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 sensitivityin 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 Koreab Research Institute of Life Science, Gyeongsang National University, South Korea

a r t i c l e i n f o

Article history:Received 16 March 2012Received in revised form 13 May 2012Accepted 25 June 2012Available online 3 July 2012

Keywords:SaffronGlucose uptakeSkeletal muscle cellsAMPKInsulin sensitivity

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.06.092

⇑ Corresponding author at: Department of Pharmacof Veterinary Medicine, Gyeongsang National Unive660-701, South Korea. Tel.: +82 55 772 2355; fax: +8

E-mail address: ekim@gnu.ac.kr (E. Kim).1 Department of Pharmacology and Toxicology, Bu

Sciences, Bushehr, Iran.

a b s t r a c t

Saffron (Crocus sativus Linn.) has been an important subject of research in the past two decades because ofits various biological properties, including anti-cancer, anti-inflammatory, and anti-atherosclerotic activ-ities. On the other hand, the molecular bases of its actions have been scarcely understood. Here, we elu-cidated the mechanism of the hypoglycemic actions of saffron through investigating its signalingpathways associated with glucose metabolism in C2C12 skeletal muscle cells. Saffron strongly enhancedglucose uptake and the phosphorylation of AMPK (AMP-activated protein kinase)/ACC (acetyl-CoA car-boxylase) and MAPKs (mitogen-activated protein kinases), but not PI 3-kinase (Phosphatidylinositol 3-kinase)/Akt. Interestingly, the co-treatment of saffron and insulin further improved the insulin sensitivityvia both insulin-independent (AMPK/ACC and MAPKs) and insulin-dependent (PI 3-kinase/Akt andmTOR) pathways. It also suggested that there is a crosstalk between the two signaling pathways of glu-cose metabolism in skeletal muscle cells. These results could be confirmed from the findings of GLUT4translocation. Taken together, AMPK plays a major role in the effects of saffron on glucose uptake andinsulin sensitivity in skeletal muscle cells. Our study provides important insights for the possible mech-anism of action of saffron and its potential as a therapeutic agent in diabetic patients.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Natural compounds with insulin-like activity (insulin mimetics)have been proposed as potential therapeutic agents in the preven-tion and/or treatment of diabetes (Lee et al., 2006; Pinent et al.,2008). They would act by promoting glucose transport and glucosemetabolism (Alberti, Zimmet, & Shaw, 2006). As the incidence andprevalence of diabetes increase worldwide, there is a considerabledemand for therapeutic reagents which have a high efficacy andlow side effect profile. Therefore, screening for novel insulinmimetics from various natural sources, such as plants and herbs,are still attractive and some of them might be safely used on dia-betes without causing serious side effects.

Saffron, the dry stigmas of the plant Crocus sativus Linn., belongto the Iridaceae family has been used as an important dietaryingredient in various parts of the world since ancient times. Fur-thermore, saffron is one of the highest priced and the most used

ll rights reserved.

ology and Toxicology, Collegersity, 900 Gajwa-dong, Jinju2 55 772 2349.

shehr University of Medical

spices around the world for flavoring and coloring food (Sampathu,Shivashankar, Lewis, & Wood, 1984). It has also been applied in tra-ditional medicine in the treatment of cramps, asthma and bron-chospasms, menstruation disorders, liver disease and pain(Schmidt, Betti, & Hensel, 2007). Among the estimated more than150 volatile and several nonvolatile compounds of saffron, majorbiologically active compounds are crocin, picocrocin, crocetin, car-otene and safranal (Rios, Recio, Giner, & Manez, 1996). Good qual-ity saffron contains about 30% crocin, 5–15% picocrocin, andusually up to 2.5% volatile compounds including safranal (Schmidtet al., 2007). The major constituents of saffron have attracted aconsiderable amount of interest during the past two decades andshown both in vitro and in vivo to possess antioxidant, anti-cancer,anti-inflammatory, anti-atherosclerosis and memory-improvingproperties (Abdullaev & Espinosa-Aguirre, 2004; Abe & Saito,2000; He et al., 2005; Hosseinzadeh & Younesi, 2002). Recently,accumulating evidence has suggested the possibility of saffronbeing used as a candidate drug for diabetes. The hypoglycemic ef-fects of saffron were reported in a study involving diabetic rats(Mohajeri, Mousavi, & Doustar, 2009), but the molecular mecha-nisms underlying this effect remain obscure.

The glucose transport in skeletal muscle is regulated by two dis-tinct pathways including phosphatidylinositol-3 kinase (PI 3-ki-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.

C. Kang et al. / Food Chemistry 135 (2012) 2350–2358 2351

3-kinase pathway includes activation of Akt leading to activation ofglycogen synthesis and the other enzymes/proteins necessary forthe acute metabolic effects of insulin, which promotes GLUT4translocation from an intracellular pool to plasma membrane(Smith & Muscat, 2005). On the other hand, AMPK is a phylogenet-ically conserved intracellular energy sensor that plays a centralrole in the regulation of glucose and lipid metabolism (Carling,2004). Activation of AMPK leads to the phosphorylation and regu-lation of a number of downstream targets that are involved in di-verse pathways, including acetyl-CoA carboxylase (ACC) in thebody (Fryer & Carling, 2005). Both distinct pathways also increasethe phosphorylation and activity of mitogen-activated protein ki-nase (MAPK) family components, which participates in the fullactivation of insulin-stimulated glucose uptake via GLUT4 translo-cation (Konrad et al., 2001).

In the present study, we showed that saffron activates AMPK/ACC and stimulate glucose uptake in C2C12 skeletal muscle cells.In addition, saffron enhanced insulin sensitivity in glucose metab-olism via activation of AMPK/ACC, MAPKs, PI 3-kinase/Akt andGLUT4 translocation to the plasma membrane. We further showedthat mTOR and its downstream pathway are also involved in thisprocess. Taken together, our results provide new insights into pos-sible mechanism of action by which saffron may contribute to thepharmacological intervention of diabetes.

2. Materials and methods

2.1. Chemicals and reagents

Dulbecco’s Modified Eagle’s Medium (DMEM), foetal bovine ser-um (FBS), bovine serum albumin (BSA), penicillin, streptomycin,trypsin were obtained from Gibco-BRL (Grand Island, NY, USA). Di-methyl sulphoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sig-

ma–Aldrich Inc. (St. Louis, MO, USA). Antibodies for Phospho-Acet-yl-CoA Carboxylase (Ser79), Phospho-AMPKa (Thr172), Phospho-Akt(Ser473), Phospho-Erk (Thr202), Phospho-p38 (Thr180), Phospho-SAPK/JNK (Thr183), AMPK, Akt and GLUT4 were obtained from CellSignaling Technology (Beverly, MA). Compound C and LY294002were purchased from Calbiochem (Merck, Darmstadt, Germany).2-Deoxy-D [3H] glucose was obtained from Perkin-Elmer Life Sci-ences (Boston, MA, USA). All other reagents used were of the purestgrade available.

2.2. Plant material and extraction

The saffron was obtained from Saharkhiz Co. (Mashhad, Iran).After grinding the saffron stigmas, 2 g of dried stigma was ex-tracted with 100 ml of methanol (80%) for 2 h in an ultrasonic bath,and then shaken (110 rpm) for 12 h at room temperature. After theextraction, the methanol was evaporated using rotary vacuumevaporator (Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The final aque-ous part was lyophilized and kept at �20 �C until use. The yield ofextraction was around 47% (w/w).

2.3. Cell culture

C2C12 (mouse myoblasts cell line) were maintained in DMEMsupplemented with 10% heat inactivated FBS and 1% antibiotics(100 U/ml penicillin and 100 lg/ml streptomycin), at 37 �C in ahumidified atmosphere with 5% CO2. For differentiation into myo-tubes, cells were reseeded in 6-well plates (for immunoblotting),and 12-well plates (for glucose uptake) at a density of2 � 104 cells/ml. After 48 h (over 80% confluence), the mediumwas switched to DMEM with 2% (v/v) FBS and was replaced after2, 4 and 6 days of culture. Experiments were initiated on day 7when myotube differentiation was complete.

2352 C. Kang et al. / Food Chemistry 135 (2012) 2350–2358

2.4. Cytotoxicity assay

Cytotoxicity experiments were conducted to determine themaximal nontoxic dose of saffron. Cell morphology was assessedby using phase-contrast microscopy, and viability was evaluatedby means of MTT assay (Mosmann, 1983). The cells were seededin 24-well plates at a density of 0.5 � 104 cells/well and culturedwith or without saffron for 24 h. Briefly, 100 ll of MTT solution(5 mg/ml MTT in PBS) was added to each well and incubated for3 h at 37 �C. After removing the supernatant, the formazan crystalgenerated was dissolved by adding 150 ll/well of dimethyl sulfox-ide (DMSO) and the absorbance was detected at 540 nm using aspectrophotometric microplate reader (BioTek Instruments, Inc.,Winooski, USA). Relative cell viability of treatment was calculatedas a percentage of vehicle-treated control ([OD of treated cells/ODof control cells] � 100).

2.5. Glucose uptake assay

Glucose uptake activity was analyzed by measuring the uptakeof 2-Deoxy-D [3H] glucose in differentiated C2C12 myotubes. Cellswere rinsed twice with warm PBS (37 �C), and then starved in ser-um free DMEM for 3 h. After saffron treatment, the cells were incu-bated in KRH buffer (20 mM HEPES, pH 7.4, 130 mM NaCl, 1.4 mMKCl, 1 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4) containing0.5 lCi of 2-Deoxy-D [3H] glucose for 10 min at 37 �C. The reactionwas terminated by placing the plates on ice and washing twicewith ice-cold PBS. The cells were lysed in 1% SDS and 400 ll ofthe lysate was mixed with 3.5 ml of scintillation cocktail and radio-activity was determined by scintillation counting. Non-specific up-take was determined in the presence of 20 lM cytochalasin B. Theresults were expressed as fold-increase.

2.6. Preparation of the cytosolic and the plasma membrane proteinfractions

The subcellular fractionation of myotubes was carried outaccording to the method (Sato, Iemitsu, Aizawa, & Ajisaka, 2008)with slight modification. Briefly, the cells from 10-cm dishes weregently scraped in buffer A (20 mM Tris pH 7.4, 1 mM EDTA,0.25 mM EGTA, 0.25 M sucrose, 1 mM DTT, 50 mM NaF, 25 mM so-dium pyrophosphate and 40 mM b-glycerophosphate). The result-ing homogenates were clarified 400 g for 15 min. The supernatantwas centrifuged at 50,000 rpm for 1 h. The resulting pellet washomogenized in buffer A and then sampled as a cytosol proteinfraction. Proteins from different fractions were solubilized for 1 hat room temperature in buffer B (20 mM Tris pH 7.4, 1 mM EDTA,0.25 mM EGTA, 2% Triton X-100, 50 mM NaF, 25 lM sodium pyro-phosphate and 40 mM b-glycerophosphate). The homogenate wascentrifuged, and the supernatant was spun for 1 h at 5000 rpm; itwas then sampled as a plasma membrane fraction. The GLUT4 pro-tein level was measured in both total and plasma membrane frac-tions. Translocation was evaluated by the difference in proteinlevels in the cytosol and plasma membrane fractions.

2.7. Protein extract and Western blotting

Cells were rinsed twice with ice-cold PBS, and then scrapedwith 100 ll of lysis buffer (50 mM Tris–HCl, pH 7.4, 1% NP-40,0.25% sodium deoxycholate, 150 mM EDTA, 1 mM PMSF, 1 mM so-dium orthovanadate, 1 mM NaF, 1 mM Na3VO4, 1 lg/ml aprotinin,1 lg/ml leupeptin, 1 lg/ml pepstatin). The plate was rocked on icefor 3 min, each samples were allowed to lyse for additional 30 minon ice with periodic vortexing. Cell debris was removed by centri-fugation (22,000g, at 4 �C for 30 min) and the resulting superna-tants were collected for immunoblot analyses. Protein

concentrations in cell lysates were measured using a Bio-Rad pro-tein assay reagent (Bio-Rad, CA, USA). Proteins in cultured cell ly-sates (30–40 lg) were separated on 12% SDS–polyacrylamide gel,transferred to PVDF membranes (Bio-Rad, CA, USA), and subse-quently subjected to immunoblot analysis using specific primaryantibodies. After incubation overnight at 4 �C with the primaryantibody, the membranes were incubated with horseradish perox-idase-conjugated secondary antibody (Cell Signaling Technology,Beverly, MA) for 1 h at room temperature. The blots were visual-ized by using the enhanced chemiluminescene method (ECL,Amersham Biosciences, Buckinghamshire, UK) on blue light-sensi-tive film (Fujifilm Corporation, Tokyo, Japan). In some cases, theblotted membranes were stripped and reprobed using other anti-bodies. Densitometric analysis was performed with a Hewlett–Packard scanner and NIH Image software (Image J).

2.8. Statistical analysis

The results are expressed as a mean ± standard deviation (SD). Apaired Student’s t-test was used to assess the significance of differ-ence between two mean values. P < 0.05 was considered to be sta-tistically significant.

3. Results

3.1. Saffron stimulates glucose uptake and AMPK/ACC pathways indifferentiated C2C12 cells

In order to assess the non-cytotoxic concentration of saffron,the viability of C2C12 cells was evaluated at the dose ranges be-tween 0 and 200 lg/ml, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Within the tested con-centrations, saffron only showed negligible cytotoxicity (data notshown), and the concentrations up to 2.5 lg/ml of saffron wereused in subsequent experiments. For determining the role of saf-fron in glucose metabolism of muscle cells, differentiated C2C12

myotube cells were treated with saffron and their glucose uptakeswere measured by 2-Deoxy-D [3H] glucose uptake assay. Interest-ingly, the glucose uptake of myotubes was considerably stimulatedby the treatment of saffron in a concentration-dependent mannerwith a maximal increase occurring at 2.5 lg/ml (Fig. 2A) for thepresent study. This result suggests that saffron may act on the pro-teins being associated with glucose uptake signaling pathways inmuscle cells. To further elucidate the possible mechanism of actionof saffron, the signaling of AMPK and ACC were examined fromC2C12 myotubes that were exposed to saffron at 2.5 lg/ml for theindicated time periods (Fig. 2B and C) or alternatively at the vari-ous concentrations for 1 h (Fig. 2D and E). From this, it was foundthat Saffron can induce a time- and dose-dependent increase ofAMPK phosphorylation in C2C12 cells. The phosphorylation of AMP-Ka (Thr172) significantly increased from 1 h of saffron treatmentand the phosphorylation of ACC-Ser79, which is the best-character-ized downstream molecule of AMPK, also showed a similar activa-tion response. More surprisingly, the stimulation of AMPK and ACCcould be observed even at the lowest concentration (0.125 lg/ml)tested in the present study (Fig 2C and E).

3.2. Saffron-induced glucose uptake is mediated by AMPK/ACC andMAPKs pathway, but not by PI 3-kinase/Akt pathway

Akt is a serine/threonine protein kinase that leads to a translo-cation of insulin-sensitive glucose transporter (GLUT4) to plasmamembrane via the activation of signal transduction cascade involv-ing insulin, insulin receptor substrate (IRS) family, and PI 3-kinase(Alessi & Cohen, 1998). To determine whether the basal activity ofPI 3-kinase/Akt is also involved in the effect of saffron on glucose

Insulin (nM)

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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) for1 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 independentexperiments, �P < 0.05, #P < 0.01, as compared with the control value.

C. Kang et al. / Food Chemistry 135 (2012) 2350–2358 2353

metabolism, we investigated the effects of saffron on PI 3-kinase/Akt pathway in comparison with those of insulin. As shown inFig. 3, Insulin caused a robust phosphorylation of Akt, while saffrontreatment had no stimulatory effect on Akt. These results indicatethat mechanism of action underlying the saffron-induced glucoseuptake in muscle cells is linked to the activation of AMPK/ACC

- + + + + -- - - - - +0 5 15 30 60 30

Saffron 2.5 μg/ml

Insulin 100 nM

Time (min)

P-AKT

GAPDH

Fig. 3. Effect of saffron on insulin signaling pathway in C2C12 cells. DifferentiatedC2C12 cells were treated with either 2.5 lg/ml of saffron for different time periods(5, 15, 30 and 60 min) or 100 nM of insulin for 30 min. Protein extracts wereprepared and subjected to Western blot assay using the primary antibodies forphospho-Akt (Ser473). GAPDH protein levels were used as a control for equalloading. The results are representative of three independent experiments.

but not to the PI 3-kinase/Akt pathway. We next corroboratedthe roles of AMPK/ACC and MAPKs in the saffron-induced glucosemetabolism of muscle cells. For this, the effect of saffron onAMPK/ACC and MAPKs pathways was investigated using the cellsignal specific inhibitors (compound C, an AMPK inhibitor, andLY294002, a PI 3-kinase inhibitor). The results showed that com-pound C, but not LY294002, potently suppressed saffron-stimu-lated AMPK/ACC and MAPKs phosphorylation (Fig. 4A–D).Similarly, the saffron-induced glucose uptake, from 2-deoxyglu-cose uptake assay, was also significantly inhibited in muscle cellsby the pretreatment of compound C, but not by LY294002(Fig. 4E). From these findings, it could be concluded that saffronby itself can induce glucose uptake in skeletal muscle cells viathe mechanisms of AMPK/ACC and MAPKs, but not through PI-3K/Akt signaling pathway.

3.3. Saffron improves insulin sensitivity via activation of AMPK/ACC,MAPKs, PI 3-kinase/Akt and mTOR pathway

Insulin has a major regulatory function for glucose metabolismin liver, adipocyte and skeletal muscle by redistributing GLUT4

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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 compoundC (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 theprimary 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 describedin 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.

2354 C. Kang et al. / Food Chemistry 135 (2012) 2350–2358

from intracellular vesicles to the cell surface. Hence, it was utmostimportant for the present study to investigate the interaction be-tween saffron and insulin on glucose metabolism-associated cellsignaling. We first attempted to study whether or not saffron hasan insulin-sensitizing potential. To this end, C2C12 cells were trea-ted with 100 nM insulin in the presence or the absence of saffron.The treatment of saffron alone did increase both AMPK/ACC andMAPKs phosphorylations without any effect on Akt (Fig. 4). Sur-prisingly, when saffron and insulin were co-treated to C2C12 cells,the phosphorylation levels of AMPK/ACC, MAPKs and Akt were fur-ther increased to the levels, which were much higher than the indi-

vidual effects of each reagent (Fig. 5A–D). To further develop thisconcept, we performed a GLUT4 Western blotting for the cytosoland membrane fractions of the C2C12 cells. The co-treatment of saf-fron and insulin further increased the translocation of and the plas-ma membrane abundance of GLUT4, without much changingGLUT4 content in the cytosol (Fig. 5E and F). On the other hand,the co-treatment of saffron and insulin slightly inhibited the phos-phorylations of mammalian target of rapamycin (mTOR) and itsdownstream targets (p70S6K and 4EBP1) (Fig. 6). Insulin-stimula-tion of PI 3-kinase/Akt is a key step in insulin signaling mechanismand negatively regulated by mTOR pathway (Tzatsos & Kandror,

p-AMPKα

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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 orabsence of insulin (100 nM). Phospho-acetyl-CoA carboxylase (Ser79), phospho-AMPKa (Thr172), phospho-Akt (Ser473), phospho-JNK, phospho-p38, phospho-ERK and GLUT4were 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 assayusing 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 independentexperiments, �P < 0.05, #P < 0.01, as compared with the control value.

C. Kang et al. / Food Chemistry 135 (2012) 2350–2358 2355

2006), which also inhibited the activation of AMPK (Inoki, Zhu, &Guan, 2003). These results suggest that saffron can increase thesensitivity of muscle cells to insulin via the glucose metabolism-associated cell signaling pathways, such as AMPK/ACC, MAPKs,PI-3/Akt pathway, and mTOR pathway.

The present findings were further confirmed by investigatingthe effects of saffron and insulin co-treatment in the presence orabsence of pharmacological inhibitors for AMPK or PI 3-kinase/Akt pathways. Saffron-enhanced insulin sensitivity on AMPK/ACC,JNK and p38 were almost completely obliterated by compound C(an AMPK inhibitor), but mostly not by LY294002 (a PI 3-kinaseinhibitor) (Fig. 7A–D). On the other hand, the activation of Akt un-der the co-treatment was significantly modulated only byLY294002 but not by compound C (Fig. 7A and B). In contrary toother MAPKs, the Erk stimulation was only slightly attenuated by

compound C but not by LY294002 (Fig. 7C and D). Consistent withother results, the induction of glucose uptake could be effectivelyattenuated by either compound C or LY294002 (Fig. 7E). These re-sults provide another evidence for a crosstalk between AMPK/ACCand MAPKs in the glucose metabolism, which eventually leads toactivation of PI-3/Akt signal pathway.

4. Discussion

Diabetes mellitus is a chronic metabolic disorder affectingabout 6% of population worldwide. Chronic hyperglycemia is amain contributor of the associated disorders, which can exacerbatedefective glucose disposal by interfering with insulin action ininsulin-target tissues such as skeletal muscle, liver and adipose tis-

- - + +- + - +

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Insulin

p-p70 S6K

p-mTOR

GAPDH

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(B)

Fig. 6. Inhibition of mTOR pathway is involved in the effect of saffron on insulinsignaling. Differentiated C2C12 cells were treated with saffron (2.5 lg/ml) for 1 h inthe presence of absence of insulin (100 nM). Protein extracts were prepared andsubjected to Western blot assay (A) using the primary antibodies for phospho-mTOR, phospho-p70S6 K (Thr389), and phospho-4EBP1(Thr37/46), then they werequantified (B). GAPDH protein levels were used as a control for equal loading. Theresults 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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sue (Schenk, Saberi, & Olefsky, 2008). Among the insulin-target tis-sues, skeletal muscle is responsible for more than 75% of glucosedisposal in response to insulin in the post-prandial state (DeFronzoet al., 1981). Regulation of glucose metabolism of skeletal muscle istherefore quantitatively most important in energy balance, and isthe primary tissue of insulin-stimulated glucose uptake, disposaland storage (Kang et al., 2010). It demonstrated that a brown algaof polyphenol-rich, Ecklonia cava possesses insulinomimetic prop-erties, since it decrease hyperglycemia in streptozotocin-diabeticrats. Furthermore, it stimulates glucose uptake in skeletal musclecell line mediated by the activation of both AMPK and PI 3-ki-nase/Akt pathways. From the reports on the potential effectivenessof traditional remedies against diabetes, it is believed that the nat-ural products may play a major role in the management of diabetes(Srinivasan, 2005).

The principal finding of present study is that saffron stimulatesglucose uptake and increase insulin sensitivity in skeletal musclecells via multiple mechanisms. These findings indicate that thehypoglycemic effect of saffron is attributable to the metabolicactivity of saffron in skeletal muscle. Results of the glucose uptakeassay demonstrated that saffron can enhance glucose uptake in adose-dependent manner in differentiated C2C12 cells (Fig 2A), indi-cating its possible regulatory role in the glucose metabolism ofskeletal muscle cells. In an effort to understand the signaling path-ways involved in saffron-mediated glucose uptake, we carried outWestern blot analyses using specific antibodies for the signalingmolecules associated with glucose metabolism. Saffron was shownto activate AMPK in a time- (Fig. 2B and C) and dose- (Fig. 2D andE) dependent manner. Accordingly, ACC-Ser79, the best-character-

ized phosphorylation site by AMPK, was also significantly phos-phorylated by the treatment of saffron. In insulin signalingpathway, PI 3-kinase is a key molecule, and its main downstreamtarget is Akt, that is important in insulin-stimulated glucose trans-port and metabolism (Ueki et al., 1998). Concurrently, we examinewhether PI 3-kinase/Akt pathway is involved in this stimulatory ef-fect of saffron. From this, insulin caused a robust phosphorylationof Akt while saffron treatment had no significant effect (Fig. 3),suggesting saffron alone does not stimulate PI 3-kinase/Aktpathway.

Recent studies have demonstrated a correlation between theAMPK and MAPKs signaling pathways; for example, p38 MAPKactivation was shown to have been completely abolished in skele-tal 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 possi-ble target in glucose metabolism. In order to confirm therelationship between AMPK and MAPKs in the skeletal musclecells, the C2C12 cells were pre-incubated with either compound Cor LY294002. Our results demonstrated that compound C potentlyattenuated the phosphorylation of AMPK/ACC, MAPKs and glucoseuptake stimulated by saffron, whereas LY294002 did not signifi-cantly inhibit saffron-induced activation of glucose metabolism(Fig. 4). These facts revealed that AMPK/ACC and MAPKs could bethe key factor in saffron-activated glucose uptake.

Several findings indicated there is a crosstalk between AMPKand insulin signaling pathways (Kang & Kim, 2010; Kovacic et al.,2003). Consistent with these previous reports, when the cells wereexposed to saffron and insulin simultaneously, both of the AMPKand PI 3-kinase/Akt pathways were more greatly stimulated com-paring with those of their individual treatments (Fig. 5A and B).Concurrently, the MAPKs were also further activated by the co-treatment of saffron and insulin from those observed by the eachtreatment (Fig. 5C and D), which is probably due to the internaliza-tion of insulin signaling is also required to phosphorylate and acti-vate the MAPKs pathway (Montagut et al., 2010). Additionally, theactivation of AMPK on glucose uptake occurs independently ofinsulin, and promotes the translocation of GLUT4 from an intracel-lular pool to the cell surface, which in turn can lead to the improve-ment of insulin stimulated glucose uptake (Zheng et al., 2001). TheGLUT4 protein levels in skeletal muscle cells showed that saffroncan increase insulin sensitivity by promoting the translocation ofGLUT4 protein to the plasma membrane (Fig 5E and F). In additionto the insulin signaling pathway, mammalian target of rapamycin(mTOR) was also well-known to be activated by insulin and exertthe physiological feedback regulation on insulin signaling throughincreasing the phosphorylation of serine residue on IRS-1 (Umet al., 2004). Moreover, activation of AMPK by treatment with AI-CAR (an AMPK activator) is associated with enhanced phosphory-lation of MAPKs and decreased phosphorylation of mTOR,suggesting that the repressive action of AMPK on mTOR signalingis dominant to the stimulatory action of MAPKs pathway (William-son, Bolster, Kimball, & Jefferson, 2006a,2006b). In the presentstudy, saffron slightly inhibited the phosphorylations of mTORand its downstream targets of p70S6K and 4EBP1 (Fig. 6). The aug-mentation of insulin sensitivity by saffron was further confirmedusing compound C and LY294002, which abrogated saffron-in-duced glucose uptake and AMPK/ACC, MAPKs and PI 3-kinase/Aktactivation (Fig. 7). Therefore, it can be concluded that saffron en-hances insulin sensitivity via multiple mechanisms. Consistentwith our study, recent evidences provide that insulin-like naturalcompounds can improve insulin sensitivity by activating AMPKand PI 3-kinase/Akt pathways (Huang et al., 2010; Kang & Kim,2010; Li, Ge, Zheng, & Zhang, 2008).

In conclusion, saffron plays a beneficial role in glucose metabo-lism of differentiated C2C12 skeletal muscle cells. The activation of

p-Akt

p-ACC

p-AMPKα

Com.C

- + + + +- - + + +- - - + -- - - - +

Saffron

LY294002

Insulin

GAPDH

Com.C

- + + + +- - + + +- - - + -- - - - +

Saffron

LY294002

Insulin

p-ERK

p-p38

GAPDH

p-JNK

Com.C

- - + + + +- + - + + +- - - - + -- - - - - +

Saffron

LY294002

Insulin

0

1

2

3

4

5

6

2-de

oxyg

luco

se u

ptak

e (fo

ld in

duct

ion)

#

##

#

#

(A)

(B)

(C)

(D)

(E)

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 subcellularmembrane 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 inMaterials 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.

C. Kang et al. / Food Chemistry 135 (2012) 2350–2358 2357

AMPK/ACC and MAPKs pathways is directly associated with saf-fron-induced glucose uptake. Saffron also increases insulin sensi-tivity which is coupled to basal glucose translocation of GLUT4through both of insulin-independent (AMPK/ACC and MAPKs)and insulin-dependent (PI 3-kinase/Akt and mTOR) pathways.These findings support a therapeutic potential of saffron in thetreatment of diabetes and its complications.

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