitoterapia 2002, 73, S53−S63. (21) Jung, W. K.; Choi, I.; Lee, D. Y.; Yea, S. S.; Choi, Y. H.; Kim, M. M.; Park, S. G.; Seo, S. K.; Lee, S. W.; Lee, C. M.; Park, Y. M.; Choi, I. W. Caffeic acid phenethyl ester protects mice from lethal endotoxin shock and inhibits lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW 264.7 macrophages via the p38/ERK and NF-kappaB pathways. Int. J. Biochem. Cell Biol. 2008, 40, 2572−82. (22) Mu, J.; Woods, J.; Zhou, Y. P.; Roy, R. S.; Li, Z.; Zycband, E.; Feng, Y.; Zhu, L.; Li, C.; Howard, A. D.; Moller, D. E.; Thornberry, N. A.; Zhang, B. B. Chronic inhibition of dipeptidyl peptidase-4 with a sitagliptin analog preserves pancreatic beta-cell mass and function in a rodent model of type 2 diabetes. Diabetes 2006, 55, 1695−704. (23) Luo, J.; Quan, J.; Tsai, J.; Hobensack, C. K.; Sullivan, C.; Hector, R.; Reaven, G. M. Nongenetic mouse models of noninsulindependent diabetes mellitus. Metab., Clin. Exp. 1998, 47, 663−668. (24) Bezerra, R. M.; Veiga, L. F.; Caetano, A. C.; Rosalen, P. L.; Amaral, M. E.; Palanch, A. C.; de Alencar, S. M. Caffeic acid phenethyl ester reduces the activation of the nuclear factor kappaB pathway by high-fat diet-induced obesity in mice. Metab., Clin. Exp. 2012, 61, 1606−14.

antidiabetic and anti-inflammatory mechanisms of CAPE in NIDDM mouse model and HepG2 cells as IR model. In conclusion, our results suggest that CAPE, extracted from propolis, significantly improved hyperglycemia, glucose tolerance, and hyperlipidemia and inhibited proinflammatory cytokine contents in vivo. In in vitro study, CAPE treatment improved glucose consumption, glucose uptake, glycogen content, and oxidative stress and inhibited the expression of G6Pase. Both in vivo and in vitro, CAPE also activated IRS-1 tyrosine phosphorylation and Akt phosphorylation but inhibited phosphorylation of JNK and NF-κB p65 and nuclear translocation of NF-κB p65. Therefore, CAPE as a novel potential therapeutic agent ameliorates T2DM and insulin resistance by regulating JNK and NF-κB signaling pathways. Further studies would be interesting to evaluate the other effects of CAPE, such as amelioration of Alzheimer’s disease, which is related to the JNK pathway.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-21-64252257. Fax: 86-21-37524228. E-mail: [email protected]. ORCID

Jiarui Nie: 0000-0001-9379-7079 Yaning Chang: 0000-0003-4415-8064 Funding

This research work was supported by China Spark Program of Shanghai Municipal Agricultural Commission [Grant (2014)-61-2]. Notes

The authors declare the following competing financial interest(s): Haibin Li serves as a consultant to Bee Forest Industry Co., Ltd.

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ACKNOWLEDGMENTS We thank Xiao-Ye Du and Pei-Jun Kong for helpful discussions in animal experiments. REFERENCES

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