Acacetin enhances glucose uptake through insulin-independent GLUT4 translocation in L6 myotubes

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Acacetin enhances glucose uptake through insulin-independent GLUT4 translocation in L6 myotubes Eun-Bin Kwon , Myung-Ji Kang , Hyung Won Ryu , Seoghyen Lee , Jae-Won Lee , Mi Kyeong Lee , Hyun-Sun Lee , Su Ui Lee , Sei-Ryang Oh , Mun-Ock Kim PII: DOI: Reference:

S0944-7113(20)30011-8 https://doi.org/10.1016/j.phymed.2020.153178 PHYMED 153178

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Phytomedicine

Received date: Revised date: Accepted date:

5 September 2019 23 December 2019 30 January 2020

Please cite this article as: Eun-Bin Kwon , Myung-Ji Kang , Hyung Won Ryu , Seoghyen Lee , Jae-Won Lee , Mi Kyeong Lee , Hyun-Sun Lee , Su Ui Lee , Sei-Ryang Oh , Mun-Ock Kim , Acacetin enhances glucose uptake through insulin-independent GLUT4 translocation in L6 myotubes, Phytomedicine (2020), doi: https://doi.org/10.1016/j.phymed.2020.153178

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Original Research

Acacetin enhances glucose uptake through insulin-independent GLUT4 translocation in L6 myotubes

Eun-Bin Kwon a,b,1, Myung-Ji Kang a,b,1, Hyung Won Ryu a, Seoghyen Lee a, Jae-Won Lee a

a

, Mi Kyeong Lee b, Hyun-Sun Lee a, Su Ui Lee a, Sei-Ryang Oh a and Mun-Ock Kim a,*

Natural Medicine Research Center, Korea Research Institute of Bioscience and

Biotechnology (KRIBB), Cheongju, Chungbuk 28116, Republic of Korea b

College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 28644,

Republic of Korea

1

These authors contributed equally to this work.

* Corresponding author Mun-Ock Kim, Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk 28116, Republic of Korea Tel.: +82-43-240-6148; Fax: +82-43-240-6119. E-mail address: [email protected] (M.O. Kim).

Keywords: Acacetin, Agastache rugosa, AMPK, GLUT4, Type 2 diabetes 1

Abbreviations: 2-DG, 2-deoxy-D-glucose; AMPK, AMP-activated protein kinase; GLUT4, glucose transporter type 4; NAFLD, non-alcoholic fatty liver diseases; OA, Oleic acid; ROS, reactive oxygen species; TG, Triglyceride.

Word count: 4542 Number of figures: 7 Number of supplementary figures: 9

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ABSTRACT Background: Lowering blood glucose levels by increasing glucose uptake in insulin target tissues, such as skeletal muscle and adipose tissue, is one strategy to discover and develop antidiabetic drugs from natural products used as traditional medicines. Purpose: Our goal was to reveal the mechanism and activity of acacetin (5,7-dihydroxy4’-methoxyflavone), one of the major compounds in Agastache rugose, in stimulating glucose uptake in muscle cells. Methods: To determine whether acacetin promotes GLUT4-dependent glucose uptake in cultured L6 skeletal muscle cells, we performed a [14C] 2-deoxy-D-glucose (2-DG) uptake assay after treating differentiated L6-GLUT4myc cells with acacetin. Results: Acacetin dose-dependently increased 2-DG uptake by enhancing GLUT4 translocation to the plasma membrane. Our results revealed that acacetin activated the CaMKII-AMPK pathway by increasing intracellular calcium concentrations. We also found that aPKCλ/δ phosphorylation and intracellular reactive oxygen species (ROS) production were involved in acacetin-induced GLUT4 translocation. Moreover, acacetinactivated AMPK inhibited intracellular lipid accumulation and increased 2-DG uptake in HepG2 cells. Conclusion: Taken together, these results suggest that acacetin might be useful as an antidiabetic functional ingredient. Subsequent experiments using disease model animals are needed to verify our results.

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INTRODUCTION In the early stages of type 2 diabetes, impaired glucose uptake in muscles is a major contributor to insulin resistance. Because skeletal muscle is responsible for approximately 85% of postprandial glucose disposal (DeFronzo et al., 1985), it is not surprising that a change in insulin sensitivity within muscle cells has a significant impact on whole-body glucose–insulin metabolism. Diabetes can be caused by a decrease in skeletal muscle mass due to sarcopenia or cachexia (Park et al., 2009). Insulin resistance is also a major cause of diabetes. However, the molecular mechanism underlying the altered insulin response remains unclear. Insulin resistance includes insulin production by beta cells and impairment of insulin sensitivity in peripheral tissues. Therefore, increasing glucose uptake in insulin target tissues can be a therapeutic strategy to treat diabetes. Glucose uptake in skeletal muscle is primarily mediated by glucose transporter type 4 (GLUT4). There are two major types of intracellular signal transduction pathways that promote the translocation of GLUT4. One pathway involves insulin binding to the insulin receptor, which leads to activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway (Cantley, 2002). The other pathway involves the activation of AMP-activated protein kinase (AMPK) (Treebak et al., 2006). Both signaling pathways are attractive targets for developing therapeutic agents for type 2 diabetes. Acacetin (5,7-dihydroxy-4’-methoxyflavone) was identified in a screening process for novel compounds that can increase glucose uptake in cultured L6 skeletal muscle cells. Acacetin, one of the main compounds of the leaves of Agastache rugosa, was isolated and characterized in our previously published article (Lee et al., 2017). A. rugosa (known as Baechohyang or Banga in the Korean language) is a fragrant herb that is native to East 4

Asia (Korea, China, and Japan). It is used as a supplement for various foods. Acacetin is known to have anti-inflammatory (Sun et al., 2017), antioxidant (Sun et al., 2017), antiobesity (Liou et al., 2017), and anticancer (Salimi et al., 2016) effects. For the first time, we demonstrated that acacetin had an antidiabetic effect in cultured L6 myotubes and HepG2 hepatocytes in the present study. Specifically, we showed that acacetin promoted GLUT4 translocation in L6 myotubes. Additionally, we found that acacetin decreased intracellular triglyceride accumulation and increased glucose uptake in HepG2 hepatocytes.

MATERIALS AND METHODS Reagents Dimethyl sulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), N-acetyl-L-cysteine (NAC), and compound C (CC) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Minimum Essential Medium Eagle-Alpha Modification (α-MEM), Opti-MEM, fetal bovine serum (FBS), and antibiotic-antimycotic were purchased from Gibco (Grand Island, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Welgene Inc. (Daegu, Korea). Wortmannin (WTM) was purchased from Tocris (Cookson, Bristol, UK). Blasticidin-HCl, o-phenylenediamine dihydrochloride (OPD) reagent, enhanced chemiluminescence (ECL) kit, Lipofectamine 2000 and Fluo-4 AM were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). PRO-PREP protein extraction solution was purchased from Intron Biotechnology (Seoul, Korea). Bradford solution, a reagent required for protein quantification, was obtained from Bio-Rad (Richmond, CA, USA). The polyvinylidene difluoride membrane was purchased from Merck Millipore (Billerica, MA, USA). Antibodies against c-Myc 5

(Cat# sc-789), sterol regulatory element-binding protein 1 (SREBP-1C, Cat# sc-13551), fatty acid synthesis (FAS, Cat# sc-8009), diglyceride acyltransferase 2 (DGAT2, Cat# sc293211), and CD36 (Cat# sc-7309) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Cat# ab97051) was purchased from Abcam (Cambridge, MA, USA). Antibodies against AMPKα/β (Cat# 2532), p-AMPK (Thr172, Cat# 2535), acetyl-CoA carboxylase (ACC, Cat# 3662), p-ACC (Ser79, Cat# 3661), p-Akt substrate 160 (Thr642, Cat# 4288), and pCaMKII (Thr28, Cat# 12716) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Isolation of acacetin A. rugosa seeds for cultivation were collected at Sacheon, Gyeongsangnam-do, Korea in September 2014. The collected plants were identified, and a voucher specimen (KRIB 0059128) was deposited at Korea Research Institute of Bioscience and Biotechnology (KRIBB). A. rugosa Kuntze (Fisch. & C.A.Mey.) was cultivated under well-controlled glasshouse conditions at KRIBB in Cheong-ju, Korea in 2018. The aerial parts (leaves and stems) were harvested for optimal raw materials at 12–13th weeks (Sep 03–Sep 10). Acacetin was isolated from the dried leaves of A. rugosa as described previously (An et al., 2017; Lee et al., 2017). Briefly, the 80% EtOH extract (1.2 g) was subjected to prep-HPLC (Gilson, Middleton, WI, USA) using an Atlantis T3 column (Waters Corporation, Milford, MA, USA) to isolate acacetin (5.2 mg). This prep-HPLC procedure with an auto injector (GX271, Gilson) was repeated 80 times using the same conditions before further isolation. of the resulting quantitative analysis of the obtained substance indicated that acacetin was used at a purity of more than 95.0% as determined by ultra-performance liquid chromatography (Supplementary Fig. S4). Acacetin isolated from 6

leaves and stems at 12–13 weeks had the highest content, with an average of 1.27 mg/g (d.w.). UPLC-QTof-MS and NMR spectral data of sufficiently isolated compounds are shown in Figure 1A and Supplementary Figures S1–S3. Cell culture Rat L6 myoblasts and human hepatocellular carcinoma HepG2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in DMEM (high glucose) supplemented with 10% FBS and 1% penicillinstreptomycin. L6-GLUT4myc cells (stably expressing GLUT4 with a Myc epitope) were obtained from Dr. Amira Klip (The Hospital for Sick Children, Toronto, Canada). L6GLUT4myc cells were maintained in α-MEM supplemented with 10% FBS, 1% antibioticantimycotic, and 2 μg/ml blasticidin-HCl under an atmosphere of 5% CO2 at 37°C. For differentiation, cells were seeded into 24-well plates at a density of 1×104 cells/ml. The medium was changed to α-MEM containing 2% FBS once every 2 days for 7 days. Differentiated myotubes were incubated in serum-free medium for 6 h before any experiment. Measurement of glucose uptake L6-GLUT4myc myotubes were treated with various concentrations of acacetin, 10 μM rosiglitazone (Rosi), and 100 nM porcine insulin for 15 min. Thereafter, 0.2 μCi/ml 2deoxy-D-[14C]glucose was added to each well, followed by incubation at 37°C for 5 min. Cells were washed twice with Krebs-Ringer buffer (118.4 mM NaCl, 4.7 mM KCl, 1.3 mM MgSO4, 1.2 mM KH2PO4, 2 mM CaCl2, 10 mM NaHCO3, and 10 mM HEPES, pH 7.4) and lysed with 0.5 N NaOH. After mixing with scintillation cocktail, radioactivity was measured with a liquid scintillation counter (Tri-Carb 2900TR Liquid Scintillation 7

Analyzer, MA, Perkin Elmer). GLUT4 translocation assay Cell surface GLUT4myc was quantified using an antibody-coupled colorimetric assay as described previously (Mitsumoto et al., 1991). Briefly, L6-GLUT4myc cells were fixed with 3% paraformaldehyde in PBS at room temperature for 20 min and then incubated with 0.1 M glycine in PBS for 10 min. Thereafter, the cells were blocked with 5% skim milk at room temperature for 1 h and incubated in the presence of polyclonal antiMyc antibody-containing 5% skim milk for 2 h. Next, the cells were incubated with HRPconjugated goat anti-rabbit IgG for 2 h and incubated with OPD reagent at room temperature for 30 min in the dark. The absorbance at 492 nm was measured with a microplate reader. Isolation of the plasma membrane Cell lysates were prepared as described previously (Nishiumi and Ashida, 2007). Briefly, cells were collected by using a cell scraper. After centrifugation at 15,000 ×g for 3 min, the pellet was homogenized with a hand homogenizer and 25-gauge needle after adding buffer A (50 mM Tris-HCl, 0.5 mM DTT, 1% NP-40 (v/v), protease inhibitor and phosphatase inhibitor). The homogenate was centrifuged at 16,000 ×g for 20 min at 4°C. Once again, buffer A was added to the pellet, incubated on ice for 60 min with occasional mixing, and centrifuged at 13,200 ×g for 20 min at 4°C. The supernatant was collected as the plasma membrane fraction. AMPKα2 silencing Cells were grown to 60% confluence for 24 h. siRNA for AMPKα2 (Ambion, 8

Austin, TX, USA) and nontarget control siRNA (Dharmacon, Lafayette, CO, USA) were purchased. Transient transfections were performed using Lipofectamine 2000 according to the manufacturer’s protocol. Briefly, 5 μl of siRNA and 5 μl of Lipofectamine 2000 were mixed with 95 μl of Opti-MEM. The mixture was incubated at room temperature for 20 min. After the reaction, the mixture was added dropwise to culture well plates containing Opti-MEM. After 4 h, the medium was changed to fresh DMEM. Cells were incubated for another 48 h and used for glucose uptake and immunoblot analysis. Western blot analysis Cells were lysed with PRO-PREP protein extraction solution on ice for 30 min. After centrifugation at 13,200 ×g for 30 min at 4°C, the supernatant was collected, and the protein concentration was quantified by the Bradford method. Equal amounts of protein samples were separated by SDS-PAGE (7.5% to 10% gel) and transferred to PVDF membranes. The membranes were blocked with blocking solution at room temperature for 1 h, incubated with primary antibodies at 4°C overnight, and washed three times with Trisbuffered saline containing 0.1% Tween 20. The secondary antibodies were then incubated with the membranes for 2 h at room temperature. These blots were developed using an ECL substrate kit. Measurement of free cytoplasmic Ca2+ concentration Intracellular Ca2+ concentration was determined using the Fluo-4 AM calcium indicator. L6 myoblasts were treated with various concentrations of acacetin for 15 min. After the reaction, the cells were incubated with 1 μM Fluo-4 AM for 30 min. Following washes with Dulbecco's phosphate-buffered saline, the cells were analyzed by using a FACSCalibur flow cytometer (Becton Dickinson, CA, USA). 9

Measurement of ROS ROS (O2- and H2O2) in L6 myoblasts were measured using hydroethidine (HE) and 2’7’-dichlorofluorescein diacetate (H2DCF-DA). Cells were pretreated with NAC for 30 min and then incubated in the absence or presence of acacetin for 15 min. Following washes with PBS, the cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson, CA, USA). Determination of de novo synthesized triglyceride (TG) HepG2 cells were incubated with various concentrations of acacetin in the presence of [14C]-glycerol (0.6 μCi) for 6 h. At the end of the incubation, intracellular lipids were extracted with a mixture of hexane:isopropanol (3:2, v/v) and separated on a thin layer chromatography plate using a hexane:diethyl ether:acetic acid (80:20:1, v/v/v) solution. Isotope-labeled TGs were detected and quantified with a bioimaging analyzer (FLA-7000, Fujifilm, Japan). Determination of TG content HepG2 cells were pretreated with various concentrations of acacetin for 1 h before exposure to 250 µM oleic acid (OA) for 24 h. After incubation, intracellular TG levels were measured using a triglyceride colorimetric assay kit (Cayman Chemical, MI, USA) according to the manufacturer’s instructions. Statistical Analysis Data are presented as the mean ± standard deviation (SD). All statistical analyses were performed using Student’s t-test. Differences were considered significant at p < 0.05.

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RESULTS Acacetin stimulates glucose uptake by enhancing the translocation of GLUT4 in differentiated L6-GLUT4myc cells The effect of acacetin on glucose uptake and GLUT4 translocation in cultured L6GLUT4myc myotubes was determined. 2-DG is a glucose analog that is not metabolized intracellularly after phosphorylation by hexokinase. This property allows quantitative tracing of absorbed glucose in cells. Acacetin induced an increase in glucose uptake in L6 myotubes in a dose-dependent manner (Fig. 1B). Insulin (INS, 100 nM) and rosiglitazone (Rosi, 10 μM) were used as positive controls. GLUT4-independent glucose uptake was excluded by cytochalasin B treatment in this experiment. Insulin and rosiglitazone increased intracellular glucose uptake by 1.5- and 1.4-fold, respectively, compared to that of the control group. Treatment with 40 μM acacetin increased glucose inflow by 1.3-fold compared to that of the control. We then examined the expression of GLUT4 on the cell surface by western blot analysis. In addition, we used antibodies to detect the myc epitope of GLUT4myc on the surface of living cells. As shown in Figure 1C, acacetin at 20 and 40 μM increased the level of Myc on the plasma membrane by 1.1-fold and 1.2-fold, respectively. In addition, plasma GLUT4 protein expression was dose-dependently increased by acacetin treatment compared to that in control cells. These results demonstrate that acacetin stimulates glucose uptake by activating the trafficking of intracellular GLUT4 vesicles to the cell surface.

Acacetin increases AMPK phosphorylation Based on previous findings that acacetin activates AMPK in 3T3-L1 cells (Liou et 11

al., 2017), we determined whether acacetin also increased the activation of AMPK in L6 myotubes. Western blot analysis revealed that acacetin dose-dependently increased AMPK phosphorylation in differentiated L6 cells. Additionally, phosphorylation of ACC, a downstream target of AMPK, was increased by acacetin (Fig. 2A). Pretreatment with compound C, an AMPK inhibitor, before acacetin treatment significantly blocked the phosphorylation of AMPK and glucose uptake (Figs. 2B and 2C). To provide direct evidence of the role of AMPK in acacetin-mediated glucose uptake and to validate the low selectivity of compound C, we used siRNA to knock down AMPKα2. Acacetin-mediated AMPK phosphorylation was abruptly reduced by AMPKα2-targeted siRNA (Fig. 2D). Increased glucose uptake by acacetin treatment was also decreased by knockdown of AMPKα2 (Fig. 2E). Overall, acacetin-induced increases in glucose uptake in L6 myotubes seemed to be dominated by phosphorylation of AMPK.

Atypical PKC is partially involved in acacetin-mediated glucose uptake In addition to activation of the PI3K/Akt axis, aPKCλ/δ serves as a major distal regulator of glucose transport in response to insulin (Kotani et al., 1998). To determine whether acacetin activates aPKCλ/δ in L6 myotubes, we performed western blot analysis using antibodies against p-aPKCλ/δ. Consistent with previous studies, phosphorylation of aPKCλ/δ was slightly increased by insulin treatment. Surprisingly, treatment with acacetin significantly increased the phosphorylation of aPKCλ/δ (Fig. 3A). Pretreatment with sotrastaurin, a pan-PKC inhibitor, significantly reduced the acacetin-mediated increase in glucose uptake (Fig. 3B). Interestingly, acacetin did not seem to activate the PI3K/Akt pathway. Pretreatment with wortmannin, a PI3K inhibitor, almost completely inhibited 12

insulin-induced glucose uptake but did not significantly reduce that of acacetin (Fig. 3C). Akt activation involves the phosphorylation of two residues, namely, Thr308 by 3phosphoinositide-dependent kinase-1 (PDK1) and Ser473 by the rictor-mammalian target of rapamycin (mTOR) complex. Phosphorylation of Akt at regulatory residues Ser473 and Thr308 leads to its full activation. Thr308 is a more reliable biomarker than Ser473 for Akt activity (Vadlakonda et al., 2013). Western blot results showed that treatment with acacetin slightly increased the phosphorylation of the Ser473 residue of Akt, but the phosphorylation of the Thr308 residue was hardly detected. Collectively, these results suggest that the phosphorylation of aPKCλ/δ but not the PI3K/Akt pathway is considerably involved in acacetin-induced glucose uptake.

Acacetin dramatically increases the free cytoplasmic calcium concentration Since calcium/calmodulin-dependent protein kinase II (CaMKII) is a well-known upstream kinase that is responsible for the phosphorylation of the α subunit of AMPK (Carling et al., 2008), we examined whether calcium acts upstream of acacetin-activated AMPK. Acacetin treatment of L6 myoblasts for 15 minutes resulted in a concentrationdependent increase in intracellular calcium concentration (Fig. 4A). We investigated the origin of calcium released into the cytoplasm by pretreatment of cells with EGTA (an extracellular calcium cheater) or BAPTA-AM, which is known to be absorbed and chelated in the cell. Pretreatment with BAPTA-AM but not EGTA significantly reduced the acacetin-mediated intracellular calcium increase (Fig. 4B). We further examined the effect of acacetin on the phosphorylation of CaMKII. As shown in Figure 4C, acacetin treatment significantly increased CaMKII phosphorylation compared to that of the control. Removal 13

of intracellular calcium by BAPTA-AM reduced the acacetin-induced phosphorylation of CaMKII and AMPK. On the other hand, phosphorylation of aPKCλ/δ was not changed by pretreatment with BAPTA-AM (Fig. 4D). Pretreatment with STO-609, a specific inhibitor of CaMKKII, reduced the acacetin-induced phosphorylation of AMPK and ACC (Fig. 4E). These experimental results indicate that acacetin activates the intracellular calciumCaMKII-AMPK axis. Pretreatment with STO-609 or BAPTA-AM reduced acacetinmediated glucose uptake by 25% or 39%, respectively (Figs. 4F and 4G). Collectively, these data indicate that acacetin induces glucose uptake through the cytoplasmic calciumCaMKII-AMPK axis.

Acacetin increases intracellular ROS production Previous reports revealed that exogenous H2O2 stimulates glucose uptake in adipocytes and skeletal muscle cells (Chambers et al., 2009). We determined whether acacetin increased intracellular ROS by using H2DCFDA and HE fluorescent ROS indicators. As shown in Fig. 5A, acacetin increased the levels of H2O2 and O2- by 1.6-fold and 1.7-fold, respectively, compared with those of the control. It should be noted that H2DCFDA and HE are less sensitive and specific because they will mark all peroxides and superoxides. Treatment with N-acetylcysteine (NAC), a nonspecific antioxidant, reduced acacetin-induced H2O2 and O2- by 18% and 13%, respectively. We further investigated whether the increased ROS due to acacetin affected the glucose uptake capacity. Treatment with 40 μM acacetin increased glucose uptake by approximately 1.5-fold compared to that of the control group, while NAC pretreatment significantly decreased glucose uptake by approximately 30% (Fig. 5B). These data indicate that acacetin-stimulated intracellular 14

ROS affect the increase in glucose uptake in L6 cells.

Acacetin alleviates excessive oleic acid-induced intracellular lipid accumulation through activation of AMPK in HepG2 cells We found that acacetin increased the phosphorylation of AMPK in cultured L6 skeletal muscle cells in this study. AMPK is a therapeutic target for metabolic diseases such as diabetes, non-alcoholic fatty liver disease (NAFLD) and obesity. Therefore, we examined whether acacetin inhibited lipid accumulation and increased glucose uptake through the activation of AMPK in HepG2 hepatocytes. As shown in Fig. 6A, acacetin increased the phosphorylation of AMPK in HepG2 cells in a dose-dependent manner. Since phosphorylation of AMPK is known to reduce the expression of TG biosynthesisrelated proteins, the effect of acacetin on intracellular TG synthesis was confirmed by measuring the degree of conversion of [ 14C]glycerol to [14C]TG. The results showed that acacetin at concentrations of 10, 20, and 40 μM significantly decreased the newly synthesized TGs by 30%, 64% and 88%, respectively, compared with those of the control. Acacetin at 40 μM suppressed TG synthesis more than a DGAT2 inhibitor (5 h) as a positive control (Fig. 6B). In vivo, excessive fatty acids cause hepatocytes to increase the biosynthesis and accumulation of TGs, leading to NAFLD (Juarez-Hernandez et al., 2016). When cells were treated with 250 μM OA for 24 hours, TGs in HepG2 cells increased approximately 4 times. Pretreatment with acacetin at various concentrations for 1 hour before OA treatment significantly decreased intracellular TG accumulation in a concentration-dependent manner (Fig. 6C). In particular, pretreatment with 40 μM acacetin decreased intracellular TGs by 73% compared to that of the group treated with 15

OA alone. Lipogenic protein expression was examined under the same conditions (Fig. 6D). OA increased the expression level of SREBP-1c, a key transcription factor related to hepatic lipogenesis, and its target genes, such as FAS and DGAT2. Activation of AMPK is known to inhibit the synthesis of hepatic fatty acids by inhibiting SREBP-1c (Li et al., 2011). Therefore, acacetin attenuated the expression of lipogenic genes increased by OA treatment to control levels. Next, we examined whether acacetin increased hepatic glucose uptake. Acacetin at 40 μM increased glucose uptake by 1.5 times in HepG2 cells, similar to that in L6 myotubes (Figs. 6E and 1B). Insulin (INS, 100 nM) and rosiglitazone (Rosi, 10 μM) were used as positive controls. Pretreatment with compound C or STO-609 almost completely inhibited the increase in glucose uptake in acacetin-treated cells, suggesting that acacetin increased glucose uptake in HepG2 cells through the CaMKIIAMPK pathway (Fig. 6F). Overall, these results demonstrate that acacetin inhibits intracellular lipid accumulation and increases glucose uptake by activating AMPK in HepG2 cells.

DISCUSSION Peripheral insulin resistance is a prominent feature of Type 2 diabetes mellitus. Insulin resistance is a defect in the process of insulin-mediated signal transduction for the translocation of cytoplasmic GLUT4 proteins to the cell surface (Koistinen et al., 2003). Therefore, compounds that can enhance the translocation of GLUT4 are recognized as candidates for treating diabetes. In the present study, we found that acacetin isolated from A. rugose promotes glucose uptake in cultured L6 muscle cells and HepG2 hepatocytes. In addition, we demonstrated that the increase in glucose uptake was accompanied by GLUT4 16

translocation to the plasma membrane through the activation of AMPK and aPKCλ/δ and an increase in intracellular ROS production. This is the first study to report that acacetin promotes glucose uptake via GLUT4 translocation in insulin-sensitive peripheral tissues, even though this has been demonstrated at the cellular level. To determine the mechanism by which acacetin induces GLUT4 translocation, we investigated the effects of acacetin on cellular signaling pathways that are well known to modulate this process. Pretreatment with the selective inhibitors wortmannin and compound C confirmed that the major signal transduction factor associated with glucose uptake by acacetin is AMPK but not PI3K (Figs. 2C and 3C). Our studies showed that acacetin promoted the activation of AMPK through an increase in free cytoplasmic calcium-mediated CaMKII phosphorylation (Fig. 4). Acacetin increased the phosphorylation of the Ser473 residue in Akt, which is generally known to be phosphorylated by PI3K (Fig. 3D). Interestingly, acacetin did not induce PI3K activation in our experiments. Several reports have indicated that Akt can be activated by a PI3K-independent mechanism in response to increases in intracellular Ca2+, cAMP or heat shock (Moule et al., 1997; Sable et al., 1997). Therefore, we are considering the possibility that phosphorylation of the Ser473 residue of Akt is caused by an increase in acacetin-induced free cytoplasmic Ca2+ concentration and subsequent activation of CaMKII. This needs to be verified through further studies. It is believed that aPKCλ/δ and ROS act in response to insulin as other distal regulators for cellular glucose transport. Accumulated evidence has shown that dietary flavonoids, including rutin and EGCG, are involved in atypical PKC phosphorylation to promote glucose uptake through GLUT4 translocation (Asano et al., 2012; Kappel et al., 17

2013). Hence, we determined whether acacetin stimulated the phosphorylation of aPKCλ/δ (Thr410/403) and intracellular ROS generation (Figs. 3 and 5). PKC, also known as PDK1 substrate, is highly susceptible to PI3K activation. Since our data did not show that acacetin promoted activation of PI3K, further research is needed to determine whether other upstream effector molecules are present or whether acacetin directly activates PKC. Based on a previous study in which aPKCλ/δ was regulated by AMPK in cardiomyocytes (Habets et al., 2012), further studies are needed to determine whether acacetin-induced aPKCλ/δ phosphorylation is promoted by AMPK phosphorylation. AMPK causes GLUT4 deployment to the plasma membrane, resulting in insulinindependent glucose uptake in insulin sensitive tissues. Thus, all of the known AMPK activators are expected to result in increased glucose uptake. Metformin and berberine have already been found to activate AMPK through inhibition of the mitochondrial electron transport system. In this study, we focused whether acacetin, which is abundantly contained in A. rugosa, has a role in diabetic improvement through the activation of AMPK in muscle cells and hepatocytes rather comparing acacetin to other compounds as a glucose uptake inducer. Acacetin increased reactive oxygen species (ROS) formation and mitochondrial membrane potential (MMP) collapse in a previous study (Salimi et al., 2016). Our supplemental study also showed that acacetin treatment significantly reduced MMP (Supplementary Figure S5). Acacetin is a natural product with a flavone backbone, and various off-targets should be considered rather than specifically targeting mitochondria. The precise mechanism by which acacetin activates AMPK should be clarified through further study. In metabolic diseases, oxidative stress acts to both induce and improve disease. Pesta and colleagues described oxidative stress as a Janus head (Pesta and Roden, 2017). 18

Chronic reactive oxygen species or fructose-induced ROS generation impairs glucose utilization in muscle cells (Ding et al., 2016). However, the mechanisms by which piperine, capsaicin, egg white ovotransferrin-derived ACE inhibitory peptide, and chromium(III) ions increase glucose uptake in muscle cells are ROS production-dependent (Son et al., 2018). Our results also showed that acacetin improves muscle glucose uptake in relation to intracellular ROS production and that treatment with NAC, an antioxidant, reversed the effect of acacetin (Fig. 5). It is difficult to determine the precise location and origin, degree of increase and decrease of ROS in different pathological and physiological conditions, and the effect of specific ROS on the activation of specific signaling pathways is unclear. GLUT4 translocation to the plasma membrane is dependent on the phosphorylation of Rab GTPase-activating protein AS160. Akt, AMPK, and CaMKII are involved in the phosphorylation of AS160 (Mohankumar et al., 2012). We confirmed that acacetin phosphorylated the Thr642 residue of AS160 (Supplementary Figure S9). Although there is no direct evidence that AS160 or other signal transduction proteins are involved in the effects of acacetin in this study, we suggest that the increased intracellular calcium and increased CaMKII and AMPK activation by acacetin might be associated with AS160. Elevated lipid accumulation in peripheral tissues of obese individuals is considered a critical pathogenic factor of metabolic diseases, including NAFLD and diabetes (Zhang et al., 2014). Since AMPK activation is known to inhibit SREBP1c (Li et al., 2011), the master transcription factor of lipogenic genes, inhibition of intracellular lipid accumulation by acacetin is one of its expected effects. Acacetin inhibited intracellular neonatal TG biosynthesis and significantly decreased intracellular lipid accumulation due to excessive OA treatment. We examined the effect of acacetin on glucose uptake in HepG2 cells. Acacetin increased the glucose uptake of HepG2 cells similar to that of L6 myotubes (Fig. 19

6E). Since GLUT4 is rarely expressed in glucose uptake in the liver (Karim et al., 2012), it is worth investigating what regulatory proteins are involved in increasing glucose utilization through acacetin treatment. In conclusion, acacetin enhances glucose uptake in cultured muscle cells via GLUT4 translocation. The results of the present study showed that acacetin stimulated the cytoplasmic calcium-CaMKII-AMPK pathway, activated aPKCλ/δ, and resulted in the generation of intracellular ROS, suggesting that an insulin-independent pathway might be the major mechanism by which acacetin exerts its antidiabetic potential (Fig. 7). Acacetin also inhibited OA-induced lipid accumulation and promoted glucose uptake, both of which were mediated by AMPK activation in HepG2 cells. Thus, acacetin might be a new compound for improving NAFLD and diabetes.

Acknowledgements: This research was supported by the KRIBB Research Initiative Program funded by the Ministry of Science, ICT and Future Planning (MSIT), Republic of Korea.

Conflict of Interest Statement: The authors declare no competing financial interest.

SUPPORTING INFORMATION DESCRIPTION Spectroscopic data for acacetin: Structure (Fig. S1), MS (Fig. S2), HRESIMS (Fig. S3), and NMR spectra (Fig. S4). 20

Biological data for acacetin: MMP (Fig. S5), synergistic effect with insulin (Fig. S6), combination effect on glucose uptake (Fig. S7), effect on PPAR-γ (Fig. S8) and AS160 phosphorylation (Fig. S9).

21

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Figure captions Figure 1. Acacetin on glucose uptake and GLUT4 translocation to the plasma membrane in L6-GLUT4myc cells. (A) Chemical structure of acacetin. (B) Glucose uptake was measured by a [14C]-2-DG uptake assay. Differentiated L6-GLUT4myc cells were treated with the indicated concentrations of acacetin, insulin (INS, 100 nM), and rosiglitazone (Rosi, 10 μM) for 15 min. INS and Rosi were used as positive controls. The vehicle control group was treated with 0.1% DMSO. (C) GLUT4 in the plasma membrane fraction was detected by western blotting (upper) and antibody-coupled colorimetric absorbance assay (bottom). The data are shown as the mean ± SD (n = 3). *p < 0.05, **p < 0.01 compared to the control.

Figure 2. Acacetin on the AMPK pathway in L6 myotubes. (A) Cell lysates were analyzed by western blotting. Differentiated L6 cells were treated with acacetin (20 and 40 μM) and metformin (Met, 100 μM) for 15 min. The vehicle control group was treated with 0.1% DMSO. L6 myotubes were pretreated with 10 μM compound C (CC, a selective AMPK inhibitor) for 30 min and then incubated with 40 μM acacetin for 15 min before western blot (B) and [14C]-2-DG uptake assays (C). L6 myoblasts were transfected with siRNA against the gene encoding AMPKα2 and then stimulated with acacetin for 15 min before western blot (D) and [14C]-2-DG uptake assays (E). The data are shown as the mean ± SD (n = 3). **p < 0.01 compared to the control; # p < 0.05 compared to acacetin alone.

Figure 3. Acacetin on the phosphorylation of aPKCλ/ζ. (A) Cell lysates were subjected to western blotting. Differentiated L6 cells were treated with acacetin (20 and 40 μM) or 25

INS (100 nM) for 15 min. The vehicle control group was treated with 0.1% DMSO. The density of the protein band of p-aPKCλ/δ in the western blot was quantified using ImageJ analysis software. Relative quantification values are shown as a bar graph. (B) [ 14C]-2-DG uptake assay. L6 myotubes were stimulated with 40 μM acacetin in the presence or absence of 0.64 nM sotrastaurin, a pan-PKC inhibitor. (C) [14C]-2D-G uptake assay. The cells were treated with 40 μM acacetin in the presence or absence of 1 μM wortmannin (WTM). (D) Cell lysates were detected by western blotting using an antibody against pAkt (Ser473 or Thr308). The data are shown as the mean ± SD (n = 2).

**

p < 0.01

compared to the control; #p < 0.05, ##p < 0.01 compared to the acacetin alone group.

Figure 4. Acacetin-induced free cytoplasmic calcium on the CaMKII-AMPK axis. (A) Free cytoplasmic calcium concentration was measured by flow cytometry using Fluo-4 AM. L6 myoblasts were treated with the indicated concentrations of acacetin for 15 min. The vehicle control group was treated with 0.1% DMSO. (B) The cells were pretreated with a chelator (0.5 mM EGTA or 10 μM BAPTA-AM) and then incubated with 40 μM acacetin before Fluo-4 AM staining. (C) Cell lysates were detected by western blotting. Differentiated L6 cells were treated with acacetin (20 and 40 μM) and INS (100 nM) for 15 min. (D and E) Phospho-forms of CaMKII, p-AMPK and p-PKC were evaluated by immunoblotting. The cells were stimulated with 40 μM acacetin in the presence or absence of 10 μM BAPTA-AM or in the presence or absence of 20 μM STO-609, a CaMKII inhibitor. (F and G) A [14C]-2-DG uptake assay was performed to evaluate glucose uptake. The cells were pretreated with EGTA or BAPTA-AM and then incubated with 40 μM acacetin. The data are shown as the mean ± SD (n = 3). **p < 0.01 compared to the control; #

p < 0.05 compared to the acacetin alone group. 26

Figure 5. Acacetin on ROS production in L6 myoblasts. (A) Cellular ROS levels were measured by FACS using DCF-DA and HE probes. The cells were treated with 40 μM acacetin in the presence or absence of 0.5 mM NAC. The vehicle control group was treated with 0.1% DMSO. (B) Glucose uptake was measured by a [14C]-2-DG uptake assay. The data are shown as the mean ± SD (n = 3). *p < 0.05 and #

**

p < 0.01 compared to the control.

p < 0.05 compared to the acacetin alone group.

Figure 6. Acacetin on OA-induced lipid accumulation and glucose uptake in HepG2 cells. (A) Cell lysates were analyzed by western blotting. Cells were treated with the indicated concentrations of acacetin for 2 h. AICAR (1 mM) was used as a positive control. The vehicle control group was treated with 0.1% DMSO. (B) [14C]-TG de novo synthesis was measured by TLC. Each [14C]-TG band was quantified by using Multi-Gauge V3.0 software. Treatment with 1 μM for 5 h was used as a positive control (Lee et al., 2013). (C) Accumulated intracellular TG was determined by using a colorimetric assay kit. Cells were pretreated with the indicated concentrations of acacetin for 1 h and then exposed to 250 μM OA for another 24 h. (D) Expression levels of lipogenesis-related proteins were evaluated by immunoblotting. Cells were treated with the indicated concentrations of acacetin for 1 h before treatment with 250 μM OA. (E) A [14C]-2-DG uptake assay was performed on cells treated with various concentrations of acacetin, 10 μM Rosi, or 100 nM INS for 15 min. (F) A [14C]-2-DG uptake assay was performed on cells treated with 40 μM acacetin for 15 min in the presence or absence of 10 μM compound C (CC) or 20 μM STO-609. All western blot bands were analyzed by using ImageJ software. The data are 27

shown as the mean ± SD (n = 3). *p < 0.05 and

**

p < 0.01 compared to the control; #p <

0.05 and ##p < 0.01 compared to OA alone.

Figure 7. Summary of signaling pathways that acacetin promotes GLUT4 translocation to the plasma membrane. Acacetin increases glucose uptake into cultured L6 muscle cells by increasing free cytoplasmic calcium-mediated phosphorylation of CaMKII-AMPK, phosphorylation of aPKCλ/δ, and production of intracellular ROS, resulting in increased translocation of GLUT4 to the cell surface.

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Figure 5.

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Graphic abstract

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