Saffron (Crocus Sativus L.) Increases Glucose Uptake and Insulin Sensitivity in Muscle Cells Via Multipathway Mechanisms

Food Chemistry 135 (2012) 2350–2358

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Food Chemistry

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Saffron (Crocus sativus L.) increases glucose uptake and insulin sensitivity in muscle cells via multipathway mechanisms

Changkeun Kang a, Hyunkyoung Lee a, Eun-Sun Jung a, Ramin Seyedian a,1, MiNa Jo a, Jehein Kim a, ⇑ Jong-Shu Kim a, Euikyung Kim a,b, a College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, South Korea b Research Institute of Life Science, Gyeongsang National University, South Korea article info abstract

Article history: Saffron (Crocus sativus Linn.) has been an important subject of research in the past two decades because of Received 16 March 2012 its various biological properties, including anti-cancer, anti-inflammatory, and anti-atherosclerotic activ- Received in revised form 13 May 2012 ities. On the other hand, the molecular bases of its actions have been scarcely understood. Here, we elu- Accepted 25 June 2012 cidated the mechanism of the hypoglycemic actions of saffron through investigating its signaling Available online 3 July 2012 pathways associated with glucose metabolism in C2C12 skeletal muscle cells. Saffron strongly enhanced glucose uptake and the phosphorylation of AMPK (AMP-activated protein kinase)/ACC (acetyl-CoA car- Keywords: boxylase) and MAPKs (mitogen-activated protein kinases), but not PI 3-kinase (Phosphatidylinositol 3- Saffron kinase)/Akt. Interestingly, the co-treatment of saffron and insulin further improved the insulin sensitivity Glucose uptake Skeletal muscle cells via both insulin-independent (AMPK/ACC and MAPKs) and insulin-dependent (PI 3-kinase/Akt and AMPK mTOR) pathways. It also suggested that there is a crosstalk between the two signaling pathways of glu- Insulin sensitivity cose metabolism in skeletal muscle cells. These results could be confirmed from the findings of GLUT4 translocation. Taken together, AMPK plays a major role in the effects of saffron on glucose uptake and insulin sensitivity in skeletal muscle cells. Our study provides important insights for the possible mechanism of action of saffron and its potential as a therapeutic agent in diabetic patients. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction spices around the world for flavoring and coloring food (Sampathu, Shivashankar, Lewis, & Wood, 1984). It has also been applied in tra- Natural compounds with insulin-like activity (insulin mimetics) ditional medicine in the treatment of cramps, asthma and bron- have been proposed as potential therapeutic agents in the preven- chospasms, menstruation disorders, liver disease and pain tion and/or treatment of diabetes (Lee et al., 2006; Pinent et al., (Schmidt, Betti, & Hensel, 2007). Among the estimated more than 2008). They would act by promoting glucose transport and glucose 150 volatile and several nonvolatile compounds of saffron, major metabolism (Alberti, Zimmet, & Shaw, 2006). As the incidence and biologically active compounds are crocin, picocrocin, crocetin, car- prevalence of diabetes increase worldwide, there is a considerable otene and safranal (Rios, Recio, Giner, & Manez, 1996). Good qual- demand for therapeutic reagents which have a high efficacy and ity saffron contains about 30% crocin, 5–15% picocrocin, and low side effect profile. Therefore, screening for novel insulin usually up to 2.5% volatile compounds including safranal (Schmidt mimetics from various natural sources, such as plants and herbs, et al., 2007). The major constituents of saffron have attracted a are still attractive and some of them might be safely used on dia- considerable amount of interest during the past two decades and betes without causing serious side effects. shown both in vitro and in vivo to possess antioxidant, anti-cancer, Saffron, the dry stigmas of the plant Crocus sativus Linn., belong anti-inflammatory, anti-atherosclerosis and memory-improving to the Iridaceae family has been used as an important dietary properties (Abdullaev & Espinosa-Aguirre, 2004; Abe & Saito, ingredient in various parts of the world since ancient times. Fur- 2000; He et al., 2005; Hosseinzadeh & Younesi, 2002). Recently, thermore, saffron is one of the highest priced and the most used accumulating evidence has suggested the possibility of saffron being used as a candidate drug for diabetes. The hypoglycemic effects of saffron were reported in a study involving diabetic rats ⇑ Corresponding author at: Department of Pharmacology and Toxicology, College (Mohajeri, Mousavi, & Doustar, 2009), but the molecular mecha- of Veterinary Medicine, Gyeongsang National University, 900 Gajwa-dong, Jinju nisms underlying this effect remain obscure. 660-701, South Korea. Tel.: +82 55 772 2355; fax: +82 55 772 2349. The glucose transport in skeletal muscle is regulated by two dis- E-mail address: [email protected] (E. Kim). 1 Department of Pharmacology and Toxicology, Bushehr University of Medical tinct pathways including phosphatidylinositol-3 kinase (PI 3-ki- Sciences, Bushehr, Iran. nase) and 50-AMP-activated protein kinase (AMPK) (Fig. 1). The PI

Fig. 1. Schematic diagram of signaling pathways for the glucose metabolism of skeletal muscle cells and their pharmacological regulators.

3-kinase pathway includes activation of Akt leading to activation of ma–Aldrich Inc. (St. Louis, MO, USA). Antibodies for Phospho-Acet- glycogen synthesis and the other enzymes/proteins necessary for yl-CoA Carboxylase (Ser79), Phospho-AMPKa (Thr172), Phospho-Akt the acute metabolic effects of insulin, which promotes GLUT4 (Ser473), Phospho-Erk (Thr202), Phospho-p38 (Thr180), Phospho- translocation from an intracellular pool to plasma membrane SAPK/JNK (Thr183), AMPK, Akt and GLUT4 were obtained from Cell (Smith & Muscat, 2005). On the other hand, AMPK is a phylogenet- Signaling Technology (Beverly, MA). Compound C and LY294002 ically conserved intracellular energy sensor that plays a central were purchased from Calbiochem (Merck, Darmstadt, Germany). role in the regulation of glucose and lipid metabolism (Carling, 2-Deoxy-D [3H] glucose was obtained from Perkin-Elmer Life Sci- 2004). Activation of AMPK leads to the phosphorylation and regu- ences (Boston, MA, USA). All other reagents used were of the purest lation of a number of downstream targets that are involved in di- grade available. verse pathways, including acetyl-CoA carboxylase (ACC) in the body (Fryer & Carling, 2005). Both distinct pathways also increase the phosphorylation and activity of mitogen-activated protein ki- 2.2. Plant material and extraction nase (MAPK) family components, which participates in the full activation of insulin-stimulated glucose uptake via GLUT4 translo- The saffron was obtained from Saharkhiz Co. (Mashhad, Iran). cation (Konrad et al., 2001). After grinding the saffron stigmas, 2 g of dried stigma was ex- In the present study, we showed that saffron activates AMPK/ tracted with 100 ml of methanol (80%) for 2 h in an ultrasonic bath,

ACC and stimulate glucose uptake in C2C12 skeletal muscle cells. and then shaken (110 rpm) for 12 h at room temperature. After the In addition, saffron enhanced insulin sensitivity in glucose metab- extraction, the methanol was evaporated using rotary vacuum olism via activation of AMPK/ACC, MAPKs, PI 3-kinase/Akt and evaporator (Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The ﬁnal aque- GLUT4 translocation to the plasma membrane. We further showed ous part was lyophilized and kept at 20 °C until use. The yield of that mTOR and its downstream pathway are also involved in this extraction was around 47% (w/w). process. Taken together, our results provide new insights into possible mechanism of action by which saffron may contribute to the pharmacological intervention of diabetes. 2.3. Cell culture

C2C12 (mouse myoblasts cell line) were maintained in DMEM 2. Materials and methods supplemented with 10% heat inactivated FBS and 1% antibiotics (100 U/ml penicillin and 100 lg/ml streptomycin), at 37 °Cina

2.1. Chemicals and reagents humidified atmosphere with 5% CO2. For differentiation into myotubes, cells were reseeded in 6-well plates (for immunoblotting), Dulbecco’s Modified Eagle’s Medium (DMEM), foetal bovine ser- and 12-well plates (for glucose uptake) at a density of um (FBS), bovine serum albumin (BSA), penicillin, streptomycin, 2 104 cells/ml. After 48 h (over 80% confluence), the medium trypsin were obtained from Gibco-BRL (Grand Island, NY, USA). Di- was switched to DMEM with 2% (v/v) FBS and was replaced after methyl sulphoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5- 2, 4 and 6 days of culture. Experiments were initiated on day 7 diphenyltetrazolium bromide (MTT) were purchased from Sig- when myotube differentiation was complete. 2352 C. Kang et al. / Food Chemistry 135 (2012) 2350–2358

2.4. Cytotoxicity assay concentrations in cell lysates were measured using a Bio-Rad protein assay reagent (Bio-Rad, CA, USA). Proteins in cultured cell ly- Cytotoxicity experiments were conducted to determine the sates (30–40 lg) were separated on 12% SDS–polyacrylamide gel, maximal nontoxic dose of saffron. Cell morphology was assessed transferred to PVDF membranes (Bio-Rad, CA, USA), and subse- by using phase-contrast microscopy, and viability was evaluated quently subjected to immunoblot analysis using specific primary by means of MTT assay (Mosmann, 1983). The cells were seeded antibodies. After incubation overnight at 4 °C with the primary in 24-well plates at a density of 0.5 104 cells/well and cultured antibody, the membranes were incubated with horseradish perox- with or without saffron for 24 h. Briefly, 100 ll of MTT solution idase-conjugated secondary antibody (Cell Signaling Technology, (5 mg/ml MTT in PBS) was added to each well and incubated for Beverly, MA) for 1 h at room temperature. The blots were visual- 3 h at 37 °C. After removing the supernatant, the formazan crystal ized by using the enhanced chemiluminescene method (ECL, generated was dissolved by adding 150 ll/well of dimethyl sulfox- Amersham Biosciences, Buckinghamshire, UK) on blue light-sensi- ide (DMSO) and the absorbance was detected at 540 nm using a tive film (Fujifilm Corporation, Tokyo, Japan). In some cases, the spectrophotometric microplate reader (BioTek Instruments, Inc., blotted membranes were stripped and reprobed using other anti- Winooski, USA). Relative cell viability of treatment was calculated bodies. Densitometric analysis was performed with a Hewlett– as a percentage of vehicle-treated control ([OD of treated cells/OD Packard scanner and NIH Image software (Image J). of control cells] 100). 2.8. Statistical analysis 2.5. Glucose uptake assay The results are expressed as a mean ± standard deviation (SD). A Glucose uptake activity was analyzed by measuring the uptake paired Student’s t-test was used to assess the significance of differ- 3 ence between two mean values. P < 0.05 was considered to be sta- of 2-Deoxy-D [ H] glucose in differentiated C2C12 myotubes. Cells were rinsed twice with warm PBS (37 °C), and then starved in ser- tistically significant. um free DMEM for 3 h. After saffron treatment, the cells were incubated in KRH buffer (20 mM HEPES, pH 7.4, 130 mM NaCl, 1.4 mM 3. Results

KCl, 1 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4) containing 0.5 lCi of 2-Deoxy-D [3H] glucose for 10 min at 37 °C. The reaction 3.1. Saffron stimulates glucose uptake and AMPK/ACC pathways in was terminated by placing the plates on ice and washing twice differentiated C2C12 cells with ice-cold PBS. The cells were lysed in 1% SDS and 400 llof the lysate was mixed with 3.5 ml of scintillation cocktail and radio- In order to assess the non-cytotoxic concentration of saffron, activity was determined by scintillation counting. Non-speciﬁc up- the viability of C2C12 cells was evaluated at the dose ranges be- take was determined in the presence of 20 lM cytochalasin B. The tween 0 and 200 lg/ml, using 3-(4,5-dimethylthiazol-2-yl)-2,5- results were expressed as fold-increase. diphenyltetrazolium bromide (MTT) assay. Within the tested concentrations, saffron only showed negligible cytotoxicity (data not 2.6. Preparation of the cytosolic and the plasma membrane protein shown), and the concentrations up to 2.5 lg/ml of saffron were fractions used in subsequent experiments. For determining the role of saf-

fron in glucose metabolism of muscle cells, differentiated C2C12 The subcellular fractionation of myotubes was carried out myotube cells were treated with saffron and their glucose uptakes according to the method (Sato, Iemitsu, Aizawa, & Ajisaka, 2008) were measured by 2-Deoxy-D [3H] glucose uptake assay. Interest- with slight modification. Briefly, the cells from 10-cm dishes were ingly, the glucose uptake of myotubes was considerably stimulated gently scraped in buffer A (20 mM Tris pH 7.4, 1 mM EDTA, by the treatment of saffron in a concentration-dependent manner 0.25 mM EGTA, 0.25 M sucrose, 1 mM DTT, 50 mM NaF, 25 mM so- with a maximal increase occurring at 2.5 lg/ml (Fig. 2A) for the dium pyrophosphate and 40 mM b-glycerophosphate). The result- present study. This result suggests that saffron may act on the pro- ing homogenates were clarified 400 g for 15 min. The supernatant teins being associated with glucose uptake signaling pathways in was centrifuged at 50,000 rpm for 1 h. The resulting pellet was muscle cells. To further elucidate the possible mechanism of action homogenized in buffer A and then sampled as a cytosol protein of saffron, the signaling of AMPK and ACC were examined from fraction. Proteins from different fractions were solubilized for 1 h C2C12 myotubes that were exposed to saffron at 2.5 lg/ml for the at room temperature in buffer B (20 mM Tris pH 7.4, 1 mM EDTA, indicated time periods (Fig. 2B and C) or alternatively at the vari- 0.25 mM EGTA, 2% Triton X-100, 50 mM NaF, 25 lM sodium pyro- ous concentrations for 1 h (Fig. 2D and E). From this, it was found phosphate and 40 mM b-glycerophosphate). The homogenate was that Saffron can induce a time- and dose-dependent increase of centrifuged, and the supernatant was spun for 1 h at 5000 rpm; it AMPK phosphorylation in C2C12 cells. The phosphorylation of AMP- was then sampled as a plasma membrane fraction. The GLUT4 pro- Ka (Thr172) significantly increased from 1 h of saffron treatment tein level was measured in both total and plasma membrane frac- and the phosphorylation of ACC-Ser79, which is the best-character- tions. Translocation was evaluated by the difference in protein ized downstream molecule of AMPK, also showed a similar activa- levels in the cytosol and plasma membrane fractions. tion response. More surprisingly, the stimulation of AMPK and ACC could be observed even at the lowest concentration (0.125 lg/ml) 2.7. Protein extract and Western blotting tested in the present study (Fig 2C and E).

Cells were rinsed twice with ice-cold PBS, and then scraped 3.2. Saffron-induced glucose uptake is mediated by AMPK/ACC and with 100 ll of lysis buffer (50 mM Tris–HCl, pH 7.4, 1% NP-40, MAPKs pathway, but not by PI 3-kinase/Akt pathway 0.25% sodium deoxycholate, 150 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM Na3VO4,1lg/ml aprotinin, Akt is a serine/threonine protein kinase that leads to a translo- 1 lg/ml leupeptin, 1 lg/ml pepstatin). The plate was rocked on ice cation of insulin-sensitive glucose transporter (GLUT4) to plasma for 3 min, each samples were allowed to lyse for additional 30 min membrane via the activation of signal transduction cascade involv- on ice with periodic vortexing. Cell debris was removed by centri- ing insulin, insulin receptor substrate (IRS) family, and PI 3-kinase fugation (22,000g,at4°C for 30 min) and the resulting superna- (Alessi & Cohen, 1998). To determine whether the basal activity of tants were collected for immunoblot analyses. Protein PI 3-kinase/Akt is also involved in the effect of saffron on glucose C. Kang et al. / Food Chemistry 135 (2012) 2350–2358 2353

4 (A) #

3 #

2 #

(fold induction) 1 deoxyglucose uptake 2-

0 Saffron (μg/ml) --0.5 1 2.5 Insulin (nM) - 100 --- (B) (D)

P-ACC p-ACC

P-AMPKα p-AMPKα

P-AMPKβ p-AMPKβ GAPDH GAPDH -+++++Saffron (2.5 μg/ml) 0 0.5 1 3 6 9 Time (hr) 0 0.125 0.25 0.5 1 2.5 Saffron (μg/ml)

Fig. 2. Saffron induces glucose transport and phosphorylates AMPK/ACC in C2C12 cells. (A) The glucose uptake was measured as described in Materials and methods.

Differentiated C2C12 cells were incubated with saffron at 2.5 lg/ml for the indicated time periods (B and C), or at various concentrations (0, 0.125, 0.25, 0.5, 1 or 2.5 lg/ml) for 1 h (D and E). Protein extracts were prepared and subjected to Western blot assay using the primary antibodies for phospho-acetyl-CoA carboxylase (Ser79), phospho-AMPKa (Thr172), and phospho-AMPKb1 (Ser108). GAPDH protein levels were used as a control for equal loading. The results are expressed as mean ± S.D. for three independent experiments, P < 0.05, #P < 0.01, as compared with the control value. metabolism, we investigated the effects of saffron on PI 3-kinase/ but not to the PI 3-kinase/Akt pathway. We next corroborated Akt pathway in comparison with those of insulin. As shown in the roles of AMPK/ACC and MAPKs in the saffron-induced glucose Fig. 3, Insulin caused a robust phosphorylation of Akt, while saffron metabolism of muscle cells. For this, the effect of saffron on treatment had no stimulatory effect on Akt. These results indicate AMPK/ACC and MAPKs pathways was investigated using the cell that mechanism of action underlying the saffron-induced glucose signal specific inhibitors (compound C, an AMPK inhibitor, and uptake in muscle cells is linked to the activation of AMPK/ACC LY294002, a PI 3-kinase inhibitor). The results showed that compound C, but not LY294002, potently suppressed saffron-stimulated AMPK/ACC and MAPKs phosphorylation (Fig. 4A–D). Similarly, the saffron-induced glucose uptake, from 2-deoxyglu- P-AKT cose uptake assay, was also significantly inhibited in muscle cells by the pretreatment of compound C, but not by LY294002 GAPDH (Fig. 4E). From these findings, it could be concluded that saffron -++++-Saffron 2.5 μg/ml by itself can induce glucose uptake in skeletal muscle cells via -----+Insulin 100 nM the mechanisms of AMPK/ACC and MAPKs, but not through PI- 3K/Akt signaling pathway. 0 5 15 30 60 30 Time (min)

Fig. 3. Effect of saffron on insulin signaling pathway in C2C12 cells. Differentiated 3.3. Saffron improves insulin sensitivity via activation of AMPK/ACC, C2C12 cells were treated with either 2.5 lg/ml of saffron for different time periods MAPKs, PI 3-kinase/Akt and mTOR pathway (5, 15, 30 and 60 min) or 100 nM of insulin for 30 min. Protein extracts were prepared and subjected to Western blot assay using the primary antibodies for phospho-Akt (Ser473). GAPDH protein levels were used as a control for equal Insulin has a major regulatory function for glucose metabolism loading. The results are representative of three independent experiments. in liver, adipocyte and skeletal muscle by redistributing GLUT4 2354 C. Kang et al. / Food Chemistry 135 (2012) 2350–2358

(A) p-ACC (C) p-JNK

p-AMPKα p-p38

p-Akt p-ERK

GAPDH GAPDH

-+++Saffron -+++Saffron --+-Com.C --+-Com.C ---+LY294002 ---+LY294002 (B) (D)

(E) 4 #

3 # #

2 (fold induction) 1 2-deoxyglucose uptake

0 Saffron --+++ Insulin -+--- Com.C ---+- LY294002 ----+

Fig. 4. Effect of saffron on glucose transport is dependent upon AMPK/ACC and MAPKs pathways. (A–D) Differentiated C2C12 cells were pretreated with or without compound C (20 lM) or LY294002 (25 lM) for 30 min, then treated with saffron (2.5 lg/ml) for 1 h. Protein extracts were prepared and subjected to Western blot assay using the primary antibodies for AMPK/ACC pathway (phospho-acetyl-CoA carboxylase (Ser79) and phospho-AMPKa (Thr172)) (A and B) as well as MAPKs pathway (phospho-JNK, phospho-p38 and phospho-ERK) (C and D). GAPDH protein levels were used as a control for equal loading. (E) Differentiated C2C12 cells were treated with insulin (100 nM, 30 min) or saffron (2.5 lg/ml, 1 h) in the presence or absence of cell signaling speciﬁc inhibitors (compound C or LY294002), and then assayed for glucose uptake as described in Materials and methods. The results are expressed as mean ± S.D. for three independent experiments, P < 0.05, #P < 0.01, as compared with the control value.

from intracellular vesicles to the cell surface. Hence, it was utmost vidual effects of each reagent (Fig. 5A–D). To further develop this important for the present study to investigate the interaction be- concept, we performed a GLUT4 Western blotting for the cytosol tween saffron and insulin on glucose metabolism-associated cell and membrane fractions of the C2C12 cells. The co-treatment of saf- signaling. We ﬁrst attempted to study whether or not saffron has fron and insulin further increased the translocation of and the plas- an insulin-sensitizing potential. To this end, C2C12 cells were trea- ma membrane abundance of GLUT4, without much changing ted with 100 nM insulin in the presence or the absence of saffron. GLUT4 content in the cytosol (Fig. 5E and F). On the other hand, The treatment of saffron alone did increase both AMPK/ACC and the co-treatment of saffron and insulin slightly inhibited the phos- MAPKs phosphorylations without any effect on Akt (Fig. 4). Sur- phorylations of mammalian target of rapamycin (mTOR) and its prisingly, when saffron and insulin were co-treated to C2C12 cells, downstream targets (p70S6K and 4EBP1) (Fig. 6). Insulin-stimula- the phosphorylation levels of AMPK/ACC, MAPKs and Akt were fur- tion of PI 3-kinase/Akt is a key step in insulin signaling mechanism ther increased to the levels, which were much higher than the indi- and negatively regulated by mTOR pathway (Tzatsos & Kandror, C. Kang et al. / Food Chemistry 135 (2012) 2350–2358 2355

(A) p-ACC (C) p-JNK

p-AMPKα p-p38

p-Akt p-ERK

GAPDH GAPDH

--++Saffron --++Saffron -+-+Insulin -+-+Insulin

(B) (D)

(F) 12 (E) PM Glut4 # 10 Cytosol Glut4 ) 8 # -+-+Saffron 6 --++Insulin # 4 (fold increase

GLUT 4 levels (PM) GLUT 4 levels 2

0 Saffron -+-+ Insulin --++

Fig. 5. Saffron improves insulin sensitivity via multiple pathways in C2C12 cells. Differentiated C2C12 cells were treated with saffron (2.5 lg/ml) for 1hr in the presence or absence of insulin (100 nM). Phospho-acetyl-CoA carboxylase (Ser79), phospho-AMPKa (Thr172), phospho-Akt (Ser473), phospho-JNK, phospho-p38, phospho-ERK and GLUT4 were detected (A, C, and E) and quantiﬁed (B, D, and F). Protein extracts of cytosol and/or plasma membrane fractions were prepared and subjected to Western blot assay using the primary antibodies described above. GAPDH protein levels were used as a control for equal loading. The results are expressed as mean ± S.D. for three independent experiments, P < 0.05, #P < 0.01, as compared with the control value.

2006), which also inhibited the activation of AMPK (Inoki, Zhu, & compound C but not by LY294002 (Fig. 7C and D). Consistent with Guan, 2003). These results suggest that saffron can increase the other results, the induction of glucose uptake could be effectively sensitivity of muscle cells to insulin via the glucose metabolism- attenuated by either compound C or LY294002 (Fig. 7E). These re- associated cell signaling pathways, such as AMPK/ACC, MAPKs, sults provide another evidence for a crosstalk between AMPK/ACC PI-3/Akt pathway, and mTOR pathway. and MAPKs in the glucose metabolism, which eventually leads to The present findings were further confirmed by investigating activation of PI-3/Akt signal pathway. the effects of saffron and insulin co-treatment in the presence or absence of pharmacological inhibitors for AMPK or PI 3-kinase/ Akt pathways. Saffron-enhanced insulin sensitivity on AMPK/ACC, 4. Discussion JNK and p38 were almost completely obliterated by compound C (an AMPK inhibitor), but mostly not by LY294002 (a PI 3-kinase Diabetes mellitus is a chronic metabolic disorder affecting inhibitor) (Fig. 7A–D). On the other hand, the activation of Akt un- about 6% of population worldwide. Chronic hyperglycemia is a der the co-treatment was significantly modulated only by main contributor of the associated disorders, which can exacerbate LY294002 but not by compound C (Fig. 7A and B). In contrary to defective glucose disposal by interfering with insulin action in other MAPKs, the Erk stimulation was only slightly attenuated by insulin-target tissues such as skeletal muscle, liver and adipose tis- 2356 C. Kang et al. / Food Chemistry 135 (2012) 2350–2358

ized phosphorylation site by AMPK, was also significantly phos- (A) p-mTOR phorylated by the treatment of saffron. In insulin signaling pathway, PI 3-kinase is a key molecule, and its main downstream p-p70 S6K target is Akt, that is important in insulin-stimulated glucose transport and metabolism (Ueki et al., 1998). Concurrently, we examine p-4EBP1 whether PI 3-kinase/Akt pathway is involved in this stimulatory ef- GAPDH fect of saffron. From this, insulin caused a robust phosphorylation of Akt while saffron treatment had no significant effect (Fig. 3), suggesting saffron alone does not stimulate PI 3-kinase/Akt --++Saffron pathway. -+-+Insulin Recent studies have demonstrated a correlation between the AMPK and MAPKs signaling pathways; for example, p38 MAPK (B) activation was shown to have been completely abolished in skeletal muscle cells expressing the dominant-negative AMPK mutant (Kim et al., 2010; Pelletier, Joly, Prentki, & Coderre, 2005). There- fore, MAPKs is likely a downstream molecule of AMPK and a possible target in glucose metabolism. In order to confirm the relationship between AMPK and MAPKs in the skeletal muscle

cells, the C2C12 cells were pre-incubated with either compound C or LY294002. Our results demonstrated that compound C potently attenuated the phosphorylation of AMPK/ACC, MAPKs and glucose uptake stimulated by saffron, whereas LY294002 did not significantly inhibit saffron-induced activation of glucose metabolism (Fig. 4). These facts revealed that AMPK/ACC and MAPKs could be the key factor in saffron-activated glucose uptake. Several findings indicated there is a crosstalk between AMPK and insulin signaling pathways (Kang & Kim, 2010; Kovacic et al., 2003). Consistent with these previous reports, when the cells were Fig. 6. Inhibition of mTOR pathway is involved in the effect of saffron on insulin exposed to saffron and insulin simultaneously, both of the AMPK signaling. Differentiated C2C12 cells were treated with saffron (2.5 lg/ml) for 1 h in and PI 3-kinase/Akt pathways were more greatly stimulated com- the presence of absence of insulin (100 nM). Protein extracts were prepared and paring with those of their individual treatments (Fig. 5A and B). subjected to Western blot assay (A) using the primary antibodies for phospho- mTOR, phospho-p70S6 K (Thr389), and phospho-4EBP1(Thr37/46), then they were Concurrently, the MAPKs were also further activated by the co- quantified (B). GAPDH protein levels were used as a control for equal loading. The treatment of saffron and insulin from those observed by the each results are expressed as mean ± S.D. for three independent experiments, P < 0.05, treatment (Fig. 5C and D), which is probably due to the internaliza- # P < 0.01, as compared with the control value. tion of insulin signaling is also required to phosphorylate and activate the MAPKs pathway (Montagut et al., 2010). Additionally, the activation of AMPK on glucose uptake occurs independently of sue (Schenk, Saberi, & Olefsky, 2008). Among the insulin-target tis- insulin, and promotes the translocation of GLUT4 from an intracel- sues, skeletal muscle is responsible for more than 75% of glucose lular pool to the cell surface, which in turn can lead to the improve- disposal in response to insulin in the post-prandial state (DeFronzo ment of insulin stimulated glucose uptake (Zheng et al., 2001). The et al., 1981). Regulation of glucose metabolism of skeletal muscle is GLUT4 protein levels in skeletal muscle cells showed that saffron therefore quantitatively most important in energy balance, and is can increase insulin sensitivity by promoting the translocation of the primary tissue of insulin-stimulated glucose uptake, disposal GLUT4 protein to the plasma membrane (Fig 5E and F). In addition and storage (Kang et al., 2010). It demonstrated that a brown alga to the insulin signaling pathway, mammalian target of rapamycin of polyphenol-rich, Ecklonia cava possesses insulinomimetic prop- (mTOR) was also well-known to be activated by insulin and exert erties, since it decrease hyperglycemia in streptozotocin-diabetic the physiological feedback regulation on insulin signaling through rats. Furthermore, it stimulates glucose uptake in skeletal muscle increasing the phosphorylation of serine residue on IRS-1 (Um cell line mediated by the activation of both AMPK and PI 3-ki- et al., 2004). Moreover, activation of AMPK by treatment with AI- nase/Akt pathways. From the reports on the potential effectiveness CAR (an AMPK activator) is associated with enhanced phosphory- of traditional remedies against diabetes, it is believed that the nat- lation of MAPKs and decreased phosphorylation of mTOR, ural products may play a major role in the management of diabetes suggesting that the repressive action of AMPK on mTOR signaling (Srinivasan, 2005). is dominant to the stimulatory action of MAPKs pathway (William- The principal finding of present study is that saffron stimulates son, Bolster, Kimball, & Jefferson, 2006a,2006b). In the present glucose uptake and increase insulin sensitivity in skeletal muscle study, saffron slightly inhibited the phosphorylations of mTOR cells via multiple mechanisms. These findings indicate that the and its downstream targets of p70S6K and 4EBP1 (Fig. 6). The aug- hypoglycemic effect of saffron is attributable to the metabolic mentation of insulin sensitivity by saffron was further confirmed activity of saffron in skeletal muscle. Results of the glucose uptake using compound C and LY294002, which abrogated saffron-in- assay demonstrated that saffron can enhance glucose uptake in a duced glucose uptake and AMPK/ACC, MAPKs and PI 3-kinase/Akt dose-dependent manner in differentiated C2C12 cells (Fig 2A), indi- activation (Fig. 7). Therefore, it can be concluded that saffron en- cating its possible regulatory role in the glucose metabolism of hances insulin sensitivity via multiple mechanisms. Consistent skeletal muscle cells. In an effort to understand the signaling path- with our study, recent evidences provide that insulin-like natural ways involved in saffron-mediated glucose uptake, we carried out compounds can improve insulin sensitivity by activating AMPK Western blot analyses using specific antibodies for the signaling and PI 3-kinase/Akt pathways (Huang et al., 2010; Kang & Kim, molecules associated with glucose metabolism. Saffron was shown 2010; Li, Ge, Zheng, & Zhang, 2008). to activate AMPK in a time- (Fig. 2B and C) and dose- (Fig. 2D and In conclusion, saffron plays a beneficial role in glucose metabo- 79 E) dependent manner. Accordingly, ACC-Ser , the best-character- lism of differentiated C2C12 skeletal muscle cells. The activation of C. Kang et al. / Food Chemistry 135 (2012) 2350–2358 2357

(A) p-ACC (C) p-JNK

p-AMPKα p-p38

p-Akt p-ERK

GAPDH GAPDH

-++++Saffron -++++Saffron --+++Insulin --+++Insulin ---+-Com.C ---+-Com.C ----+LY294002 ----+LY294002

(B) (D)

(E) 6 # 5

4 #

# 3 # # 2 (fold induction) 1 2-deoxyglucose uptake 0 Saffron --++++ Insulin -+-+++ Com.C ----+- LY294002 -----+

Fig. 7. Effect of saffron on insulin sensitivity is down-regulated by speciﬁc inhibitors. Differentiated C2C12 cells were pretreated with either compound C (20 lM) or LY294002 (25 lM) for 30 min, then treated with saffron (2.5 lg/ml) for 1 h in the presence or absence of insulin (100 nM). (A–D) Protein extracts of cytosol and/or subcellular membrane fractions were prepared and subjected to Western blot assay (A and B) using the primary antibodies for phospho-acetyl-CoA carboxylase (Ser79), phospho-AMPKa (Thr172), phospho-Akt (Ser473), phospho-JNK, phospho-p38, and phospho-ERK, then they were quantiﬁed (B and D). (E) Glucose uptake was measured as described in Materials and methods. GAPDH protein levels were used as a control for equal loading. The results are expressed as mean ± S.D. for three independent experiments, P < 0.05, #P < 0.01, as compared with the control value.

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