Promotion of Glucose Uptake in C2C12 Myotubes by Cereal Flavone

Article pubs.acs.org/JAFC

Promotion of Glucose Uptake in C2C12 Myotubes by Cereal Flavone Tricin and Its Underlying Molecular Mechanism Sohyun Kim, Gwang-Woong Go, and Jee-Young Imm* Department of Foods and Nutrition, Kookmin University, 861-1, Jeongnung-dong, Seongbuk-gu, Seoul 136-702, Korea ABSTRACT: The effect of tricin, a methylated flavone widely distributed in cereals, on glucose uptake and the underlying molecular mechanism was investigated using C2C12 myotubes. Tricin significantly increased glucose uptake in C2C12 myotubes, regardless of the absence (1.4-fold at 20 μM) or presence (1.6-fold at 20 μM) of insulin. The GLUT4 expression on the plasma membrane was increased 1.6-fold after tricin treatment (20 μM) in the absence of insulin. Tricin treatment significantly activated the insulin-dependent cell signaling pathway, including the activation of insulin receptor substrate-1 (IRS1), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and AKT substrate of 160 kDa (AS160). The oral administration of tricin (64 and 160 mg kg−1 of body weight day−1) also significantly lowered blood glucose levels in glucoseloaded C57BL/6 mice (p < 0.05). These results suggest that tricin has great potential to be used as a functional agent for glycemic control. KEYWORDS: tricin, glucose uptake, C2C12 myotubes, GLUT4, insulin-dependent pathway

1. INTRODUCTION Failure of glucose homeostatic mechanisms plays an important role in the development of metabolic disorders, such as type 2 diabetes and obesity.1 Impaired insulin secretion or insulin resistance causes diabetes, which is characterized by hyperglycemia (fasting plasma glucose > 126 mg/dL).2 Diabetic incidence has increased over the last few decades, and it has now become a global health concern. Consequently, molecular mechanisms regulating blood glucose levels and the development of safe antidiabetic agents are a major research focus. Skeletal muscle is the primary peripheral tissue responsible for blood glucose control, and more than 70% of insulinmediated glucose uptake occurs here.3 Because muscle is a key player of glucose utilization in the body, the promotion of glucose uptake in muscle tissues is an efficient strategy for glycemic control.4 Glucose uptake in skeletal muscle is mediated by two major signals, i.e., insulin and exercise.5 The insulin-dependent pathway involves an insulin-receptor substrate (IRS)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signal. On the other hand, the insulin-independent glucose uptake mechanism involving the 5′-adenosine-monophosphate-activated protein kinase (AMPK) signal is stimulated by exercise. Some dietary polyphenols and flavonoids have beneficial effects on type 2 diabetes mellitus and have received considerable attention as potential hypoglycemic agents.6,7 Tricin (5,7,4′-trihydroxy-3′,5′-dimethoxyflavone) is a cereal flavone and widely distributed in the bran and hull of wheat, oat, maze, etc.8 A conjugated form of tricin [tricin 4′-O-(threo-β-guaiacylglyceryl) ether (TTGE)] was isolated from traditional medicinal Njavara rice, and TTGE exerted potent antiinflammatory activity through suppression of nuclear factorκB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) pathways.9,10 In addition, the colon and breast cancer preventative activity of tricin was demonstrated in a © XXXX American Chemical Society

mouse model. The presence of two methoxy groups in the B ring of the flavonoid was closely related to its chemopreventive efficacy.11−13 Tricin also showed a more favorable safety profile than genistein and quercetin in acute toxicity evaluation.14 Recently, we identified tricin as the active compound in oat hull extract possessing antiadipogenic activity in 3T3-L1 adipocytes.15 Up to now, studies on the antidiabetic effect of flavones and their underlying molecular mechanisms of this effect were somewhat limited in comparison to other classes of flavonoids and antidiabetic potential of tricin has never been investigated. The objective of this study was to evaluate the antidiabtic effect of tricin and to elucidate the molecular mechanism leading to improved glucose uptake in C2C12 skeletal muscle cells.

2. MATERIALS AND METHODS 2.1. Chemicals. The C2C12 myoblasts were purchased from the American Type Culture Collection (Manassas, VA, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), horse serum (HS), and penicillin−streptomycin (PS) were obtained from WelGENE (Daegu, Korea). Tricin was purchased from Dalton PharmaServices (Toronto, Ontario, Canada). 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) and LY294002 were purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.) and Tocris (Avonmouth, Bristol, U.K.), respectively. Both anti-GLUT4 and the secondary antibody fluorescein isothiocyanate (FITC)−IgG were obtained from Abcam (Cambridge, MA, U.S.A.), and mounting medium with 6,4′-diamidino-2-phenylindole (DAPI) was purchased from Vectashield (Burlingame, CA, U.S.A.). The radioimmunoprecipitation assay (RIPA) buffer, the protease inhibitor, and the phosphatase inhibitor were obtained from Atto (Tokyo, Japan). The IRS-1, phospho-PI3K (Tyr199), PI3K, phospho-AKT (Thr308), AKT, phospho-AS160 (Thr642), AS160, GLUT4, β-actin, and Received: February 7, 2017 Revised: April 28, 2017 Accepted: April 28, 2017

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DOI: 10.1021/acs.jafc.7b00578 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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sulfoxide (DMSO)], (2) tricin [16 mg kg−1 of body weight (BW) day−1], (3) tricin (64 mg kg−1 of BW/day−1), and (4) tricin (160 mg kg−1 of BW day−1). The animals had free access to the basal diet and water, and the indicated dose of tricin was orally administrated daily for 7 days using an oral gavage. After 5 h of fasting, a glucose solution (1 g/kg of BW) was orally administered to the mice and the blood glucose level were measured from the tail vein using a blood glucose analyzer (AccuCheck Performa, Roche, Mannheim, Germany) at 0, 15, 30, 60, and 120 min after glucose administration. 2.8. Statistical Analysis. Data were presented as the mean ± standard error, and all quantitative determinations were carried out in triplicate. Statistical analysis was performed using one-way analysis of variance (ANOVA) and SPSS statistical software (SPSS, Inc., Chicago, IL, U.S.A.). Duncan’s multiple comparison test was carried out when a significant difference (p < 0.05) was found.

horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.). The Na+/K+-ATPase α was purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.A.), and the phospho-IRS-1 (Tyr612) was purchased from Abcam. Rosiglitazone and all other solvents were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). 2.2. Cell Culture. The C2C12 myoblasts were cultured in DMEM containing 10% FBS and 1% PS at 37 °C in humidified air containing 5% CO2. When the myoblasts were confluent, they were treated with differentiation inducers (DMEM containing 2% HS) for 5 days. The medium was replaced every 2 days. 2.3. Cell Viability. Cell viability was evaluated using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytotoxicity was determined 5 days after differentiation in the presence of 5, 10, and 20 μM tricin (20 μM rosiglitazone) as previously described.15 2.4. Glucose Uptake Assay. The confluent myotubes in glucosefree DMEM were incubated for 24 h with tricin (5, 10, or 20 μM) and 2-NBDG (100 μM). After removal of the culture medium, the cells were washed 3 times with cold phosphate-buffered saline (PBS). The fluorescence intensity of cellular 2-NBDG was measured at an excitation wavelength of 485 nm and emission wavelength of 535 nm using a microplate reader (Biotek Instruments, Inc., Winooski, VT, U.S.A.). 2.5. Measurement of Cell Surface GLUT4 Levels. The detection of GLUT4 on the cell surface was performed according to the previously published method, with a slight modification.16 Briefly, cells were cultured and differentiated on the coverslip. After sample treatment, cells were fixed with formaldehyde/PBS containing Triton X-100 (0.1%) and blocked with PBS containing 3% bovine serum albumin (BSA) for 1 h. The cells were incubated with anti-GLUT4 (1:200 dilution in 3% BSA/PBS) for 90 min at 4 °C and washed with PBS. The cells were then incubated with the secondary antibody labeled with FITC (1:200 dilution in 3% BSA/PBS) and washed with PBS. Cells were treated with mounting medium with DAPI. Representative images were taken using a confocal microscope LSM 700 (Carl Zeiss, Germany) and ZEN 2.1 lite software. GLUT4 expression on the plasma membrane (PM) was measured by a biotinylation assay using a Pierce cell surface protein isolation kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.) by the method of Cunningham et al.17 Western blot analysis was performed to quantify the expression level of GLUT4 in the cell membrane. GLUT4 levels were normalized by Na+/K+-ATPase, which is a known PM marker.18 2.6. Western Blot Analysis. After sample treatment, cells were harvested by centrifugation at 17 000 rpm for 1 min. The cell pellets were lysed using RIPA buffer (Atto, Tokyo, Japan) containing 1% protease inhibitor and 1% phosphatase inhibitor. The protein concentration of cell lysates was determined using the Bradford assay, and an equal quantity of protein was separated by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) (10%). The protein was transferred to a polyvinylidene fluoride membrane (Milllipore, Billerica, MA, U.S.A.) at 0.2 A for 90 min, and the membrane was blocked with 5% BSA in Tris-buffered saline and 0.1% Tween 20 (TBST) for 50 min at room temperature. The membrane was incubated for 2 days at 4 °C with the primary antibody solution (1:1000 dilution with 5% BSA) (p-IRS, IRS, p-PI3K, PI3K, pAKT, AKT, p-AS160, AS160, GLUT4, Na+/K+-ATPase α, and β-actin) and rinsed with TBST. The membrane was incubated with the HRPconjugated secondary antibody and rinsed again. The protein bands were detected by an enhanced chemiluminescence (ECL) kit and quantified using the Image Lab software (BioRad, Hercules, CA, U.S.A.). 2.7. Oral Glucose Tolerance Test (OGTT). All animal experiments were approved by Kookmin University Institutional Animal Care and Use Committee (KMUIACUC-2016-05). Four-week-old male C57BL/6 mice were purchased from Dae-han Bio Link (ChungBuk, Korea). The animals were maintained at 22 ± 1 °C and 50 ± 10% relative humidity, with a 12 h light (from 7 am to 7 pm)/dark cycle. After 7 days of acclimatization, animals (21 ± 1 g) were randomly assigned to four groups (n = 8) as follows: (1) control [0.2% dimethyl

3. RESULTS AND DISCUSSION 3.1. Tricin-Stimulated Glucose Uptake in C2C12 Myotubes. Tricin treatment at a concentration up to 20 μM for 24 h did not produce any sign of cytotoxicity in C2C12 myotubes (data not shown). To evaluate the effect of tricin treatment on glucose uptake, cells were treated with 2-NBDG, a fluorescently labeled glucose analogue, with or without tricin, for 24 h. In the absence of insulin, glucose uptake of C2C12 myotubes was significantly increased by tricin. The extent of this increase was comparable to that observed with rosiglitazone, the positive control (Figure 1). The tricin-

Figure 1. Effect of tricin on glucose uptake in C2C12 myotubes. Differentiated C2C12 myotubes were treated with 5, 10, and 20 μM tricin (20 μM rosiglitazone) alone or with 100 nM insulin for 24 h. Each data set is expressed as the mean ± standard error (SE) of three independent experiments (n = 3). Bars with different letters are significantly different (p < 0.05).

mediated promotion of glucose uptake was further augmented in the presence of insulin (1.6-fold at 20 μM). These results suggest that the promotion of glucose uptake by tricin occurred via a complementary pathway to the insulin-signaling cascade. A variety of flavonoids increase glucose uptake in muscle cells. However, the efficacy of this effect and the underlying mechanisms are somewhat unclear, and multiple mechanisms could be involved depending upon experimental conditions (e.g., presence of insulin, sample concentration, and type of cell culture model).6 Rosiglitazone, a member of the thiazolidinedione class, is a currently available antidiabetic drug. However, some concerns B

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Figure 2. (A) Effect of tricin on GLUT4 translocation and (B) expression level of GLUT4 in PM of C2C12 myotubes. Differentiated C2C12 myotubes were treated with 100 nM insulin or 20 μM tricin. (A) In the images, the green color indicates GLUT4 bound with the FITC-labeled antibody, while the blue color indicates a nucleus stained with DAPI. Arrows indicate where the green fluorescence appears relatively strong. Scale bar = 20 μm. (B) Expression level of GLUT4 on PM was normalized with that of Na+/K+-ATPase. Each data set is expressed as the mean ± SE of three independent experiments (n = 3). Bars with different letters are significantly different (p < 0.05).

1.6-fold after tricin treatment (Figure 2B). This result suggests that the elevated glucose uptake in C2C12 myotubes induced by tricin was mediated by GLUT4 translocation. Although impaired glucose uptake in muscle cells via GLUT4 translocation has been found in type 2 diabetes, the roles of different regulatory compartments on the GLUT4 traffic remain to be fully elucidated.25 The effect of the flavonoid structure on glucose uptake in muscle cells has not been clearly demonstrated. Several antidiabetic compounds were identified in Cleome droserifolia herbal extract based on their ability to promote glucose uptake in C2C12 myotubes.26 The three highly active compounds, namely, isorhamnetin-3-O-β-D-glucoside, quercetin-3′-methoxy-3-O-(4″-acetylrhamnoside)-7-O-α-rhamnoside, and kaempferol-4′-methoxy-3,7-dirhamnoside, possess 3′ or 4′ methoxy groups at the B ring. The authors speculated that the presence of a methoxy group could be related to higher activity of the compounds. In this context, the two methoxy moieties at the 3′ and 5′ positions of the B ring in tricin possibly contribute to the enhancement of glucose uptake via GLUT4 translocation. 3.3. Tricin Activated the Insulin-Dependent PI3K/AKT/ AS160 Signaling Pathway. To identify the mechanism underlying tricin-stimulated glucose uptake, the involvement of the insulin-mediated pathway was examined. The insulinmediated glucose uptake is initiated by activation of the insulin receptor by insulin, which leads to sequential activation of docking proteins, such as IRS, PI3K, AKT, and AS160.27 Activation of the insulin-signaling cascade was determined following tricin treatment using western blotting. Tricin increased phosphorylation of IRS by 1.7-fold. This increase was higher than that of the positive control,

have been raised regarding its side effects, including weight gain, hepatotoxicity, and cardiovascular problems.19,20 In our previous study, tricin treatment resulted in the antiadipogenic effect mediated via the AKT/mTOR/SREBP-1 pathway in 3T3-L1 adipocytes.21 This suggests that tricin is able to promote glucose uptake in skeletal muscles without inducing fat accumulation in adipocytes. Similarly, kaempferitrin (kaempferol 3,7-dirhamnoside) found in medicinal extract, Bauhindia acuminate, increased glucose uptake in skeletal muscle cells while inhibiting GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes.22,23 3.2. Tricin Enhanced GLUT4 Translocation in C2C12 Myotubes. Insulin-stimulated glucose uptake is primarily mediated by GLUT4. GLUT4 vesicles are normally located in the cytoplasm, and they are translocated to the PM in response to insulin. Thus, GLUT4 principally contributes to the absorption of glucose into cells.24 To investigate whether the tricin-induced increase in glucose uptake was mediated by GLUT4 translocation, immunofluorescence staining was used to observe the extent of GLUT4 translocation. Treatment with Triton X-100 during sample preparation facilitated penetration of anti-GLUT4 through the cell membranes, which bound to GLUT4 located in both intracellular storage and the PM. Thus, FITC-labeled fluorescence intensity (green) was proportional to the amount of anti-GLUT4 binding, while nuclei were stained blue by DAPI staining. As shown in Figure 2A, GLUT4 was mainly located close to nucleic acids in the control sample, whereas a GLUT4 fluorescence signal close to the PM was substantially increased by either insulin or tricin treatment. The GLUT4 expression on the PM was also quantified by a biotinylation assay. In the absence of insulin, GLUT4 expression increased C

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Figure 3. Effect of tricin on phosphorylation of (A) IRS1, (B) PI3K, (C) AKT, and (D) AS160 in C2C12 myotubes. Differentiated C2C12 myotubes were treated with 100 nM insulin or 5, 10, 20 μM tricin (20 μM rosiglitazone), and the phosphorylation of IRS1, PI3K, AKT, and AS160 was analyzed. Each data set is expressed as the mean ± SE of three independent experiments (n = 3). Bars with different letters are significantly different (p < 0.05).

from the insulin binding site.28 The IR is a tetramer consisting of two α and two β subunits, and the binding of insulin to the α subunit causes conformational changes, leading to activation of the intercellular tyrosine kinase domain. This, in turn, phosphorylates other cellular signaling agents, including PI3K/AKT and the mitogen-activated protein kinase (MAPK) pathway.29 Molecular docking studies of banana flower flavonoids using a three-dimensional (3D) structure of the IR tyrosine kinase suggest that hesperitin triacetate, naringenin, and pelagonidin are IR tyrosine kinase activators.30

rosiglitazone, at the same concentration (Figure 3A). Phosphorylation of PI3K (Tyr199) was increased >2-fold by treatment with 20 μM tricin compared to treatment with insulin alone (p < 0.05) (Figure 3B). Tricin treatment also significantly increased phosphorylation of AKT and AS160 by 1.36- and 1.38-fold, respectively (p < 0.05) (panels C and D of Figure 3). These results suggest that tricin acts as an insulin agonist. Similarly, procyanidin extracted from grape seed activated the insulin receptor (IR) and other key targets of insulin-signaling molecules, but its activation site was different D

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Figure 4. Effect of LY294002, a PI3K inhibitor, on (A) tricin-stimulated glucose uptake and tricin-activated phosphorylation of (B) PI3K, (C) AKT, and (D) AS160 in C2C12 myotubes. Differentiated C2C12 myotubes were pretreated with LY294002 (40 μM) for 12 h. Cells were treated with 100 nM insulin or 20 μM tricin. Glucose uptake was then determined, and the phosphorylation of PI3K, AKT, and AS160 was analyzed. Each data set is expressed as the mean ± SE of three independent experiments (n = 3). Bars with different letters are significantly different (p < 0.05).

The synergistic effect of tricin and insulin on glucose uptake is possibly brought about in a similar way, but a more detailed investigation is required to identify the exact nature of the

synergistic action. AS160, a direct AKT downstream molecule, contains a Rab GTPase domain, which regulates several GLUT4 translocation steps, including vesicle budding, motility, E

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Journal of Agricultural and Food Chemistry and membrane fusion.31 It has been demonstrated that insulinstimulated AS160 phosphorylation was impaired in the skeletal muscle of type 2 diabetic patients.32 However, in the absence of insulin, tricin treatment did not increase phosphorylation of AMPK in C2C12 myotubes (data not shown). Myricetin, a hexahydroxyflavone found in berries and herbs, stimulated glucose uptake via both insulindependent and insulin-independent pathways. However, the activation of PI3K and the AMPK pathway by myricetin varied depending upon the concentration of insulin in the media.33 Similar to our findings, rutin enhanced glucose uptake by activation of IR and AKT but did not affect phosphorylation of AMPK in C2C12 myotubes.34 Saffron (Crocus sativus L.) extract increased glucose uptake via activation of AMPK in the absence of insulin, but insulin sensitivity was increased in muscle cells upon co-treatment with insulin and saffron through AMPK and PI3K/AKT pathways. 35 Tricin could also potentially recruit other mechanisms to increase insulin sensitivity. In comparison to other flavonoid subclasses, the studies regarding the antidiabetic effect of dietary flavones are relatively limited. Apigenin (4′,5,7-trihydroxyflavone) exerts a positive effect on alloxan-induced type 1 diabetic mice,36 but its effect on type 2 diabetic animal models has not been proven. Luteolin (3′,4′,5,7-tetrahydroxyflavone) failed to show a significant effect on plasma glucose controls up to 200 mg kg−1 in OGTTs, although it had a strong α-glucosidase inhibitory activity, with IC50 of 2.3 mM.37 The involvement of an insulin-dependent pathway in tricininduced glucose uptake in muscle cells was confirmed using the cell signal-specific PI3K inhibitor, LY294002. As shown in Figure 4, LY294002 (40 μM) significantly suppressed the glucose uptake seen following both tricin and insulin treatment to control levels. In addition, LY294002 significantly decreased tricin-stimulated phosphorylation of PI3K, AKT, and AS160. On the basis of these results, the insulin-dependent PI3K/ AKT/AS160 signaling pathway is a major contributor to the tricin-associated increase in glucose uptake. 3.4. Tricin Lowered Blood Glucose Levels after Glucose Load. The management of postprandial blood glucose levels is important in practical terms. The OGTT was conducted in C57BL/6 mice to evaluate the efficacy of tricin with respect to the glycemic control. The dose was selected on the basis of the chemopreventive effect of tricin on colon carcinogenesis in mice.38 Blood glucose levels had rapidly increased 15 min after administration of the glucose load. However, medium (64 mg kg−1 of BW day−1) and high (160 mg kg−1 of BW day−1) doses of tricin significantly attenuated the elevated blood glucose levels seen following glucose loading at this time point (p < 0.05) (Figure 5A). The calculated area under the curve also demonstrated that the medium and high doses of tricin significantly lowered the blood glucose response, indicating enhanced insulin sensitivity, compared to the control (p < 0.05) (Figure 5B). In the OGTT, tricin treatment increased utilization of peripheral glucose in mice, resulting in improved glucose tolerance. The extent of improved glucose tolerance may vary depending upon the dose of samples and the insulin sensitivity of animals. In our experimental conditions, blood glucose tolerance significantly improved by tricin treatment with 64 mg kg−1 of BW day−1, although further improvement was not observed at 160 mg kg−1 of BW day−1. These results suggest that tricin is also effective with respect to blood glucose control in animal models. It was reported that

Figure 5. (A) Changes in blood glucose levels and (B) area under curve after oral administration of tricin in glucose-loaded C57BL/6 mice. After oral administration of a glucose solution (1 g/kg of BW), blood glucose levels were measured at 0, 15, 30, 60, and 120 min. Each data set is expressed as the mean ± SE (n = 6/group). (∗) p < 0.05 compared to the control.

methylated flavonoids displayed greater resistance to metabolic modification than non-methylated flavonoids in intestine and liver. Tricin was detected in an unmetabolized form in the feces of rats, while quercetin was extensively metabolized and excreted in the urine.39 The two methoxy groups located on either side of the 4′-hydroxyl group in the B ring might be responsible for the higher metabolic stability of tricin. In conclusion, tricin significantly increased glucose uptake in skeletal muscle cells, and this stimulatory action was associated with the insulin-dependent signaling cascade involving PI3K, AKT, and AS160. The improved glucose uptake induced by tricin probably contributed to its glucose-lowering action, as seen in the OGTT. This study provides good evidence that tricin widely distributed in cereal bran has excellent potential to be used as a functional agent for glycemic control.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 82-2-910-4772. Fax: 82-2-910-5249. E-mail: [email protected]. ORCID

Jee-Young Imm: 0000-0003-3152-7051 F

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putative cancer chemopreventive agent tricin, a naturally occurring flavone. Cancer Chemother. Pharmacol. 2006, 57, 1−6. (15) Lee, D.; Park, H. Y.; Kim, S.; Park, Y.; Bang, M. H.; Imm, J. Y. Anti-adipogenic effect of oat hull extract containing tricin on 3T3-L1 adipocytes. Process Biochem. 2015, 50, 2314−2321. (16) Ariga, M.; Nedachi, T.; Katagiri, H.; Kanzaki, M. Functional role of sortilin in myogenesis and development of insulin-responsive glucose transport system in C2C12 myocytes. J. Biol. Chem. 2008, 283, 10208−10220. (17) Cunningham, M. R.; McIntosh, K. A.; Pediani, J. D.; Robben, J.; Cooke, A. E.; Nilsson, M.; Gould, G. W.; Mundell, S.; Milligan, G.; Plevin, R. Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4). J. Biol. Chem. 2012, 287, 16656−16669. (18) Zierath, J. R.; He, L.; Guma, A.; Wahlström, E. O.; Klip, A.; Wallberg-Henriksson, H. Insulin action on glucose transport and plasma membrane GLUT4 content in skeletal muscle from patients with NIDDM. Diabetologia 1996, 39, 1180−1189. (19) Nissen, S. E.; Wolski, K. Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes. N. Engl. J. Med. 2007, 356, 2457−2471. (20) Hollander, P. Anti-diabetes and anti-obesity medications: Effects on weight in people with diabetes. Diabetes Spectrum 2007, 20, 159− 165. (21) Lee, D.; Go, G. W.; Imm, J. Y. Tricin, a methylated cereal flavone, suppresses fat accumulation by downregulating AKT and mTOR in 3T3-L1 preadipocytes. J. Funct. Foods 2016, 26, 548−556. (22) Vishnu Prasad, C. N.; Suma Mohan, S.; Banerji, A.; Gopalakrishnapillai, A. Kaempferitrin inhibits GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes. Biochem. Biophys. Res. Commun. 2009, 380, 39−43. (23) Cazarolli, L. H.; Pereira, D. F.; Kappel, V. D.; Folador, P.; Figueiredo, M. D. S. R. B.; Pizzolatti, M. G.; Silva, F. R. M. B. Insulin signaling: A potential signaling pathway for the stimulatory effect of kaempferitrin on glucose uptake in skeletal muscle. Eur. J. Pharmacol. 2013, 712, 1−7. (24) Rowland, A. F.; Fazakerley, D. J.; James, D. E. Mapping insulin/ GLUT4 circuitry. Traffic 2011, 12, 672−681. (25) Ishiki, M.; Klip, A. Minireview: Recent developments in the regulation of glucose transporter-4 traffic: New signals, locations and partners. Endocrinology 2005, 146, 5071−5078. (26) Motaal, A. A.; Ezzat, S. M.; Haddad, P. S. Determination of bioactive markers in Cleome droserifolia using cell-based bioassays for antidiabetic activity and isolation of two novel active compounds. Phytomedicine 2011, 19, 38−41. (27) Zaid, H.; Antonescu, C. N.; Randhawa, V. K.; Klip, A. Insulin action on glucose transporters through molecular switches, tracks and tethers. Biochem. J. 2008, 413, 201−215. (28) Montagut, G.; Onnockx, S.; Vaque, M.; Blade, C.; Blay, M.; Fernandez-Larrea, J.; Pujadas, G.; Salvado, M. J.; Arola, L.; Pirson, I.; Ardevol, A.; Pinent, M. Oligomers of grape-seed procyanidin extract activate the insulin receptor and key targets of the insulin signaling pathway differently from insulin. J. Nutr. Biochem. 2010, 21, 476−481. (29) Saltiel, A. R.; Kahn, C. R. Insulin signaling and the regulation of glucose and lipid metabolism. Nature 2001, 414, 799−806. (30) Ganugapati, J.; Baldwa, A.; Lalani, S. Molecular docking studies of banana flower flavonoids as insulin receptor tyrosine kinase activators as cure for diabetes mellitus. Bioinformation 2012, 8, 216− 220. (31) Sano, H.; Kane, S.; Sano, E.; Miinea, C. P.; Asara, J. M.; Lane, W. S.; Garner, C. W.; Lienhard, G. E. Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation. J. Biol. Chem. 2003, 278, 14599−14602. (32) Karlsson, H. K. R.; Zierath, J. R.; Kane, S.; Krook, A.; Lienhard, G. E.; Wallberg-Henriksson, H. Insulin-stimulated phosphorylation of the Akt substrate AS160 is impaired in skeletal muscle of type 2 Diabetic subjects. Diabetes 2005, 54, 1692−1697.

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, Information and Communications Technology (ICT) and Future Planning (MSIP, 2014R1A2A2A01007169). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.7b00578 J. Agric. Food Chem. XXXX, XXX, XXX−XXX