Journal of Ethnopharmacology 153 (2014) 344–351

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Research paper

Cyclocarya paliurus extract modulates adipokine expression and improves insulin sensitivity by inhibition of inflammation in mice Cuihua Jiang a,b, Nan Yao a, Qingqing Wang a,b, Jinghua Zhang b, Yan Sun b, Na Xiao b, Kang Liu b, Fang Huang b, Shengzuo Fang c, Xulan Shang c, Baolin Liu b, Yicheng Ni a,d, Zhiqi Yin b,n, Jian Zhang a,nn a Laboratory of Translational Medicine, Jiangsu Academy of Traditional Chinese Medicine, No. 100, Shizi Street, Hongshan Road, Nanjing 210028, Jiangsu Province, PR China b Department of Natural Medicinal Chemistry & State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, Jiangsu Province, PR China c College of Forest Resources and Environment, Nanjing Forestry University, Nanjing 210037, PR China d KU Leuven, Faculty of Medicine, Herestraat 49, 3000 Leuven, Belgium

art ic l e i nf o

a b s t r a c t

Article history: Received 22 April 2013 Received in revised form 10 January 2014 Accepted 7 February 2014 Available online 14 February 2014

Ethnopharmacological relevance: Cyclocarya paliurus Batal., a Chinese native plant, is the sole species in its genus and its leaves have been widely used as a remedy for diabetes in traditional folk medicine. The study was undertaken to evaluate the effects of Cyclocarya paliurus leaves extracts (CPE) on adipokine expression and insulin sensitivity in mice. Materials and methods: Mice were stimulated with conditioned medium (prepared from activated macrophages, Mac-CM) to induce adipose dysfunction and insulin resistance. Then mice were treated with CPE (100, 200 and 500 mg/kg, ig.) or metformin (200 mg/kg, ig.), followed by glucose and insulin intolerance, adipokine expression, phosphorylation of insulin receptor substrate (IRS-1) and glucose consumption measurement. Results: CPE, as well as metformin effectively promoted glucose disposal in oral glucose tolerance test in normal mice. Mac-CM challenge induced glucose and insulin intolerance, but CPE reversed these alternations with increased glycogen content in muscle and liver, well demonstrating its beneficial effects on glucose homeostasis. RT-qPCR analysis showed that CPE inhibited TNF-a, IL-6, MCP-1 and resistin overexpression and effectively enhanced adiponectin expression in adipose tissue when mice were exposed to Mac-CM stimulation. Inflammation impaired insulin signaling in muscle, whereas CPE inhibited inflammation-induced serine phosphorylation of IRS-1 and effectively restored the phosphorylation of both IRS-1 at tyrosine residues and downstream Akt phosphorylation in response to insulin. Moreover, independently of insulin, CPE promoted glucose consumption in adipocytes under normal and inflammatory conditions. Conclusion: Above-mentioned results demonstrated that CPE beneficially regulated adipokines expression and ameliorated insulin resistance through inhibition of inflammation in mice. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cyclocarya paliurus Macrophages-derived conditioned medium Adipokine expression Insulin resistance Inflammation

1. Introduction Obesity is reasoned as a chronic low-grade inflammatory disease, partly because it is characterized by an enhanced infiltration of macrophages in obese adipose tissue. Insulin resistance is n

Corresponding author. Tel.: þ 86 25 86185371. Corresponding author. Tel.: þ 86 25 52 3621 16; fax: þ 86 25 85 63 78 17. E-mail addresses: [email protected] (Z. Yin), [email protected] (J. Zhang). nn

http://dx.doi.org/10.1016/j.jep.2014.02.003 0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.

characterized by specific impairment of insulin PI3K signaling with decreased sensitivity to metabolic actions of insulin in skeletal muscles, liver and adipose tissue(Accili, 2004). It is well established that dysregulation of adipokine expression is tightly associated with the development of insulin resistance (Shoelson et al., 2007; Waki and Tontonoz, 2007; Guilherme et al., 2008; Matsubara et al., 2012). Proinflammatory adipokines, such as TNF-a, IL-6 and resistin, which are mainly derived from adipocytes and/or recruited macrophage collaboratively, decrease insulin sensitivity by targeting insulin signaling (Hotamisligil et al., 1995; Dandona et al., 2004; Gual

C. Jiang et al. / Journal of Ethnopharmacology 153 (2014) 344–351

et al., 2005). Inflammatory mediators induce insulin resistance and anti-inflammatory agents can reverse the alternation, indicating the link between inflammation and insulin resistance. Cyclocarya paliurus (Batal.) Iljinsk (Cyclocarya paliurus), locally known as ‘sweet tea tree’, is native to China and the sole species in its genus (Fang et al., 2006). Cyclocarya paliurus is a traditional Chinese medicinal herb with Qingrejiedu (to clear heat and toxin) and Shengjinzhike (to help produce saliva and slake thirst) efficacies recorded in Zhong Hua Ben Cao. According to the traditional Chinese medicine theory, dampness-heat obstruction and toxin stasis are two of the most important pathological factors associated with metabolic disorders, such as obesity and type 2 diabetes. Over the years, leaves of Cyclocarya paliurus have been widely used by folks for the treatment of diabetes mellitus and identified with its anti-diabetic activity by more recent research. The leaves of Cyclocarya paliurus inhibited glucosidase and glycogen phosphorylase activities (Li et al., 2011) and some studies demonstrate its hypoglycemic effects in diabetic mice (Wang, 2003; Kurihara et al., 2003b; Xu et al., 2004). Moreover, extracts from leaves of Cyclocarya paliurus were shown to strongly inhibit protein tyrosine phosphatase 1B to maintain a good anti-diabetic effect (Zhang et al., 2010). But up to now, little is yet known about its regulation of insulin action under insulin resistance conditions. In the present research, we observed the effect of Cyclocarya paliurus leaves extracts (CPE) on adipokine expression and insulin sensitivity under inflammatory conditions in mice. Our work showed that CPE beneficially regulated adipokine expression against inflammatory insult and ameliorated insulin resistance by improvement of insulin signaling in mice. Better understanding of CPE action in regulation of insulin sensitivity and identification of the possible molecular targets would be beneficial for its possible therapeutic applications in the management of insulin resistance and diabetes.

2. Materials and methods 2.1. Animals Kun Ming (KM) mice (male, weighing 18–22 g, SPF grade, certification no. SCXK2007-004) were supplied by the Experimental Animal Center of Academy of Military Medical Sciences. The Institutional Animal Care and Use Committee (IACUC) approved the project. The care and treatment of these mice were maintained in accordance with NIH publication no. 85-23 (revised in 1996) on “Principles of laboratory animal care”. 2.2. Reagents Lipopolysaccharide (E. coli serotype 055:B5, LPS) and insulin were purchased from Sigma (St. Louis, MO, USA). Metformin is a product from Sino-American Shanghai Squibb Pharma (Shanghai, China). Maxima First Strand cDNA Synthesis Kit for RT-qPCR, Maxima SYBR Green/ROX qPCR Master Mix (2X) was the product of Thermo Fisher Scientific Co., Ltd. Antiphospho-IRS-1 (Ser307) (BS4104), anti-IRS-1 (R301) (BS1408), anti-phospho-Akt (T308) (BS4008), and anti-Akt (A444) (BS1810) were supplied by Bioworld Technology (St. Paul, MN, USA); horseradish-peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA); PY99 (sc-7020) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). TRIzol Plus and 2-NBD-glucose (N13195, lot: 873337) were offered by Invitrogen (Eugene, Oregon, USA). ELISA kits for TNF-α, IL-6 were purchased from R&D Systems (Minneapolis, MN, USA).

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2.3. Cell culture and differentiation 3T3-L1 cells, a preadipocyte cell line, were purchased from the cell bank of the Chinese Academy of Sciences. Cells were cultured in a high glucose Dulbecco's Modified Eagle Medium (H-DMEM) supplemented with 10% (vol/vol) FBS, streptomycin (100 U/ml) and penicillin (100 U/ml) at 37 1C under an atmosphere of 5% CO2 and 95% humidified air. The medium was renewed every 2 days until the cells were grown to confluence. Then cells were differentiated via incubation in DMEM containing 0.5 mmol/L isobutylmethylxanthine (IBMX), 1 μM dexamethasone, 10 μg/ml insulin for 48 h, and next in DMEM (10% FBS) containing 10 μg/ml insulin alone for 2 days. After that cells were maintained in and refreshed with DMEM, 25 mmol/L glucose, and 10% FBS. More than 80% of cells exhibited the adipocyte phenotype with large lipid droplets apparent in the cytoplasm, when cells were initiated 8–12 days follow the differentiation protocol as previously described (Kang et al., 2010). 2.4. Preparation of Cyclocarya paliurus extract Leaves of Cyclocarya paliurus were air dried and ground into fine powder before extraction, which were provided by Nanjing Forestry University and authenticated by Professor Qin Mingjian from China Pharmaceutical University, and a voucher specimen (No. L20100033) was deposited in the Department of Pharmacology of Chinese Materia Medica, of the university. Qualities of the crude drugs mentioned above meet the standards of “Pharmacopoeia of the People's Republic of China” (2010 Edition). Leaves of Cyclocarya paliurus were air dried and ground into fine powder before extraction and the leaves powder (2.5 kg) was extracted three times with 10 volumes (v/w) of 80% ethanol for 2 h.Then the combined organic layer was evaporated to get crude residue (466 g, 18.64%), which was stored at 4 1C before the use. 2.5. Preparation of macrophages-derived conditioned medium (macCM) Mouse was sacrificed by cervical dislocation and PBS (5 ml) was injected intraperitoneally. Peritoneal fluid containing peritoneal macrophages was collected, after gentle abdominal massage for 2 min, then washed with PBS and cultured in six-well plates (2  106/well) for 4 h. Unadherent cells were removed by washing with PBS, while adherent cells were cultured in serum-free DMEM and stimulated by 5 μg/ml of LPS for 24 h. The supernatant was collected as conditioned medium (Mac-CM) in which TNF-α and IL-6 were assayed with enzyme-linked immunosorbent assay (ELISA) kits. A significant increase of TNF-α and IL-6 in the supernatant was considered as the activation of macrophages (Liu et al., 2011). Mac-CM was filtered through a 0.22 μm filter and stored at  70 1C. 2.6. Determination of glucose and insulin tolerance in mice After 12 h of food deprivation, mice were orally administrated with CPE (100, 200, and 500 mg/kg) or metformin (200 mg/kg) respectively. Then, mice were intraperitoneally injected with or without 0.1 ml/10 g of diluted Mac-CM (Mac-CM: PBS ¼1:2, v/v). 30 min later, mice were orally administered with glucose solution (4 g/kg). Blood were collected from orbital sinus at regular time intervals and blood glucose was measured with a commercial kit based on the glucose oxidase method. To determine insulin tolerance, mice were administrated with insulin (0.5 U/kg, s.c) 30 min after Mac-CM treatment. Blood AUC (Area Under Curve) was calculated for blood glucose as follows: 0.5  [Bg0þ Bg30]/ 2 þ0.5  [Bg30 þBg60]/2 þ1  [Bg60 þBg120]/2 (Bg0, Bg30, Bg60

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and Bg120 referred to the blood glucose concentration at 0, 30, 60 and 120 min after glucose load) (Shao et al., 2013). 2.7. Measurement of liver and muscle glycogen in mice Mice were treated as mentioned previously in Mac-CM-induced glucose intolerance test. 30 min after oral glucose load, mice were executed by cervical dislocation and the liver and skeletal muscles (gastrocnemius) were immediately removed. The dissected organization was homogenized with ice-cold PBS and glycogen (mg/g wet weight of tissue) in the suspension was determined with a commercial kit provided by Nanjing Jiancheng Bioengineering Institute, China. 2.8. RNA isolation, cDNA synthesis and real-time RT-PCR Mice fasted overnight were pretreated with CPE or metformin at given doses for 30 min, before intraperitoneally injected with 0.1 ml/ 10 g of diluted Mac-CM. Mice were killed at 1 h post-injection and the fat tissue located in epididymis was immediately removed. 100 mg fat tissue was chopped into small pieces and homogenized with 1 ml Trizol on ice. Total RNA from tissues was isolated using Trizol reagent (Invitrogen). cDNA was synthesized with the iScript Synthesis kit from Bio-Rad. Quantitative PCR was performed in 20 μl reactions using SYBR-Green Quantitative PCR Kit (Thermo). PCR reagents were offered by Thermo and used according to the instructions of the manufacturer. Amplification was performed with the ABI 7500 sequence detection system using the following protocol: 40 cycles (30 s at 95 1C and 30 s at 60 1C) after an initial activation step for 10 min at 95 1C. Primer sequences are shown as follows: β-actin [127 base pairs (bp)], forward primer: 50 -ACGGATGTCAACGTCACACT-30 , reverse primer: 50 -CACTATTGGCAACGAGCGGTTCCG-30 ; TNF-α (111 bp), forward primer: 50 -GGGACAGTGACCTGGACTGT-30 , reverse primer: 50 -CTCCCTTTGC-AGAAC TCAGG-30 ; IL-6 (173 bp), forward primer: 50 -AACCCAAGGGCATTTCAATC-30 , reverse primer: 50 -CACCGCATCTATCACCACAG-30 ; MCP-1 (129 bp), forward primer: 50 -TTAAGGCATCACAGTCCGAG-30 , reverse primer: 50 -TGAATGTGAAGTTGACCCGT-30 ; resistin (219 bp), forward

primer: 50 -GCATC-CTTACCCACTGAACCAT-30 , reverse primer: 50 -TCAAGCCTGTCTTTCACCTC-TG-30 ; adiponectin (96 bp), forward primer: 50 -CTGGTGAACACTCAGCTTGCTT-30 , reverse primer: 50 -AGACTGATGTGAGAAGGCCCAT-30 .The mRNA level of individual gene was normalized and represented as a ratio against β-actin. 2.9. Western blot analysis After 12 h of food deprivation, mice were pretreated with tested agents at given concentrations for 30 min, followed by addition of diluted Mac-CM or insulin as mentioned above in the insulin tolerance test. After 30 min, mice were put to death and the muscle tissue located in mice gastrocnemius was immediately removed and placed on ice. Muscle tissue (100 mg) was lysed with 0.5 ml cold Western blot lysis buffer [1% Triton X-100, 150 mM NaCl, 25 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na2EDTA, 1 mM Na3VO4, 1 μg/ml leupeptin, and 1 mM PMSF]. Homogenates were centrifuged at 13,000g for 15 min at 4 1C and supernatants were collected. The protein content was determined with a Bicinchoninic Acid Protein Assay kit. Protein samples (50 mg) were separated by10% SDS-PAGE and then transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes (0.22 μm). After blocked in 5% skim milk in TBST buffer (Tris–HCl 5 mM, pH 7.6, NaCl 136 mM, 0.05% Tween-20) for 2 h at room temperature, and probed with primary antibodies (1:800 dilution in TBST) overnight at 4 1C, the proteins were detected by enhanced chemiluminescence using horseradish peroxidase-labeled secondary antibodies (1:2000 dilution in TBST) at room temperature for 1 h. The signals were detected by enhanced chemiluminescence (ECL) and quantized by densitometry with Image-Pro plus 6.0 (IPP 6.0) software. 2.10. Detection of glucose consumption in adipocytes Serum-starved differentiated adipocytes (1  105/well) were cultured in 48-well plates for 4 h in Krebs–Ringer phosphate– HEPES buffer (KRHB, containing 118 mmol/L NaCl, 5 mmol/L KCl,

Fig. 1. CPE ameliorated oral glucose tolerance under normal and inflammatory conditions in mice. (A and B) Mice were orally administrated with 100, 200, and 500 mg/kg CPE or 200 mg/kg metformin (Met), then challenged with or without diluted Mac-CM (0.1 ml/10 g) for 30 min. After another 30 min, mice were orally administered with glucose (2 g/kg) and blood glucose was assayed at different time points. Metformin was taken as a positive control. (a and b) Glucose AUCs also showed the beneficial regulation of glucose tolerance by CPE in mice. Results were expressed as the mean 7 S.D. (n ¼10). #po 0.05 vs. blank 2; np o 0.05 vs. control (Blank1 group without exposure to glucose and Mac-CM, Blank2 with glucose load and without Mac-CM treatment, control group treated with Mac-CM and glucose.).

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1.3 mmol/L CaCl2, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, and 30 mmol/L HEPES, pH 7.4) containing 11 mM glucose and then treated with CPE (100, 200, 500 μg/ml) or metformin (1 mmol/L) for 30 min. In the case of Mac-CM challenge, KRH was replaced with Mac-CM (diluted in KRH, 1:1, v/v). The glucose concentration in KRH at 0 h and 4 h after culture were quantified with a glucose assay kit, and the difference between the two concentrations was regarded as the amount of consumed glucose (Ha et al., 2012).

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3.2. CPE restored insulin tolerance in mice Mac-CM stimulation induced insulin intolerance as glucoselowing action of insulin was attenuated in Mac-CM-treated mice. Oral administration of CPE reversed insulin intolerance, as blood glucose concentrations at 0.5 h and 1 h post-injection of insulin were significantly reduced compared with the control. Metformin showed a similar modulation effect as CPE. The AUCs for insulin tolerance were significantly decreased by CPE treatment. Results were shown in Fig. 2.

2.11. Statistical analysis Results were expressed as mean7S.D. Differences were analyzed by one-way analysis of variance followed by Student–Newman–Keuls test. A value of Po0.05 was regarded as significant.

3. Results 3.1. CPE ameliorated oral glucose tolerance in mice To assess the effect of CPE on glucose homeostasis in mice, we first observed its effect on glucose tolerance. As shown in Fig. 1A, oral glucose administration resulted in an increase of blood glucose, reaching its Cmax at 30 min, followed by gradually returning to the basal level at 2 h after glucose load. Treatment of mice with CPE increased glucose disposal significantly in a dose-dependent manner at 30 min and 60 min after glucose load. Mac-CM challenge induced glucose intolerance evidenced by decreased glucose disposal, whereas CPE administration effectively reversed glucose intolerance (Fig. 1B). Glucose AUCs also showed the beneficial regulation of glucose tolerance by CPE in mice (Fig. 1a and b).

3.3. CPE administration increased liver and muscle glycogen in mice Glucose load increased glycogen contents in liver and muscle, but Mac-CM challenge attenuated this action, as glycogen contents in liver and muscle were greatly reduced by 24.4% and 16.9%, respectively. As shown in Fig. 3, administration of CPE effectively restored sugar load-induced glycogen synthesis in a dosedependent manner evidenced by increased glycogen contents. Metformin also demonstrated a similar modulation as CPE.

3.4. CPE modulated adipokine expression in adipose tissue Dysregulation of adipokines in mice adipose tissue was manifested by overexpression of TNF-α, IL-6, MCP-1 and resistin and downregulation of adiponecin, when mice were exposed to MacCM. CPE treatment effectively attenuated TNF-α, IL-6, MCP-1 and resistin mRNA expression and restored mRNA expression of adiponecin (Fig. 4). Metformin also showed similar action as CPE in the modulation of the gene expression for inflammation-related adipokines.

Fig. 2. CPE improved insulin tolerance in mice. Mice were treated with CPE or metformin by gavages and then stimulated with diluted Mac-CM as above. After 30 min, mice were administered with insulin (0.5 U/kg, s.c) and blood glucose was determined at regulated times. Metformin was taken as a positive control. Glucose AUCs also showed the beneficial regulation of insulin tolerance by CPE in mice. Results were expressed as the mean 7S.D. (n ¼10). #p o0.05 vs. blank 2; np o 0.05 vs. control (Blank1 group without exposure to insulin and Mac-CM, Blank2 with insulin load and without Mac-CM treatment, control group treated with Mac-CM and insulin.).

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3.5. CPE facilitated insulin signaling in muscle IRS-1 phosphorylation is a key regulator of insulin signal transduction. As shown in Fig. 5, Mac-CM stimulated IRS-1 serine phosphorylation and thereby attenuated insulin-mediated IRS-1 tyrosine phosphorylation and subsequent Akt phosphorylation in

muscle tissue. Administration of CPE significantly decreased serine phosphorylation of IRS-1 and effectively restored IRS-1 tyrosine phosphorylation in response to insulin. Meanwhile, CPE also enhanced Akt phosphorylation in response to insulin in a dosedependent manner. Metformin demonstrated a similar regulation on phosphorylating modification in IRS-1and AKT as CPE.

3.6. CPE promoted glucose consumption in adipocytes Meanwhile, we also investigate the regulation of glucose homeostasis without the presence of insulin. As shown in Fig. 6, CPE effectively promoted glucose consumption by adipocytes in a concentration-dependent manner. Mac-CM stimulation reduced glucose consumption, whereas this alteration was counteracted by CPE. As same as CPE, metformin also promoted glucose consumption under basal and inflammatory conditions.

4. Discussion

Fig. 3. CPE restored glucose load-induced liver and muscle glycogen synthesis in mice. Mice were treated with CPE or metformin at given doses and then intraperitoneally injected with diluted Mac-CM. 30 min later, mice were orally administered with 2 g/kg glucose.(A and B) Glycogen content in liver and muscle were determined with a commercial kit following the glucose loaded at 30 min. Metformin was taken as a positive control. Results were expressed as the mean 7 S. D. (n¼10).#p o 0.05 vs. blank 2; *p o 0.05 vs. control (Blank1 group without exposure to glucose and Mac-CM, Blank2 with glucose load and without Mac-CM treatment, control group treated with Mac-CM and glucose.).

In obesity, dysfunction of adipose tissue leads to increased production of pro-inflammatory cytokines, which play an important role in the initiation and development of local and systemic insulin resistance through paracrine or circulating hormonal effects. Qingrejiedu herbs can effectively reduce the inflammatory damage and oxidative stress, and thereby improve insulin sensitivity and/or insulin secretion (Lu, 2009). As a kind of Qingrejiedu herbs, leaves of Cyclocarya paliurus have been widely used for drug formulations in traditional Chinese medicine and as an ingredient in functional foods in China. Previous pharmacological investigation showed that leaves of Cyclocarya paliurus exhibited the antihyperglycemic, antihyperlipidemic and antioxidant activities (Kurihara et al., 2003a, 2003b; Xie et al., 2010). Nevertheless, the mechanism of its regulation of insulin action under inflammatoryinduced insulin resistance conditions is not fully understood. In the present study, we stimulated mice with Mac-CM and successfully established insulin resistance. Our work showed that CPE inhibited inflammatory response in adipose tissue and ameliorated insulin resistance by facilitating insulin signaling, well demonstrating its beneficial regulation of insulin sensitivity under inflammatory conditions.

Fig. 4. CPE regulated inflammation-related adipokines mRNA expression in mice adipose tissue. Mice were pretreated with CPE or metformin at given doses followed by stimulation with diluted Mac-CM for 30 min as above. mRNA expression of related adipokines in fat was determined by real-time qPCR. Metformin was taken as a positive control. Results were expressed as mean7S.D. of 3 independent experiments. *po0.05 vs. control (Blank group received no Mac-CM treatment, Control received Mac-CM injection.).

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Fig. 6. CPE increased insulin-stimulated glucose uptake in adipocytes. Differentiated adipocytes were pretreated with CPE at the concentration of 100, 200, and 500 μg/ml or metformin (1 mmol/L) for 30 min and then co-cultured with 11 mM glucose and with or without diluted Mac-CM for 1 h. Glucose in the culture medium were quantified with a commercial kit. Results were expressed as mean7SD of 3 independent experiments. *po0.05 vs. control. (Blank group received no Mac-CM treatment, Control received Mac-CM treatment.).

Fig. 5. CPE modulated serine/tyrosine phosphorylation of IRS-1 and restored insulin-mediated Akt phosphorylation in mice muscle tissue. Mice were intraperitoneally injected with diluted Mac-CM for 30 min after oral administration of CPE or metformin. Serine phosphorylation of IRS-1 was assayed by Western blot. Mice were treated with insulin (100 nmol/L) for 30 min after stimulation with diluted Mac-CM, and then tyrosine phosphorylation of IRS-1 and Akt phosphorylation in muscle tissue were determined. Metformin was taken as a positive control. Results were expressed as mean7 S.D. of 3 independent experiments. #p o 0.05 vs. blank 2; p o 0.05 vs. control (Blank1 group without exposure to insulin and Mac-CM, Blank2 with insulin load and without Mac-CM treatment, Blank group received no Mac-CM treatment, Control received Mac-CM injection.).

CPE improved oral glucose tolerance under normal and inflammatory conditions, well demonstrating its beneficial effects on glucose homeostasis. It is well known that glucose load stimulates

insulin secretion from pancreatic islet B cells, and insulin in turn promotes glucose disposal in muscle and liver in the form of glycogen. Mac-CM challenge induced glucose intolerance and this change was reversed by CPE. To know whether CPE improved glucose tolerance by influence on insulin action, we observed the effect of CPE on insulin tolerance test. CPE effectively reversed insulin intolerance, and these results suggested that CPE promoted glucose disposal by improvement of insulin sensitivity against inflammatory insult. Consistent with its beneficial effects on glucose and insulin tolerance, CPE increased glycogen in muscle and liver when mice were challenged with inflammatory insult. In addition to its energy-storage function, adipose tissue is widely recognized as an endocrine tissue that secretes a variety of bioactive substances (adipokines or adipocytokines). In obesity, dysfunction of adipose tissue is characterized by overexpression of inflammatory cytokines and depressed secretion of beneficial ones, such as adiponectin (Guerre-Millo, 2004; Matsuzawa, 2006). MCP-1 is proposed to be a major chemokine for macrophage infiltration into adipose tissue. Among proinflammatory cytokines, TNF-α was first discovered to induce insulin resistance (Hotamisligil et al., 1993). As same as TNF-α, IL-6 was also shown to be involved in the development of insulin resistance (Senn et al., 2002). Adipocytes are the main source of secreted adiponectin which can promote insulin sensitivity, as well as resistin which can impair insulin sensitivity (Bassols et al., 2009; Galic et al., 2011). CPE inhibited MCP-1, TNF-α, IL-6 and resistin expression and upregulated adiponectin expression, well demonstrated its anti-inflammatory activity implicated in regulation of adipokine expression. Insulin resistance is characterized by the impairment of insulin PI3K signaling. CPE inhibited inflammation in adipose tissue, and therefore we wondered whether this action could attribute to attenuate insulin resistance by beneficial effects on insulin signaling.

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Because skeletal muscle accounts for the majority of glucose disposal, we observed the effect of CPE on insulin signaling in skeletal muscle. Serine phosphorylation of IRS-1 is considered to be a molecular indicator of insulin resistance and a key control linking inflammation to insulin resistance (Gual et al., 2005). Some inflammatory molecules, such as TNF-α, IL-6, FFA and IKKβ were identified to be involved in inhibition of insulin-mediated IRS-1 tyrosine phosphorylation by enhancing serine phosphorylation, and thereby impaired downstream insulin PI3K signaling in response to insulin (Feinstein et al., 1993; Yu et al., 2002; Gual et al., 2005; Thirone et al., 2006). Therefore, in the current study, we investigated the phosphorylation of Ser307 to detect an interaction effect between inflammation and insulin resistance. We observed that Mac-CM stimulation induced IRS-1 serine phosphorylation as well as attenuated tyrosine phosphorylation in response to insulin. As expected, CPE effectively reversed these alterations, well demonstrating its beneficial modification of serine/tyrosine phosphorylation. Insulin-mediated IRS-1 tyrosine phosphorylation generates docking sites for PI3K activation and downstream Akt is directly responsible for Glut-4-mediated glucose uptake (Esposito et al., 2001). Mac-CM stimulation induced parallel downregulation in tyrosine phosphorylation of IRS-1 and downstream Akt phsophorylation in response to insulin, indicating the impairment of insulin signaling along IRS-1 and PI3K. CPE beneficially regulated IRS-1 function, leading to improvement of PI3K signaling evidenced by restored Akt phosphorylation in response to insulin. The resultant improvement of insulin signaling should be responsible for its positive effects on glucose or insulin tolerance when mice were exposed to Mac-CM challenge. Cyclocarya paliurus leave extract was reported to contain many phytochemicals constituents, including polysaccharides, triterpenoids, flavonoids, protein, steroids, saponins, phenolic compounds, etc. (Xie and Xie, 2008; Xiang-xiang, 2009). Among these constituents, flavonoids and polysaccharides were proved to exhibit anti-diabetic effects (Xie et al., 2006; Li et al., 2008, 2011). Polysaccharide of Cyclocarya paliurus was demonstrated to improve the capability of glucose tolerance and decrease the contents of glucose and insulin in serum in diabetic mice (Li et al., 2002; de Luca and Olefsky, 2008; Shi et al., 2009). Cyclocarya paliurus flavonoids were found to inhibit αglycosidase activity in diabetic mice (Yang et al., 2007). Phenolic compounds from the leaves of Cyclocarya paliurus exhibited strong inhibitory activity against PTP1B and could be used as a potential source for functional food ingredient with anti-diabetic effects (Zhang et al., 2010). But our previous study showed that polysaccharide and flavonoids did not appear to be the active anti-diabetic constituent in Cyclocarya paliurus (Wang et al., 2013), which suggested that there might be other active ingredients for the pharmaceutical effects. Further researches are in progress to precisely define the bioactive compound(s) responsible for its anti-diabetic properties. Above mentioned evidence showed the regulation of insulin sensitivity by CPE, whereas we wanted to know more information about its regulation of glucose homeostasis independently of insulin. CPE promoted glucose consumption in adipocytes without the presence of insulin under normal and inflammatory conditions, indicating that it regulated glucose disposal in an insulinindependent manner. Because anti-diabetic agent metformin is known an activator of AMP-kinase activity, it increased glucose consumption in adipocytes, suggesting the involvement of AMPK. To confirm the involvement of AMPK in the regulation of glucose consumption by CPE, further study is needed.

5. Conclusions In conclusion, CPE improves oral glucose tolerance by enhancing insulin sensitivity and reversed inflammation-related adverse changes in adipokine expression. CPE inhibited inflammation and

ameliorated insulin resistance by positive regulation of IRS-1 function. These presented findings may provide mechanistic explanations for drug formulations of Cyclocarya paliurus in the treatment of diabetes mellitus and its related disorders in traditional Chinese medicine.

Acknowledgments This work was partially supported by the grants awarded by the National Natural Science Foundation of China (Grant no. 81072976, 81001379, 31270673), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. ZJ11175).

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