9-O-benzoyl-substituted berberine exerts a triglyceride-lowering effect through AMPK signaling pathway in human hepatoma HepG2 cells

Accepted Manuscript Title: 9-O-benzoyl-substituted berberine exerts a triglyceride-lowering effect through AMPK signaling pathway in human hepatoma HepG2 cells Authors: Shijie Cao, Shengyang Yu, Lina Cheng, Jiankun Yan, Yan Zhu, Yanru Deng, Feng Qiu, Ning Kang PII: DOI: Reference:

S1382-6689(18)30382-X https://doi.org/10.1016/j.etap.2018.09.007 ENVTOX 3087

To appear in:

Environmental Toxicology and Pharmacology

Received date: Accepted date:

5-2-2018 14-9-2018

Please cite this article as: Cao S, Yu S, Cheng L, Yan J, Zhu Y, Deng Y, Qiu F, Kang N, 9-O-benzoyl-substituted berberine exerts a triglyceride-lowering effect through AMPK signaling pathway in human hepatoma HepG2 cells, Environmental Toxicology and Pharmacology (2018), https://doi.org/10.1016/j.etap.2018.09.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

9-O-benzoyl-substituted berberine exerts a triglyceride-lowering effect through AMPK signaling pathway in human hepatoma HepG2 cells Shijie Cao b, #, Shengyang Yu b, c, #, Lina Cheng a, Jiankun Yan d, Yan Zhu b, Yanru Deng , Feng Qiu b, c, *, Ning Kang a, *

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School of Integrative Medicine, Tianjin University of Traditional Chinese Medicine,

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c

b

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Tianjin 300193, PR China

Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of

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Traditional Chinese Medicine, Tianjin 300193, PR China c

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College of Science and Technology, Agricultural University of Hebei, Huanghua

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061100, PR China #

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Medicine, Tianjin 300193, PR China

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School of Chinese Materia Medica, Tianjin University of Traditional Chinese

These authors contributed equally to this work.

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*Author to whom correspondence should be addressed.

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Postal address: Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Tianjin, 300193, PR China; E-mail: [email protected] (Ning Kang); [email protected] (Feng Qiu)

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Tel.: +86-22-59596223

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Graphical abstract

Highlights

9-O-benzoyl-substituted berberine (A13) has strong binding to AMPK.

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A13 significantly up-regulates p-AMPK in HepG2 cells.

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A13 reduces the cellular triglyceride levels in HepG2 cells.

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A13 mediates mRNA levels of genes involved in fatty acid synthesis and oxidation.

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Abstract Berberine is an isoquinoline alkaloid extracted from Rhizoma coptidis and shows antihyperlipidemia effect in vivo and in vitro. We previously found that berberine could decrease the intracellular triglyceride content in human hepatoma HepG2 cells through

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activation of AMP-activated protein kinase (AMPK), a major regulator of lipid metabolism. Herein, to find a more effective agent, several berberine analogues (A1-

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A13) were isolated and synthesized, and the triglyceride-lowering effects and potential mechanisms were investigated in HepG2 cells. Among these berberine analogues, 9-O-

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benzoyl-substituted berberine (A13) showed strong affinity to AMPK and significantly

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up-regulated the levels of phospho-Thr172 AMPK α subunit. Meanwhile, A13 reduced

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the cellular triglyceride levels. Furthermore, A13 could mediate the mRNA levels of

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downstream proteins involved in triglyceride synthesis and fatty acid oxidation of

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AMPK signaling pathway. These results suggested that A13 exerts a triglyceridelowering effect via stimulation of AMPK pathway, which may be beneficial to regulate

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hyperlipidemia.

Keywords: hyperlipidemia, berberine, 9-O-benzoyl-substituted berberine, AMPK

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pathway, triglyceride, HepG2 cells

1 Introduction Hyperlipidemia is characterized by abnormal blood lipid, including elevated triglyceride (TG), cholesterol (TC), and low-density lipoprotein cholesterol (LDL-c) levels (Chu et al., 2015; Durrington, 2003). There are two types of hyperlipidemia:

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familial hyperlipidemia (primary hyperlipidemia) and acquired hyperlipidemia (secondary hyperlipidemia) (Beaumont et al., 1972). According to an abundance of

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reports and surveys, most patients are acquired hyperlipidemia due to diet,

hypotyroidism, alcohol consumption, chronic kidney and liver disease, etc (Rai and

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Bhatnagar, 2016). Nowadays hyperlipidemia has become the major risk to

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cardiovascular disease, which is the leading cause of morbidity and mortality

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worldwide, particularly in developing countries (Yusuf et al., 2001; Dixon et al., 2015).

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In addition, hyperlipidemia is also strongly associated with metabolic diseases, for

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example obesity and diabetes (Jandeleit-Dahm et al., 1999). Liver plays a key role in lipid metabolism since it is the organ where lipid

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biosynthesis and fatty acid oxidation occur. In the liver, a variety of proteins are closely

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related with the lipid metabolism, for example AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptors (PPAR), sterol-regulatory element binding proteins (SREBP), fatty acid synthase (FAS) and so on (Yin et al., 2017; Sundqvist et

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al., 2005). Among these key proteins, AMPK, a heterotrimeric complex consisting of a catalytic subunit α and two regulatory subunits β and γ, plays a core role in maintaining both lipid and glucose metabolism. Currently, AMPK has been used as a potential therapeutic target for the treatment of hyperlipidemia and diabetes (Zhang et al., 2009).

Several clinical drugs, including metformin and fenofibrate show lipid- or glucoselowering effects through AMPK activation (Cho et al., 2015). Furthermore, an abundance of active compounds from natural plants have been reported to show antihyperlipidemia effect via AMPK signaling pathway in vivo and in vitro. Seo et al

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observed that honokiol, which is isolated from Magnolia officinalis effectively, decreased hepatic TG and fat accumulation in high-fat-diet rats; meanwhile it inhibited

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the biosynthesis of TC and TG in free fatty acid-exposed HepG2 cells. All these antihyperlipidemia effects were involved with activation of AMPK (2015). Huang et al

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reported that Ginsenoside Rb2 from Panax ginseng inhibited hepatic lipid accumulation

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through AMPK activation both in db/db mice and in HepG2 cells (2017). Therefore,

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screening the AMPK activators from natural plants might be a potential method to find

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new active anti-hyperlipidemia compounds.

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Berberine is an isoquinoline quaternary alkaloid, which is isolated from Rhizoma coptidis, and has diverse pharmacological properties, including anti-diabetic, anti-

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hyperlipidemic and anti-tumor. Berberine shows anti-hyperlipidemia effect through

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AMPK activation in liver (Kim et al., 2009; Cao et al., 2013). Further development of berberine as an anti-hyperlipidemic agent is limited because of its low bioavailability (<5%) (Maeng et al., 2002). Recently, some research groups proved that 9-N- and 9-O-

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substituted berberine derivatives showed stronger effect on glucose consumption activity, down-regulation of oncogene c-myc, and up-regulation of LDLR, respectively, which are related with hypoglycemic, anti-tumor and cholesterol-lowering activity (Ma et al., 2008; Wang et al., 2012; Zhang et al., 2016). In addition, several berberine

analogues, such as Y53, 13-methylberberine and 8-cetyl-berberine showed higher antioxidant, anti-inflammatory, anti-adipogenic and anti-hyperlipidemic activities compared with berberine (Chow et al., 2016; Li et al., 2016; Ye et al., 2011), suggesting that it is beneficial to find more effective compounds through structural modification

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of berberine. In the present work, a series of berberine analogues were isolated and synthesized, and molecular docking and western blotting were used to find more active

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compounds through the binding to AMPK and activation of AMPK pathway.

Furthermore, we explored the molecular mechanism of the most active berberine

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analogues, 9-O-benzoyl-substituted berberine.

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2. Materials and Methods

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2.1 Reagents and antibodies

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Berberine chloride was supplied by the Northeast General Pharmaceutical Factory

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(Shenyang, China). Berbeine’s analogues (A1-A5) were isolated from Coptis chinensis Franch (Li et al., 2012). A6-A13 were synthesized as previously described (data not 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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shown).

bromide

(MTT),

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phenylmethylsulfonyl fluoride (PMSF) and RNA isolation reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). RT reagent kit and PCR reagent were obtained from TaKaRa (Dalian, China). Antibody against p-AMPK (Thr72) was

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obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies for AMPK and β-Actin were purchased from ProteinTech Group (Chicago, IL, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

2.2 Cell culture The HepG2 cells were obtained from ATCC (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco/BRL, Gaithersburg, MD, USA) with 100 units/mL streptomycin, and 100 μg/mL penicillin, and 10% FBS

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(TBD Biotechnology Development, Tianjin, China), at 37°C in a humidified 5% CO2 atmosphere.

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2.3 Cell viability

Cells were incubated with 15 μM berberine and its analogues (A1-A13) for 24 h,

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and the cell viability of these compounds on HepG2 cells was measured by MTT assay,

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as described previously (Cao et al., 2013).

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2.4 Western blotting

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HepG2 cells were incubated with 15 μM berberine and its analogues (A1-A13) for

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24 h, and lysed in RIPA lysis buffer (Beyotime, Shanghai, China) and 1 mM PMSF. The protein concentration was assayed using BCA protein assay kit (Solarbio Life

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Sciences, Beijing, China). The aliquots of samples containing 30 μg protein were

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subjected to 10% SDS-PAGE and then transferred to 0.45 μm PVDF membranes (Milipore, MA, USA). Immunoblotting was performed using p-AMPKα Thr-172 (1:1000), AMPK (1:2000) and anti-β-actin (1:5000) antibodies. Following incubation

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with secondary antibodies (1:6000), proteins were detected with ECL Western Blotting Substrate (Thermo Scientific, IL, USA). The bands were quantified using ImageJ software. 2.5 Molecular docking of berberine and analogues into AMPK

The structure of AMPK (PDB code 5EZV) was obtained from the Protein Data Bank (http://www.rcsb.org). Molecular Docking of berberine and its analogues to active site of AMPK, was done by Molegro Virtual Docker (MVD) program. We used a grid resolution of 0.3 Å to calculate the cavities in the protein. For docking, a grid resolution

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of 0.30 Å and a radius of 13 Å around the binding site were used. We used the MolDock SE as a search algorithm, and the number of runs was set to 10. In the parameter settings,

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population size, max iteration, energy threshold, simplex evolution max steps, neighbor distance factor for each run were set as 50, 1500, 100, 300 and 1.00, respectively. The

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2.6 Semi-quantitative reverse transcription PCR

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maximum number of poses to generate was set to default value of 5.

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The mRNA levels of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC),

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medium-chain acyl-CoA dehydrogenase (mCAD), carnitine palmitoyltransferase 1

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(CPT-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by semi-quantitative reverse transcription PCR as described previously (Zhou et al.,

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2014). The densitometry was quantitatively analyzed by ImageJ software. The

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sequences of these primers used in semi-quantitative reverse transcription PCR are listed in Table 1.

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2.7 Measurement of triglyceride (TG) content HepG2 cells were treated with berberine and A13 for 24 h, and washed one time

with PBS and then cells collected after centrifugation. Intracellular TG contents were determined by using an enzymatic reagent kit (Applygen Technology, Beijing, China). The protein concentration was assayed using BCA protein assay kit.

2.8 Statistical analysis Data are presented as the mean ± S.D. Significant differences between control and treatment groups were assessed by one-way ANOVA and Student’s t-test. P<0.05 was considered statistically significant.

3.1 Molecular modeling of berberine and it analogues into AMPK

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3 Results and Discussion

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AMPK is a well-established therapeutic target for several diseases such as hyperlipidemia, diabetes, obesity, etc. Several research groups found AMPK activators,

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such as cyclopentanone (Wu et al., 2014) and phenylamides (Ramesh et al., 2016)

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through molecular docking. Herein, in order to test whether berberine analogues could

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bind to AMPK, molecular docking analysis was performed. Table 2 shows MolDock

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score and re-rank score of selected 14 compounds. It showed clearly that A9-A13 have

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highest docking score than other compounds including berberine. The best poses of berberine and A9-A13 docked to AMPK are shown in Figure 2. Comparing the

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interactions between berberine with AMPK, A9-A13 had the different amino acid

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residues involved in the interactions to AMPK and increased the binding ability. For example, the oxygen atom of the 9-O-benzoyl group in A13 (9-O-benzoyl-substituted berberine) formed hydrogen bond interaction with amino acid residue lysine-29 (LYS-

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29), whereas berberine has a hydrogen bond interaction between 2-O and amino acid residue asparagine-48 (ASN-48). Furthermore, new Pi-sigma and carbon hydrogen interactions were formed in the A13-AMPK complex. Together, the docking analysis reveals that the structural modification of berberine might increase the binding to

AMPK with lower binding energy and more stable to AMPK. 3.2 Effects of berberine and it analogues on AMPK activation HepG2, a liver cell line derived from a human hepatoblastoma that is free of known hepatotropic viral agents, has been found to express a wide variety of proteins

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associated with liver-specific metabolic functions (Javitt, 1990). HepG2 cell line is a microcosm of liver for studying lipid and glucose metabolism, thus the cell line

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becomes an appropriate in vitro model for the metabolic study of hyperlipidemia and

diabetes (Brusq et al., 2006; Wu et al., 2011; Yang et al., 2008). In our current work,

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HepG2 cell was used to investigate the lipid-lowering effects and related mechanisms

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of berberine and its analogues. Firstly, the preliminary work is expelling the toxicity

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caused by the test compounds which might affect the anti-hyperlipidemia effect. We

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previously showed that up to 15 μM berberine for 24 h did not affect the cell growth of

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HepG-2 cells (cell viability > 95%). To determine whether 15 μM berberine analogues affect HepG2 cell viability, MTT assay was used to detect the cell viability of these

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compounds. As shown in Figure 3A, 15 μM berberine analogues did not show any

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cellular toxicity (P>0.05, compared with control group). Thus, the concentration was conducted the subsequent experiments on HepG2 cells. AMPK activation requires the phosphorylation of a catalytic a subunit at Thr172.

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Based on several recent reports, berberine could increase the protein expression of pAMPK in HepG2 cells (Choi et al., 2017; Kuang et al., 2015), which might contribute to glucose consumption and inhibition of lipid accumulation. Consistent with these reports, we previously found that berberine could lead to concentration- and time-

dependent increases on p-AMPK levels in the HepG2 cells. Herein, to investigate whether the analogues of berberine could induce the activation of AMPK signaling, HepG2 cells were incubated with the analogues of berberine for 24 h and the levels of p-AMPK were tested by western blotting. As shown in Figure 3B, berberine and its

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analogue A13 could increase the levels of p-AMPK, while other analogues did not significantly up-regulate AMPK activation. A13 showed higher effect on the up-

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regulation of p-AMPK compared with berberine. Although A9, A10 and A12 showed

stronger binding to AMPK in molecular docking than A13, they did not activate AMPK.

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Together, the results of western blotting and molecular docking show that A13 has high

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binding and activation of AMPK α submit.

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3.3 Effects of berberine and A13 on cellular TG content

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Berberine decreased TG content and inhibited the deposition TG in the liver both

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on diabetic hyperlipidemic rats and high-fat diet rats (Chang et al., 2010; Zhou et al., 2008). Our previous result is consistent with these studies that berberine decreased the

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intracellular TG contents in HepG2 cells. To evaluate whether A13 has lipid-lowering

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effect, we examined the intracellular TG content of HepG2 cells treated with 15 μM berberine and A13. As shown in Figure 4, the level of cellular TG of A13 group was markedly decreased compared to the control group. A13 showed stronger effect than

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berberine.

3.4 Effects of berberine and A13 on the mRNA expression of fatty acid oxidation and fatty acid synthesis genes AMPK regulates lipid metabolism through modulating downstream enzymes in

fatty acid synthesis and oxidation. ACC is the first enzyme shown to be downstream targets for AMPK, which are key enzymes in fatty acid synthesis (Hardie, 2004). Inhibition of ACC by AMPK leads to an increase in mitochondrial fatty acid oxidation via the allosteric regulation of CPT-1, which catalyzes the entry of long-chain fatty acyl-

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CoA into mitochondria. Moreover, the activation status of AMPK functions as a master regulator of FAS, an enzyme catalyzing eukaryotic fatty acid biogenesis (Ferre et al.,

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2003). Meng et al proved that activated AMPK increases mCAD and PPARα mRNA expression, and promotes fatty acid oxidation (Meng et al., 2009). Above all, ACC,

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FAS, CPT-1 and mCAD are the downstream of AMPK signaling. Natural products such

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as luteolin and astragaloside IV (Liu et al., 2011; Zhou et al., 2017) activated AMPK

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and mediated these genes expression so as to induce lipid-lowering effects. Therefore,

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we tested whether A13 could affect the expressions of these TG metabolism genes. As

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shown in Figure 5, berberine and A13 apparently reduced the genes related to fatty acid synthesis, ACC and FAS. In addition, A13 up-regulated the mRNA level of mCAD and

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CPT-1, which were associated with lipolysis. A13 showed higher effect on the genes of

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fatty acid oxidation.

In summary, we have demonstrated for the first time that berberine analogue 9-O-

benzoyl-substituted berberine potently reduces TG content in HepG2 cells. Specifically,

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9-O-benzoyl-substituted berberine exerts the most potent inhibitory effects, and the underlying mechanism might be involved with the binding to AMPK and the activation of AMPK pathway. Furthermore, 9-O-benzoyl-substituted berberine is able to downregulate fatty acid synthesis genes (FAS and ACC) and up-regulate oxidation genes

expression (mCAD and CPT-1). These findings together suggest that 9-O-benzoylsubstituted berberine may be a more effective compound for the prevention and

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treatment of hyperlipidemia.

Conflict of Interest

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The authors declare no conflict of interest.

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Acknowledgements

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This work was supported by National Natural Science Foundation of China (Grant

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number: 20972098 and 81703776) and Key Program of National Natural Science

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Foundation of China (Grant number: 81430095).

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Yusuf, S., Reddy, S., Ounpuu, S., Anand, S., 2001, Global burden of cardiovascular diseases: part I: general considerations, the epidemiologic transition, risk factors, and impact of urbanization. Circulation. 104, 2746-2753. Zhang, B.B., Zhou, G., Li, C., 2009, AMPK: an emerging drug target for diabetes and the metabolic syndrome. Cell. Metab. 9(5), 407-416.

Zhang, S.S., Wang, X.B., Yin, W.C., Liu, Z.B., Zhou, M., Xiao, D.P., Liu, Y.F., Peng, D.M., 2016, Synthesis and hypoglycemic activity of 9-O-(lipophilic group substituted) berberine derivatives. Bioorg. Med. Chem. Lett. 26, 4799-4803. Zhou, B., Zhou, D.L., Wei, X.H., Zhong, R.Y., Xu, J., Sun, L., 2017, Astragaloside IV

via AMPK activation. Acta. Pharmacol. Sin. 38(7), 998-1008.

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attenuates free fatty acid-induced ER stress and lipid accumulation in hepatocytes

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Zhou, J.Y., Zhou, S.W., Zhang, K.B., Tang, J.L., Guang, L.X., Ying, Y., Xu, Y., Zhang, L., Li, D.D., 2008, Chronic effects of berberine on blood, liver glucolipid

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metabolism and liver PPARs expression in diabetic hyperlipidemic rats. Biol Pharm.

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Bull. 31(6), 1169-1176.

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Zhou, Y., Cao, S.J., Wang, Y., Xu, P.X., Yan, J.K., Bin, W., Qiu, F., Kang, N., 2014,

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Berberine metabolites could induce low density lipoprotein receptor up-regulation

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to exert lipid-lowering effects in human hepatoma cells. Fitoterapia. 92, 230-237.

Figure legends Figure 1. The chemical structures of berberine (BBR) and it analogues (A1-A13). Figure 2. Binding analysis of BBR and it analogues with AMPK. Three-dimensional representation of BBR (A), A9 (B), A10 (C), A11 (D), A12 (E), A13 (F) and AMPK binding interactions.

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Figure 3. Effect of BBR and it analogues on AMPK activation. Cells were treated with 15 μM BBR and it analogues for 24 h. Cell viability was determined using the MTT assay (A). The protein expressions of p-AMPK and β-Actin were determined by western blotting (B). The β-Actin was used as a loading control and blots are representative of at least 3 repeats. Values are expressed as mean ± SD. *P < 0.05, compared to the control group.

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Figure 4. Effects of BBR and A13 on triglyceride contents in HepG2 cells. Cells were incubated with 15 μM BBR and A13 for 24 h. The intracellular triglyceride contents were determined by an enzymatic reagent kit. Values are expressed as mean ± SD. The experiments repeated four independent experiments. *P < 0.05, compared to the control group.

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Figure 5. Effects of BBR and A13 on expression in HepG2 cells. Cells were treated with 15 μM of BBR and A13 for 24 h. The mRNA levels of FAS, ACC, CPT-1 and mCAD were determined by semi-quantitative reverse transcription PCR. Values are expressed as mean ± SD of three independent experiments. * P < 0.05 and ** P < 0.01, compared to the control group.

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Table 1 Gene-specific primers used for semi-quantitative reverse transcription PCR

FAS

Forward (5’-3’)

Reverse (5’-3’)

AGCTGCCAGAGTCGGAA

CAAGAACTGCACGGAGGTGT

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Gene

CTGTAGAAACCCGGACAGTAGAAC

GGTCAGCATACATCTCCATGTG

mCAD

CTACCAAGTATGCCCTGGAAAG

TGTGTTCACGGGCTACAATAAG

CPT

AGACGGTGGAACAGAGGCTGAAG

TGAGACCAAACAAAGTGATGATGTCAG

GAPDH

TGCACCACCAACTGCTTAG

GACGCAGGGATGATGTTC

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Table 2 Docking parameters (kcal/mol) in active site of AMPK MolDock Score

Re-rank Score

Berberine

-103.366

-65.2255

A1

-95.0943

-97.0095

A2

-114.094

-113.505

A3

-91.1081

-88.7424

A4

-91.1353

-87.1177

A5

-95.9293

-72.8403

A6

-93.1888

A7

-90.3822

A8

-109.788

A9

-155.544

A10

-134.345

A11

-121.47

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A13

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-64.3611

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-117.109

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A12

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Ligand

-104.804 -74.7635

-131.948

-101.892

-129.767

-98.3301