YTAAP-13320; No of Pages 12 Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap

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Min Suk Seo, Jung Hwan Kim, Hye Jung Kim, Ki Churl Chang, Sang Won Park ⁎

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Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju 660-751, Republic of Korea

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Article history: Received 27 August 2014 Revised 14 February 2015 Accepted 21 February 2015 Available online xxxx

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Keywords: Adenosine monophosphate-activated protein kinase Hepatocytes Honokiol Lipogenesis Liver kinase B1 Non-alcoholic steatosis

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Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes

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Honokiol is a bioactive neolignan compound isolated from the species of Magnolia. This study was designed to elucidate the cellular mechanism by which honokiol alleviates the development of non-alcoholic steatosis. HepG2 cells were treated with honokiol for 1 h, and then exposed to 1 mM free fatty acid (FFA) for 24 h to simulate non-alcoholic steatosis in vitro. C57BL/6 mice were fed with a high-fat diet for 28 days, and honokiol (10 mg/kg/day) was daily treated. Honokiol concentration-dependently attenuated intracellular fat overloading and triglyceride (TG) accumulation in FFA-exposed HepG2 cells. These effects were blocked by pretreatment with an AMP-activated protein kinase (AMPK) inhibitor. Honokiol significantly inhibited sterol regulatory element-binding protein-1c (SREBP-1c) maturation and the induction of lipogenic proteins, stearoyl-CoA desaturase-1 (SCD-1) and fatty acid synthase (FAS) in FFA-exposed HepG2 cells, but these effects were blocked by pretreatment of an AMPK inhibitor. Honokiol induced AMPK phosphorylation and subsequent acetyl-CoA carboxylase (ACC) phosphorylation, which were inhibited by genetic deletion of liver kinase B1 (LKB1). Honokiol stimulated LKB1 phosphorylation, and genetic deletion of LKB1 blocked the effect of honokiol on SREBP-1c maturation and the induction of SCD-1 and FAS proteins in FFA-exposed HepG2 cells. Honokiol attenuated the increases in hepatic TG and lipogenic protein levels and fat accumulation in the mice fed with high-fat diet, while significantly induced LKB1 and AMPK phosphorylation. Taken together, our findings suggest that honokiol has an anti-lipogenic effect in hepatocytes, and this effect may be mediated by the LKB1–AMPK signaling pathway, which induces ACC phosphorylation and inhibits SREBP-1c maturation in hepatocytes. © 2015 Published by Elsevier Inc.

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Introduction

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Non-alcoholic fatty liver disease (NAFLD), a condition caused by the pathological accumulation of fat in the liver, is recognized as a highly prevalent major health issue. NAFLD affects 10–35% of the current world population (Neuschwander-Tetri and Caldwell, 2003). The severity of the disease ranges from simple steatosis to acute steatohepatitis. The pathogenesis and the molecular mechanisms controlling its progression are poorly understood.

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Abbreviations: ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; ATGL, adipose triglyceride lipase; CaMKK, calcium/calmodulin-dependent protein kinase kinase; FAS, fatty acid synthase; FFA, free fatty acid; HFD, high fat diet; Hon, honokiol; LKB1, liver kinase B1; ND, normal diet; SCD-1, stearoyl-CoA desaturase-1; SIRT1, NADdependent deacetylase sirtuin-1; SREBP-1c, sterol regulatory element-binding protein-1c; TGs, triglycerides. ⁎ Corresponding author at: Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, 816-15 Jinjudaero, Jinju 660-751, Republic of Korea. Fax: +82 55 772 8079. E-mail address: [email protected] (S.W. Park).

The main feature of NAFLD pathogenesis is the accumulation of triglycerides (TGs) in the liver. A study using a multiple-stable-isotope method demonstrated that approximately 60% of liver TG content is derived from free fatty acid (FFA) influx from adipose tissue, 26% from de novo lipogenesis, and 15% from the diet (Donnelly et al., 2005). In contrast, in healthy individuals, only b5% of hepatic TGs contribute to de novo lipogenesis (Hudgins et al., 2000). FFA influx and de novo lipogenesis are key pathogenic processes in the development of NAFLD, and modulation of any of the multiple mechanisms involved in lipid accumulation in the liver may be a useful therapeutic strategy for preventing the development of NAFLD. AMP-activated protein kinase (AMPK), a serine–threonine kinase comprised of a heterotrimeric complex, serves as a key cellular energy sensor in most tissues. Its activity is controlled by phosphorylation at Thr172 (Lee et al., 2014). Recent studies have reported that AMPK can be phosphorylated at Thr172 by both liver kinase B1 (LKB1) and calcium/calmodulin-dependent protein kinase kinase (CaMKK) in mammalian cells (Hou et al., 2008; Purushotham et al., 2009). AMPK activation inactivates the rate-limiting enzymes associated with lipolysis, such as acetyl-CoA carboxylase (ACC) by phosphorylation in the liver (Chakrabarti et al., 2011). Under fasting conditions, AMPK reduces

http://dx.doi.org/10.1016/j.taap.2015.02.020 0041-008X/© 2015 Published by Elsevier Inc.

Please cite this article as: Seo, M.S., et al., Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.020

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Animals and honokiol treatments. All animal studies were approved by the Institutional Animal Care Committee of Gyeongsang National University. Male C57BL/6 mice (20–22 g) were purchased from KOATECH (Pyungtaek, Korea). The mice were maintained on a 12:12-h light–dark cycle at 25 °C and given free access to water and normal diet (ND) for 1 week before dividing into four groups (n = 6–7 for each groups). The mice fed ND (protein 20%, carbohydrate 70%, fat 10%; D12450B, Research Diet, New Brunswick, NJ, USA) or HFD (protein 20%, carbohydrate 20%, fat 60%; D12492, Research Diet) were treated with or without honokiol. The mice were daily treated with honokiol (10 mg/kg/day) by gavage dissolved in 10% polyethylene glycol 400 (v/v). Honokiol solution was prepared freshly before treatment.

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Histological analysis of liver tissue. Liver tissues were fixed in 10% formalin and then embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosin by standard methods. Briefly, formalin-fixed paraffin embedded sections (5 μm) were incubated in hematoxylin solution for 15 min, and then were washed in 1% acidic alcohol buffer. The sections were counterstained with eosin.

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Cell viability. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. Briefly, the cells were treated with honokiol (10–500 μM) or FFA mixture (0.25–2 mM). Twenty-four hours after treatment with the honokiol or FFA mixture, 1 mg/ml MTT solution was added to each well and incubated at 37 °C in 5% CO2 for 3 h. The medium was removed, and DMSO was added to dissolve the MTT–formazan complex. The absorbance was measured at 570 nm using a microplate reader (Infinite F200, Tecan Group Ltd., Männedorf, Switzerland).

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Materials. Cell culture media and supplements were obtained from Invitrogen (Grand Island, NY). Compound C and STO609 were purchased from Calbiochem (Darmstadt, Germany). Unless otherwise specified, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

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RNA interference for LKB1. Small interfering RNA (siRNA) for LKB1 was purchased from Bioneer Corporation (Daejeon, Korea). Transfection of LKB1-specific and scrambled siRNA into HepG2 cells was performed with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, 150 pmol siRNA was diluted in 500 μl serum-free Opti-MEM medium (Invitrogen) and incubated with 5 μl Lipofectamine RNAiMAX at room temperature for 20 min. The mixture of siRNA and Lipofectamine was added to a 6-well culture plate with 2 ml medium containing the appropriate number of cells to give 30% confluence. The cells were further incubated for 72 h.

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oleate/palmitate was diluted in culture medium to reach the desired final concentrations (Gomez-Lechon et al., 2007). The control cells were treated with the corresponding concentration of bovine serum albumin.

Cell culture and FFA treatment. Human hepatoma HepG2 cells were purchased from the American Type Culture Collection (Manassas, VA) and maintained in low-glucose Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated FBS, penicillin G (100 U/ml), streptomycin (100 mg/ml), and L-glutamine (2 mM) at 37 °C in 5% CO 2 . After reaching 75% confluence, the cells were serum-starved for 16 h, and then exposed to an FFA mixture to induce fat overloading of the cells. Stock solutions of 50 mM oleate and 50 mM palmitate were prepared in culture medium containing 1% fatty acid-free bovine serum albumin. An FFA mixture at a 2:1 ratio of

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Oil Red O staining. Oil Red O was used to stain intracellular lipids. A portion of each liver was embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek USA, Torrance, CA) and was frozen on dry ice. Five-micrometer frozen sections were stained with 0.5% Oil Red O in 60% isopropanol for 15 min and were counterstained with hematoxylin. Twenty-four hours after treatment with the FFA mixture, the cells were washed once and fixed with 10% PBS-buffered formalin for 1 h at room temperature. After the removal of the formalin, cells were rinsed with 60% isopropanol and incubated with 2.1 mg/ml Oil Red O in 60% isopropanol for 10 min. The images were then obtained under an inverted microscope CKX41SF (Olympus, Tokyo, Japan) connected to a Nikon DS-Fi1c CCD camera (Nikon, Tokyo, Japan). Oil Red O was eluted by adding 100% isopropanol, and then the quantity of Oil Red O was measured at 545 nm using a microplate reader.

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Intracellular TG level. Twenty-four hours after treatment with the FFA mixture, the cells were collected with centrifugation at 1000 ×g for 10 min at 4 °C. The intracellular TG levels were measured using a commercially available TG Colorimetric Assay kit (Cayman, Ann Arbor, MI) according to the manufacturer's protocol.

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Western blot analysis. Whole-cell lysates or liver tissues were prepared with RIPA buffer (Pierce Biotechnology, Rockford, IL) containing Halt™ Protease and Phosphatase Inhibitor Cocktail (Pierce Biotechnology). The nuclear fraction was isolated using NE-PER® Nuclear and Cytoplasmic Extraction Reagents according to the manufacturer's instructions (Pierce Biotechnology). The total protein concentration was determined using the Pierce BCA protein assay kit (Pierce Biotechnology). Protein samples were separated with 5– 15% SDS-PAGE, and then transferred to PVDF membranes (Roche, Germany). The membranes were blocked for 1 h at room temperature with 5% (w/v) dry milk, and then incubated overnight at 4 °C with the following primary antibodies: adipose triglyceride lipase (ATGL), AMPK, phospho-AMPK, ACC, phospho-ACC, fatty acid synthase (FAS), LKB1, phospho-LKB1, NAD-dependent deacetylase sirtuin-1 (SIRT1), stearoyl-CoA desaturase 1 (SCD-1) (Cell Signaling Technology, Beverly, MA), SREBP-1c, proliferating cell nuclear antigen (Santa Cruz Biotechnology, Dallas, TX), and β-actin (Sigma-Aldrich).

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lipogenesis by suppressing sterol regulatory element binding protein-1c (SREBP-1c) activity in the liver. AMPK activators, including metformin and thiazolidinediones, have been shown to inhibit the expression of the SREPB-1c gene and to prevent the development of hepatic steatosis (Zhou et al., 2001). Magnolia officinalis has been used in Chinese herbal medicine to treat liver disease and other disorders such as gastrointestinal disorders, anxiety, and allergic disease (Lee et al., 2011). Honokiol, a bioactive component isolated from the stem bark of M. officinalis, shows protective effects against liver injury caused by hepatotoxicants and hypoxia/reoxygenation (Park et al., 2003; Lin et al., 2012). Recent evidence indicates that honokiol has a potential to ameliorate alcoholic steatosis by blocking fatty acid synthesis regulated by SREBP-1c (Yin et al., 2009). Honokiol combined with magnolol, another active component of M. officinalis, inhibited hepatic steatosis through the inhibition of SREBP-1c (Lee et al., 2014). However, the effects of honokiol on the non-alcoholic fatty liver have not been described. This study was designed to investigate the effect of honokiol against the development of non-alcoholic steatosis using in vitro and in vivo models and to elucidate its possible mechanism. We demonstrate that honokiol activates the LKB1–AMPK signaling pathway in FFA-exposed HepG2 cells and high-fat diet (HFD)-fed mice and attenuates the SREBP-1c maturation and induction of lipogenic enzymes.

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Please cite this article as: Seo, M.S., et al., Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.020

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Results

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Cytotoxicity of honokiol or the FFA mixture on HepG2 cells

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We first determined the concentration dependence of the cytotoxic effects of honokiol or the FFA mixture for 24 h in HepG2 cells using the MTT assay. Cell viability was not affected by honokiol up to 100 μM, but cell viability decreased to 36.6% and 15.0% with 250 μM and 500 μM honokiol, respectively (Fig. 1B). Cell viability was not affected by up to 1 mM FFA mixture in HepG2 cells, but 2 mM FFA mixture significantly reduced cell viability of HepG2 cells (Fig. 1C). In addition, the FFA mixture induced accumulation of intracellular lipid droplets in HepG2 cells in a dose-dependent manner (data not shown). Hence, 1 mM FFA mixture was used to induce the non-alcoholic fatty liver condition in HepG2 cells.

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Honokiol attenuates lipid accumulation in FFA-exposed HepG2 cells

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To investigate the effect of honokiol on FFA-induced lipid accumulation in HepG2 cells, the cells were exposed to 1 mM FFA mixture for 24 h, and intracellular lipid contents and TG levels were measured. Cells were pretreated with honokiol (25–100 μM) for 1 h prior to the addition of the FFA mixture. Exposure to 1 mM FFA mixture induced a 4.0-fold increase in lipid contents compared to the vehicle control

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We examined the activation of the lipogenic transcriptional factor SREBP-1c and its primary lipogenic target enzymes, SCD-1 and FAS. Exposure to 1 mM FFA mixture for 24 h significantly increased the level of mature SREBP-1c in the nuclear fraction and SCD-1 and FAS protein levels in whole-cell lysates, but these increases were attenuated by honokiol in a concentration-dependent manner (Figs. 2C and D).

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To examine whether honokiol induces AMPK and ACC phosphorylation in HepG2 cells, the time course of the effect of honokiol on AMPK and ACC protein levels was investigated in HepG2 cells. Total AMPK and ACC protein levels in HepG2 cells were not affected by 100 μM honokiol treatment. However, the level of phosphorylated AMPK began to increase 30 min after honokiol addition and was maintained continuously for 8 h. The level of phosphorylated ACC gradually increased 1–8 h after honokiol treatment (Fig. 3A). We also determined the concentration-dependent response to honokiol regarding the AMPK and ACC protein levels. HepG2 cells were treated with 10– 100 μM honokiol for 2 h. The levels of phosphorylated AMPK and ACC increased following the addition of honokiol in a concentrationdependent manner in HepG2 cells, and the levels of phosphorylated AMPK and ACC were highest with 100 μM honokiol. Pretreatment with compound C, an AMPK inhibitor, reduced the phosphorylation of AMPK and ACC (Fig. 3B).

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Honokiol induces AMPK and ACC phosphorylation in HepG2 cells

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Honokiol attenuates the increases in lipogenic proteins in FFA-exposed 227 HepG2 cells 228

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Statistical analysis. All results are presented as the means ± SE. The overall significance of the experimental results was determined with one-way analysis of variance. The differences between groups were considered significant at P b 0.05 with the appropriate Bonferroni correction made for multiple comparisons.

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group in HepG2 cells, but this increase was attenuated by honokiol in a dose-dependent manner (Fig. 2A). We also measured the intracellular TG level in HepG2 cell lysates. As shown in Fig. 2B, the intracellular TG level was significantly increased in HepG2 cells exposed to 1 mM FFA mixture for 24 h, but this increase was also inhibited by honokiol in a concentration-dependent manner.

The binding of all antibodies was detected using an ECL detection system (Animal Genetics, Truro, UK) according to the manufacturer's instructions. The intensity of the immunoreactive bands was determined using a GS-710 calibrated imaging densitometer (Bio-rad, Hercules, CA).

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Fig. 1. The chemical structure of honokiol and cytotoxicity of honokiol and FFA mixture in HepG2 cells. (A) The chemical structure of honokiol. (B, C) HepG2 cells were treated with various concentrations of honokiol (B) or FFA mixture (C) for 24 h and then analyzed by MTT assay. DMSO (0.1%) was used as the vehicle for honokiol, and fatty acid-free BSA (1%) was used for control cells. Data are presented as the means ± SE. **P b 0.01 vs. vehicle-treated (or control) cells.

Please cite this article as: Seo, M.S., et al., Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.020

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HepG2 cells were treated with honokiol to examine whether this compound activates LKB1, an upstream regulator of AMPK phosphorylation. The level of total LKB1 protein was not changed with honokiol treatment, but phosphorylated LKB1 was dramatically

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To evaluate whether AMPK phosphorylation mediates the inhibitory effect of honokiol on FFA-induced lipogenic processes in HepG2 cells, the cells were pretreated with 40 μM compound C 30 min prior to honokiol treatment. Intracellular lipid contents and TG levels were measured 24 h after the addition of the FFA mixture. Honokiol (100 μM) significantly inhibited the increases in intracellular lipid contents and TG levels in FFA mixture-exposed HepG2 cells, but this effect was blocked by compound C (Figs. 4A and B). In addition, we also measured the protein levels of nuclear

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SREBP-1c, SCD-1, and FAS in HepG2 cells. Honokiol significantly attenuated the increases in the level of nuclear SREBP-1c and the protein expression of SCD-1 and FAS in FFA mixture-exposed HepG2 cells, but these effects were blocked by compound C (Figs. 4C and D).

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Fig. 2. Honokiol attenuates increases in lipid contents, TG level, SREBP-1c maturation and the induction of SCD-1 and FAS proteins in FFA-treated HepG2 cells. HepG2 cells were treated with 1 mM FFA for 24 h, and honokiol (25–100 μM) was treated 1 h prior to FFA treatment. Control cells were treated with fatty acid-free BSA (1%), and DMSO (0.1%) was used as a vehicle for honokiol. (A) The cells were stained with Oil Red O dye, and the quantity of lipid contents was analyzed using a spectrometer at 545 nm. Photographs (magnification 400×) are representative images of 3 independent experiments. (B) Intracellular TG levels were measured using a commercial kit. (C) Mature SREBP-1c was measured by Western blot analysis in the nuclear fraction of HepG2 cells. (D) The protein levels of SCD-1 and FAS were measured by Western blot analysis in the HepG2 cell lysates. Data are presented as the means ± SE. **P b 0.01, compared to Veh + control cells; #P b 0.05 and ##P b 0.01, compared to Veh + FFA cells.

Please cite this article as: Seo, M.S., et al., Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.020

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increased 10–120 min after the addition of 100 μM honokiol (Fig. 5A). In addition, honokiol increased the level of phosphorylated LKB1 in a concentration-dependent manner (Fig. 5B).

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Honokiol induces AMPK phosphorylation in HepG2 cells through LKB1 277 activation, but not CaMKK 278 To investigate whether LKB1 mediates honokiol-induced AMPK phosphorylation, HepG2 cells were transfected with LKB1-specific siRNA. The efficiency of LKB1 siRNA was confirmed by marked suppression of the LKB1 protein level (Fig. 6A). Honokiol treatment increased the levels of phosphorylated AMPK and ACC in scrambled siRNAtreated cells, but the compound failed to induce AMPK and ACC phosphorylation in LKB1 siRNA-treated cells (Fig. 6B). Next, to elucidate whether LKB1 mediates the inhibitory effect of honokiol on lipogenic processes in HepG2 cells, we added the FFA mixture to LKB1 siRNAtreated cells. Honokiol attenuated the increases in nuclear SREBP-1c, SCD-1, and FAS protein levels induced by the FFA mixture in scrambled siRNA-treated HepG2 cells, but not in LKB1 siRNA-treated HepG2 cells (Figs. 6C and D). Finally, we tested whether CaMKK, another upstream signal of AMPK, mediates the effect of honokiol in HepG2 cells. HepG2 cells were pretreated with a CaMKK inhibitor, STO609, and then treated with 100 μM honokiol. STO609 pretreatment did not affect the levels of AMPK or ACC phosphorylation that was induced by honokiol in HepG2 cells (Fig. 6E).

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Fig. 3. Honokiol induces AMPK and ACC phosphorylation in HepG2 cells. (A) The HepG2 cell lysates were collected at the indicated times after honokiol treatment (100 μM). (B) The HepG2 cell lysates were collected at 2 h after honokiol treatment (10–100 μM) and compound C was pretreated 1 h prior to honokiol treatment. DMSO (0.1%) was treated as a vehicle and the changes in protein levels were determined by Western blot analysis.

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Honokiol attenuates the development of fatty liver in mice fed with a high 297 fat diet 298 To elucidate whether honokiol attenuates the fatty liver development in an in vivo model, the mice were fed with a high fat diet. There was no significant difference in food intake for 28 days, but honokiol inhibited the body weight increase in the mice fed with HFD. In addition, honokiol significantly attenuated the increases in liver weight and the hepatic TG level in the mice fed with HFD for 4 weeks (Fig. 7A). HFD feeding for 4 weeks resulted in the formation of cytoplasmic lipid droplets in the liver, as assessed by histological examination and Oil Red O staining. However, honokiol attenuated the accumulation of lipid droplets in the liver from HFD-fed mice (Figs. 7B and C).

Please cite this article as: Seo, M.S., et al., Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.020

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Fig. 4. AMPK mediates the effect of honokiol on the increases in lipid contents, TG level, SREBP-1c maturation and the induction of SCD-1 and FAS proteins in FFA-treated HepG2 cells. HepG2 cells were treated with 1 mM FFA for 24 h, and honokiol (100 μM) was treated 1 h prior to FFA treatment. Control cells were treated with fatty acid-free BSA (1%), and DMSO (0.1%) was used as a vehicle for honokiol. To inhibit the AMPK activation, compound C was pretreated 30 min before honokiol treatment. (A) The cells were stained with Oil Red O dye, and the quantity of lipid contents was analyzed using a spectrometer at 545 nm. Photographs (magnification 400×) are representative images of 3 independent experiments. (B) Intracellular TG levels were measured using a commercial kit. (C) Mature SREBP-1c was measured by Western blot analysis in the nuclear fraction of HepG2 cells. (D) The protein levels of SCD-1 and FAS were measured by Western blot analysis in the HepG2 cell lysates. Data are presented as the means ± SE. **P b 0.01, compared to Veh + control cells; ##P b 0.01, compared to Veh + FFA cells; $$P b 0.01, compared to Hon + FFA cells.

Please cite this article as: Seo, M.S., et al., Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.020

M.S. Seo et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

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steatohepatitis, a condition that can result in end-stage liver disease. Non-alcoholic fatty liver is characterized by the accumulation of TGs, and is the earliest recognizable stage of NAFLD. The accumulation of TGs results from the esterification of FFA and glycerol in the hepatocytes. At rest, about 80% of plasma FFA is transported to the liver where the FFAs are either oxidized to form ATP or reesterified for storage as TG (Niu et al., 2012). Elevated FFA levels result in intracellular lipid accumulation in the liver, causing the development of the non-alcoholic fatty liver. Patients with NAFLD display an increase in delivery of FFA to the liver (Hur et al., 2012). FFA influx and de novo lipogenesis are considered key pathogenic processes in the development of NAFLD. Honokiol, with a chemical formula of 2-(4-hydroxy-3-prop-2-enylphenyl)-4-prop-2-enyl-phenol (Fig. 1A), is a bioactive neolignan compound isolated from the medicinal herb, M. officinalis. Honokiol has antioxidant, anti-inflammatory, antithrombotic, and antitumor activities (Lee et al., 2011). Although the polyphenols that are present in many natural products have become an important subject in nutrition research, the development of polyphenolic agents as therapeutics has often been impeded by poor absorption and rapid excretion (Bar-Sela et al., 2010). Due to its small molecular weight structure, honokiol exhibits a desirable spectrum of bioavailability with relatively low toxicity, in contrast with many other natural products (Arora et al., 2012). Lin et al. (2012) suggested that honokiol crosses the blood–brain barrier and may reach therapeutic concentrations in the brain via passage through the tight junctions formed by cerebral endothelial cells. Pharmacokinetics study demonstrated that honokiol could be absorbed quickly, and maintained in plasma for longer than 10 h (Chen et al., 2004). These properties make honokiol a promising medicinal agent. A recent study showed that honokiol reduces body fat accumulation, insulin resistance, and adipose inflammation in high fat-fed mice (Kim et al., 2013). In addition, an in vivo model using the Lieber–DeCarli ethanol diet showed that honokiol improves alcoholic steatosis by blocking fatty acid synthesis (Yin et al., 2009). However, the cellular mechanism that mediates the anti-steatotic effect of honokiol in hepatocytes is unclear. Therefore, we employed an in vitro model using FFA-exposed HepG2 cells and an in vivo model using HFD-fed obese mice to elucidate the mechanism underlying protective effects of honokiol against nonalcoholic fatty liver. In the present study, we found that honokiol lowered lipid accumulation and TG levels in FFA-exposed HepG2 cells. In addition, SREBP-1c maturation and the induction of SCD-1 and FAS proteins were attenuated by honokiol. SREBP-1c is a key lipogenic transcription factor, which is abundant in the mammalian liver (Musso et al., 2009). Fatty acid delivery into hepatocytes induces de novo lipogenic processes due to the overexpression of SREBP-1c (Mendez-Sanchez et al., 2007). Upon maturation, SREBP-1c translocates into the nucleus and transcriptionally upregulates SCD-1 and FAS, the key enzymes required for de novo fatty acid and TG synthesis in hepatocytes (Horton et al., 2002). SCD-1 is a rate-limiting enzyme involved in the synthesis of unsaturated fatty acids and is responsible for forming a double bond in stearoylCoA (Gutierrez-Juarez et al., 2006). A previous study reported that genetic deletion of SCD-1 prevents the development of fatty liver and insulin resistance in mice (Postic and Girard, 2008). FAS is a multienzyme protein that catalyzes the last step of the fatty acid biosynthetic pathway (Griffin et al., 2007). Therefore, our findings indicate that honokiol targets FFA-mediated lipogenesis to attenuate lipid accumulation in hepatocytes. AMPK has emerged as a critical molecule that mediates the beneficial effects of many natural products in preventing the development of NAFLD (Wu et al., 2014). Activated AMPK stimulates ATP-producing catabolic pathways, such as fatty acid β oxidation, and inhibits ATPconsuming processes, such as lipogenesis, directly by phosphorylating regulatory proteins and indirectly by affecting expression levels of genes in these pathways (Browning and Horton, 2004). The first downstream enzymatic target of AMPK is ACC, which is involved in the

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Fig. 5. Honokiol induces LKB1 phosphorylation in HepG2 cells. (A) The HepG2 cell lysates were collected at the indicated times after honokiol treatment (100 μM). (B) The HepG2 cell lysates were collected at 30 min after honokiol treatment (10–100 μM). DMSO (0.1%) was treated as a vehicle and the changes in protein levels were determined by Western blot analysis.

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The major findings of this study are that honokiol stimulated LKB1 phosphorylation in HepG2 cells, subsequently leading to AMPK and ACC phosphorylation. In addition, honokiol attenuated the intracellular lipid accumulation and the induction of lipogenic proteins in FFAexposed HepG2 cells. These effects were mediated by the LKB1–AMPK signaling pathway because honokiol failed to attenuate FFA-induced lipogenesis in HepG2 cells that had been treated with compound C or LKB1-specific siRNA. Finally, honokiol treatment induced the phosphorylation of LKB1, AMPK and ACC, and attenuated the increases in lipid droplets, intracellular TG levels and lipogenic protein expression in the liver from the mice fed with HFD. The two major risk factors for excessive fat accumulation in liver tissue are obesity/insulin resistance and excessive alcohol intake. Although no laboratory, imaging, or histological findings can accurately distinguish between non-alcoholic steatosis and alcoholic steatosis, NAFLD that develops in the absence of alcohol abuse is gaining increasing recognition as a major health issue (Dowman et al., 2010). Non-alcoholic fatty liver (or simple steatosis) is a benign, noninflammatory condition that is associated with non-alcoholic

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To investigate the effect of honokiol on lipogenic process in an in vivo model, lipogenic and anti-lipogenic proteins were measured in the liver using Western blot analysis. The levels of total LKB1 and AMPK proteins were not changed with honokiol treatment, but those of phosphorylated LKB1 and AMPK were significantly increased in the liver from the mice fed with ND or HFD (Fig. 8A). In addition, honokiol significantly increased the levels of SIRT1, phosphorylated ACC and ATGL in the liver from the mice fed with ND or HFD (Fig. 8B). HFD feeding for 4 weeks increased the expression of lipogenic proteins, SREBP-1c, FAS and SCD-1, in the liver. But these increases were significantly attenuated by honokiol treatment (Fig. 8B).

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Honokiol induces the expression of anti-lipogenic proteins and reduces the expression of lipogenic proteins in the liver from mice fed with a high fat diet

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Please cite this article as: Seo, M.S., et al., Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.020

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found that honokiol stimulated AMPK activation followed by an increase in ACC phosphorylation. Moreover, AMPK inhibition blocked the effect of honokiol on the attenuation of intracellular lipid droplets and TG levels, SREBP-1c maturation, and protein expression of SCD-1 and FAS after the exposure of HepG2 cells to FFA. Therefore, our results suggest that the activation of AMPK and subsequent ACC phosphorylation mediates the inhibitory effect of honokiol on lipid accumulation in FFA-overloaded hepatocytes. AMPK responds to an increased cellular AMP:ATP ratio, and AMPK phosphorylation is also regulated by upstream signaling pathways including LKB1 and CaMKK. Increasing evidence has demonstrated that the extent of phosphorylation at Thr172 reflects the degree of AMPK activation, which is necessary and sufficient for AMPK activity (Stein et al., 2000). LKB1, also known as STK11, was originally identified as a tumor suppressor, is an upstream kinase capable of phosphorylating AMPK, and is a critical mediator of the cellular response to low energy (Bai et al., 2014). Bioactive compounds, such as mangiferin, stimulate AMPK phosphorylation in an LKB1-independent manner by increasing the AMP:ATP ratio (Niu et al., 2012). Another natural compound, thymoquinone, alleviates hepatic fibrosis and the inflammatory response by activating the LKB1–AMPK signaling pathway (Kim et al., 2009). Increasing evidence has shown that the phosphorylation of AMPK by natural compounds is dependent on upstream LKB1 activation for the protection of the liver from hepatotoxicity (Bai et al., 2014). Moreover, honokiol activates AMPK in breast cancer cells via an LKB1-dependent pathway and inhibits breast carcinogenesis (Nagalingam et al., 2012). As shown in our results, honokiol stimulated the LKB1 phosphorylation, and deletion of LKB1 using LKB1specific siRNA clearly blocked AMPK and ACC phosphorylation in honokiol-treated hepatocytes. Consistent with these results, honokiol did not attenuate SREBP-1c maturation or the induction of SCD-1 and FAS proteins by FFA in LKB1-deleted hepatocytes. As CaMKK is another upstream kinase of AMPK, we also tested whether this kinase mediates the anti-lipogenic action of honokiol in hepatocytes. However, our data showed that the inhibition of CaMKK did not affect AMPK or ACC phosphorylation induced by honokiol, suggesting that CaMKK does not play a role in these honokiolmediated effects in hepatocytes. Although the results from an in vitro study clearly showed the hepatocellular mechanism of honokiol, the development of non-alcoholic fatty liver is a long term process rather than an acute response. So, we conducted an in vivo study using the mice fed with HFD including large amount of fat (60%). Consistent with the results from the in vitro study, honokiol induced the phosphorylation of LKB1, AMPK and ACC in the liver of the mice fed with ND or HFD. The HFD feeding for 4 weeks increased the hepatic lipid droplets and intracellular TG levels and upregulated the lipogenic proteins, SREBP-1c, FAS and SCD-1. These changes were significantly attenuated by honokiol treatment. In addition, we found that honokiol could induce the protein expression

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synthesis of malonyl-CoA. AMPK inhibits ACC activity by phosphorylating ACC at Ser77 and Ser79, thereby stimulating fatty acid oxidation and reducing fatty acid synthesis (Ha et al., 1994). Liver-specific deletion of ACC reduces hepatic TG accumulation by decreasing de novo fatty acid synthesis (Mao et al., 2006). Although the mechanism is undefined, SREBP-1c is reduced by activated AMPK. In the present study, we

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Fig. 6. LKB1 mediates the effect of honokiol stimulating AMPK and ACC phosphorylation and inhibiting the increases in SREBP-1c maturation and the induction of SCD-1 and FAS proteins in FFA-treated HepG2 cells. HepG2 cells were transfected with scrambled or LKB1-specific siRNAs, and were treated with 100 μM of honokiol. (A) After 30 min of honokiol treatment, LKB1 protein expression was determined in the cell lysates to confirm the efficiency of LKB1 specific-siRNA. (B) AMPK and ACC protein levels were determined 2 h after honokiol treatment in HepG2 cells transfected with scrambled or LKB1 specificsiRNA. (C) After 1 h of honokiol treatment, HepG2 cells transfected with scrambled or LKB1 specific-siRNA were treated with FFA (1 mM) for 24 h. SREBP-1c levels were determined using Western blot analysis in nuclear fraction. (D) After 1 h of honokiol treatment, HepG2 cells transfected with scrambled or LKB1 specific-siRNA were treated with FFA (1 mM) for 24 h. SCD-1 and FAS protein levels were determined using Western blot analysis in the whole cell lysates. (E) To inhibit the CaMKK, HepG2 cells were treated with STO609 1 h prior to honokiol treatment. AMPK and ACC protein levels were determined 2 h after honokiol treatment.

Please cite this article as: Seo, M.S., et al., Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.020

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Fig. 7. Honokiol attenuates the development of fatty liver in mice fed with a high fat diet. The mice were fed with ND or HFD (60% fat) for 28 days and honokiol was treated at a dose of 10 mg/kg/day. (A) Body weight and food intake were observed twice a week, and liver weight and hepatic TG levels were measured at 28 days. (B) Representative photomicrographs (magnification 200×) of hematoxylin and eosin-stained liver sections. (C) Representative photomicrographs (magnification 200×) of Oil Red O dye-stained liver sections. Photographs (magnification 200×) are representative images of 6 liver tissues from each group. Data are presented as the means ± SE. *P b 0.05, compared to the Veh + ND group; #P b 0.05, compared to the Veh + HFD group.

Please cite this article as: Seo, M.S., et al., Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.020

All authors declare that there are no conflicts of interest.

This study was supported by a grant (A001100476) from the Regional Industrial Technology Development Program through the Ministry of Trade, Industry and Energy of Korea to Huons Co., Ltd., and the Gyeongsang National University of Korea, 2012. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2013R1A1A1013194).

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lipid accumulation induced by FFA in hepatocytes. Therefore, honokiol 484 treatment could be a potential therapeutic strategy for treatment of 485 non-alcoholic fatty liver. 486

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of SIRT1 and ATGL in the liver of the mice fed with ND or HFD. SIRT1 is known as a fuel-sensing molecule that modulates lipid metabolism in hepatocytes. The activation of SIRT1 was reported to activate the LKB1/AMPK signaling axis as an upstream regulator in hepatocytes (Hou et al., 2008). AMPK activation by SIRT1 alleviated the lipid accumulation by facilitating hepatic fatty acid metabolism and by inhibiting the de novo fatty acid synthase (Purushotham et al., 2009). The role of SIRT1 in lipid metabolism is established in connection with Forkhead box protein O1-mediated ATGL expression. Genetic depletion of SIRT1 inhibited TG hydrolysis by reducing ATGL expression (Chakrabarti et al., 2011). Therefore, our data suggest that SIRT1 and ATGL are involved in the protection by honokiol against the development of non-alcoholic fatty liver. Collectively, our findings demonstrate that honokiol triggers the activation of the LKB1–AMPK signaling pathway, thereby leading to the induction of lipolysis and the inhibition of lipogenesis in hepatocytes. As a result of these changes, honokiol attenuates intracellular

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Fig. 8. Honokiol changes the lipogenic and anti-steatotic protein levels in the liver from mice fed with a high fat diet. The mice were fed with ND or HFD (60% fat) for 28 days and honokiol was treated at a dose of 10 mg/kg/day. (A) Phosphorylated/total LKB1 or AMPK levels and (B) SIRT1, phosphorylated ACC, ATGL, SREBP-1c, FAS and SCD-1 protein levels were measured using Western blot analysis in the liver tissues. Data are presented as the means ± SE. *P b 0.05, compared to the Veh + ND group; #P b 0.05, compared to the Veh + HFD group.

Please cite this article as: Seo, M.S., et al., Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.020

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Please cite this article as: Seo, M.S., et al., Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.02.020

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