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Anti-adipogenic effect of the flavonoids through the activation of AMPK in palmitate (PA)-treated HepG2 cells

Background Flavonoids are natural polyphenols found widely in citrus fruit and peel that possess anti-adipogenic effects. On the other hand, the detailed mechanisms for the anti-adipogenic effects of flavonoids are unclear. Objectives The present study observed the anti-adipogenic effects of five major citrus flavonoids, including hesperidin (HES), narirutin (NAR), nobiletin (NOB), sinensetin (SIN), and tangeretin (TAN), on AMP-activated protein kinase (AMPK) activation in palmitate (PA)-treated HepG2 cells. Methods The intracellular lipid accumulation and triglyceride (TG) contents were quantified by Oil-red O staining and TG assay, respectively. The glucose uptake was assessed using 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) assay. The levels of AMPK, acetyl-CoA carboxylase (ACC), and glycogen synthase kinase 3 beta (GSK3β) phosphorylation, and levels of sterol regulatory element-binding protein 2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression were analyzed by Western blot analysis. The potential interaction between the flavonoids and the γ-subunit of AMPK was investigated by molecular docking analysis. Results The flavonoid treatment reduced both intracellular lipid accumulation and TG content in PA-treated HepG2 cells significantly. In addition, the flavonoids showed increased 2-NBDG uptake in an insulin-independent manner in PA-treated HepG2 cells. The flavonoids increased the AMPK, ACC, and GSK3β phosphorylation levels and decreased the SREBP-2 and HMGCR expression levels in PA-treated HepG2 cells. Molecular docking analysis showed that the flavonoids bind to the CBS domains in the regulatory γ-subunit of AMPK with high binding affinities and could serve as potential AMPK activators. Conclusion The overall results suggest that the anti-adipogenic effect of flavonoids on PA-treated HepG2 cells results from the activation of AMPK by flavonoids.


INTRODUCTION
AMP-activated protein kinase (AMPK) plays a vital role in controlling and regulating adipogenesis, indicating that its activation could be a promising target for anti-adipogenic strategies [1]. AMPK is a crucial energy sensor that regulates energy homeostasis by decreasing the ATP-consuming processes (fatty acid and cholesterol biosynthesis) and increasing ATP-generating processes (glucose uptake and fatty acid oxidation) [2]. AMPK activation is involved in regulating the lipid homeostasis by enhancing the phosphorylation of acetyl-CoA carboxylase (ACC), and inhibiting the sterol regulatory element-binding protein 2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which are the key proteins responsible for fatty acid and cholesterol biosynthesis [2,3]. In addition, AMPK activation also regulates the glucose homeostasis by stimulating the non-insulin mediated glucose uptake and enhancing the phosphorylation of glycogen synthase kinase 3 beta (GSK3β), a key target in glycogen synthesis [4,5]. Therefore, AMPK is a significant target for the treatment of adipogenesis.
AMPK is a serine/threonine protein kinase that is activated by the phosphorylation at Thr172 in the α-subunit [6]. AMP binding to the γ-subunit stimulates Thr172 phosphorylation by the upstream kinases, including liver kinase B1 (LKB1) and calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ) [7]. Various in silico studies showed that polyphenols act as positive modulators by interacting with the γ-subunit of AMPK, similar to AMP [8,9]. Accordingly, a direct activator of AMPK that would mimic the mechanism of AMP might be a suitable therapeutic strategy against adipogenesis.
Increasing attention has focused on natural plant flavonoids to develop novel anti-adipogenic drugs with high efficacy and few side effects. Flavonoids are natural polyphenolic compounds present in citrus fruit and peel at high concentrations [10]. A recent study showed that immature citrus fruits are rich in five flavonoids, including 2 flavone glycosides (hesperidin and narirutin) and three polymethoxylated flavones (nobiletin, sinensetin, and tangeretin) ( Fig. 1) [10]. These flavonoids exhibit a wide range of promising pharmaceutical effects, including antiadipogenic effects [11][12][13]. On the other hand, the detailed mechanisms for the anti-adipogenic effects of these flavonoids are unclear. In this context, an adipogenic model was established using palmitate (PA)-treated HepG2 cells [14] to explore the anti-adipogenic effects of the five major citrus flavonoids and the molecular mechanism underlying their effects.
The present study hypothesized that the five major citrus flavonoids possess anti-adipogenic effects by activating the AMPK pathway in PA-treated HepG2 cells. For this, the effects of the flavonoids on lipid accumulation, triglyceride (TG) content, and glucose uptake were evaluated. AMPK and its downstream substrates involved in lipid metabolism were analyzed to determine the molecular mechanisms underlying their effects. In silico analysis was performed to investigate the AMPK-activating capacity of the flavonoids and assess the primary mechanism of action. These results might offer a valuable clue for elucidating the mechanism of the anti-adipogenic effects of the flavonoids through AMPK activation.

Preparation of PA
PA-bovine serum albumin (BSA) conjugate was made by soaping of PA with sodium hydroxide (NaOH) and blending with BSA (Sigma-Aldrich), as described previously [15]. Briefly, palmitate (25 mM) was diluted in NaOH (50 mM) at 70°C for 30 min and mixed with 10% BSA (Sigma-Aldrich) at 55°C for 15 min as a 5 mM reserving solution. The reserving solution was diluted in serum-free DMEM to obtain a 0.4 mM palmitate solution.

Oil Red O staining
Intracellular lipid accumulation was determined by Oil Red O staining using a previously reported method with slight modifications [16]. HepG2 cells were plated in a 96-well plate at a density of 1×10 5 cells/well. After treatment, the HepG2 cells were rinsed with phosphate-buffered saline (PBS) and fixed with 10% formaldehyde for 1 h. The cells were rinsed with 60% isopropanol and stained with Oil Red O (Sigma-Aldrich) for 30 min at room temperature. The cells were rinsed with sterile water four-five times to remove excess staining solution. IncuCyte ZOOM (Essen BioScience, USA) was used to capture and analyze the images at 20× magnification.

TG assay
The cellular triglyceride contents were evaluated using a commercially available triglyceride colorimetric assay kit (BioAssay Systems, USA). After treatment, the cells were rinsed with PBS and collected in a cell lysis buffer containing 5% Triton X-100. The cells were homogenized, followed by centrifugation at 3,000 × g for 5 min. The triglyceride content of the supernatant was examined according to the manufacturer's instructions. The protein concentration was quantified using a Bio-Rad DC protein assay (Bio-Rad Laboratories, USA). The triglycerides contents were normalized to the protein concentration, as determined by BSA as the calibration standard.

Preparation of cell lysates and Western blot analysis
HepG2 cells were seeded in a 24-well plate at a density of 5×10 4 cells/well. After the treatments, the cells were rinsed with PBS and lysed with ice-cold RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM Na 2 EDTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM NaVO 4 , and 1 µg/mL leupeptin) containing protease inhibitor. The wholecell lysate was centrifuged at 12,000 rpm for 10 min, and the supernatants were collected and assayed for the protein concentration using the Bradford assay kit (Bio-Rad Laboratories). For Western blotting, an equal amount of protein was separated by 10% sodium dodecyl sulphatepolyacrylamide gel electrophoresis and transferred electrophoretically to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20. The membranes were incubated with the primary antibodies for AMPK (Cell Signaling Technology, USA), p-AMPK (Thr172) (Cell Signaling Technology), ACC (Cell Signaling Technology), p-ACC (Ser79) (Cell Signaling Technology), GSK3β (Cell Signaling Technology), p-GSK3β (Ser9) (Cell Signaling Technology), β-actin (Thermo Fisher, USA), SREBP-2 (Thermo Fisher), and HMGCR (Thermo Fisher), followed by incubation with the HRP-conjugated secondary antibodies. Immunoreactive bands were analyzed using a Chemiluminescence Bioimaging Instrument (NeoScience, Korea).

Molecular docking
The crystal structure of AMPK in the complexes with AMP (PDB ID; 2V8Q) was obtained from Protein Data Bank (PDB). The sitemap tool (Schrodinger Software, Germany) was used to identify the 4 cystathionine-β-synthase (CBS) domains (CBS1, CBS2, CBS3, and CBS4) in the γ-subunit of AMPK. All AMP molecules in the γ-subunit of AMPK were removed for docking flavonoids. Anti-adipogenic effect of the flavonoids minimized energetically using PyRx software (Python Prescription 0.8; The Scripps Research Institute, USA). The grid box used for focused docking was set to 26×44×46 Å to ensure the structure of the γ-subunit of AMPK. The docking experiments were carried out using the AutoDock Vina module (Molecular Graphics Lab, The Scripps Research Institute). Based on binding energy, the best-docked pose was selected, and the 3D images were produced using the PyMOL (The PyMOL Molecular Graphics System, Ver.2.5.0; Schrodinger, LLC, USA). Docked complex of AMPK was optimized further, validated, and explored using Discovery Studio visualizer (Ver.21.1.0.20298). The hydrogen bond and hydrophobic interactions between the receptor and ligand were analyzed using the Ligplot program [17].

Statistical analysis
The data are expressed as the means ± SE of 3 independent experiments and analyzed statistically using IBM SPSS Statistics (Ver.17.0; IBM Corp., USA). The statistical differences among the groups were analyzed by one-way analysis of variance (ANOVA) followed by a Tukey's test. The following were considered significant differences from the PA-treated control group: p < 0.05, p < 0.005, and p < 0.0005.

Cell viability
A MTT assay was performed to evaluate the cytotoxicities of HES, NAR, NOB, SIN, and TAN on HepG2 cells (Fig. 2). HepG2 cells were treated with different concentrations of PA, HES, NAR, NOB, SIN, or TAN for 24 h to examine the cell viability. PA did not exhibit any cytotoxicity up to 500 μM ( Fig. 2A). The flavonoids did not have any cytotoxic effects at concentrations up to 100 μM ( Fig. 2B-F). Accordingly, 400 μM of PA and 50 μM of the flavonoids were used in the following experiments.

Intracellular lipid accumulation and TG content
Oil Red O staining and a TG assay were carried out to assess the effect of HES, NAR, NOB, SIN, and TAN on intracellular lipid accumulation and TG content in HepG2 cells (Fig. 3).
HepG2 cells treated with PA increased (p < 0.0005) lipid accumulation and TG content significantly compared to the untreated control cells. The cells treated with SIM (positive control) showed significantly lower (p < 0.005) lipid accumulation and TG content than the PA-treated control cells. In addition, the cells treated with HES, NAR, NOB, SIN, or TAN showed significantly lower (p < 0.005) lipid accumulation and TG contents than the PAtreated control cells ( Fig. 3A-C). Overall, the results suggest that HES, NAR, NOB, SIN, and TAN markedly reduced the lipid accumulation and TG content in PA-treated HepG2 cells.

Glucose uptake
A 2-NBDG uptake assay was carried out to examine the effects of HES, NAR, NOB, SIN, and TAN on the glucose uptake in PA-treated HepG2 cells (Fig. 4). The HepG2 cells treated with PA showed lower glucose uptake compared to the untreated control cells. On the other hand, the cells treated with MET showed significantly higher (p < 0.005) glucose uptake. In particular, the cells treated with HES, NAR, NOB, SIN, or TAN showed significantly enhanced (p < 0.005) glucose uptake compared to the PA-treated control cells, regardless of insulin stimulation ( Fig. 4A and B). These results suggest that the HES, NAR, NOB, SIN, and TAN treatment improved the glucose uptake in an insulin-independent manner in PA-treated HepG2 cells.

AMPK signaling pathway
The effects of HES, NAR, NOB, SIN, and TAN on the phosphorylation of AMPK (Thr172) and ACC (Ser79) and the levels of SREBP2 and HMGCR expression in PA-treated HepG2 cells were analyzed by Western blot (Fig. 5). The results showed that the treatment with PA significantly decreased (p < 0.05) the AMPK (Thr172) and ACC (Ser79) phosphorylation levels and increased (p < 0.005) the SREBP2 and HMGCR expression levels. By contrast, the treatment with HES, NAR, NOB, SIN, and TAN significantly increased (p < 0.05) the AMPK (Thr172) and ACC (Ser79) phosphorylation levels in PA-treated HepG2 cells and reduced (p < 0.005) the SREBP2 and HMGCR expression levels significantly (Fig. 5A-D). These results suggest that the flavonoids activate the AMPK pathway and have an anti-adipogenic effect on PA-treated HepG2 cells.

Phosphorylation of GSK3β
The effects of HES, NAR, NOB, SIN, and TAN on the phosphorylation of GSK3β in PA-treated HepG2 cells were observed by Western blot analysis (Fig. 6). Based on densitometry analysis, the HepG2 cells treated with PA showed significantly lower (p < 0.05) phosphorylation levels of GSK3β than the untreated control cells. On the other hand, the MET treatment significantly increased (p < 0.05) the level of GSK3β phosphorylation compared to the PA-treated control cells. In addition, the treatment with HES, NAR, NOB, SIN, or TAN increased (p < 0.05) the level of GSK3β phosphorylation remarkably compared to PA-treated control cells (Fig. 6). The results show that the flavonoids increase the level of GSK3β phosphorylation in the PA-treated HepG2 cells.

DISCUSSION
In the present study, the flavonoid treatments increased the AMPK and ACC phosphorylation levels and decreased the SREBP-2 and HMGCR expression levels in PA-treated HepG2 cells. The activation of AMPK is involved in regulating liver lipogenesis, lipid oxidation, and cholesterol synthesis via protein phosphorylation or differential gene expression [19,20]. Moreover, the activation of AMPK enhances the phosphorylation of ACC, which is a crucial rate-limiting enzyme involved in fatty acid biosynthesis [21]. The phosphorylation of ACC leads to its inactivation, which inhibits the formation of malonyl CoA and de novo lipogenesis and stimulates fatty acid oxidation [22]. Nobiletin phosphorylates AMPK and inhibits lipogenesis through ACC phosphorylation in high glucose-treated HepG2 cells [23]. Furthermore, AMPK activation inhibits the transcriptional activity of SREBP-2, which functions as a major transcriptional regulator of cholesterol biosynthesis [24]. HMGCR, a rate-limiting enzyme for cholesterol biosynthesis, is a target gene of SREBP-2 [25]. Apigenin, a plant flavonoid, suppressed the TC and TG contents and decreased the SREBP-2 and HMGCR expression levels in palmitic acid-treated HepG2 cells [26]. Therefore, the flavonoids effectively inhibit lipid accumulation in PA-treated HepG2 cells via the AMPK signaling pathway.
The present study observed that citrus flavonoids increase GSK3β phosphorylation in PAtreated HepG2 cells. AMPK phosphorylates and inhibits its downstream substrate, including GSK3β, which improves glucose utilization for glycogenesis [27]. Nobiletin controls glucose homeostasis by eliciting GSK3β phosphorylation via AMPK in PA-treated HepG2 cells [11]. The activation of AMPK by the flavonoids enhances the glucose uptake in an insulinindependent manner in PA-treated HepG2 cells. Previous studies reported that the flavonoids alone could influence glucose uptake in normal HepG2 cells without PA [11,28]. On the other hand, the present work showed that the flavonoids enhanced the decreased glucose uptake by the PA-treatment significantly through the activation of AMPK in HepG2 cells. The phosphorylation of AMPK promotes the membrane translocation of GLUT2 and enhances glucose uptake in an insulin-independent manner in the liver [4]. Overall, the results show that flavonoids regulate the hepatic glucose metabolism by enhancing glucose uptake and glycogenesis via the AMPK signaling pathway.
In the present study, in silico molecular docking showed that the flavonoids bind to all the three CBS domains in the γ-subunit of AMPK. AMPK is a heterotrimer consisting of a catalytic α-subunit and 2 regulatory (β and γ) subunits [7]. The γ-subunit contains four tandem repeats of cystathionine-β-synthase (CBS) domains [29]. Among the 4 CBS domains, 3 (CBS1, CBS3, and CBS4) domains are involved in binding adenine nucleotides, including AMP, ADP, and ATP [30]. AMP binding to CBS domains releases the autoinhibitory domain from the catalytic domain in α-subunit [6]. This active conformation allows Thr172 to be phosphorylated by an upstream kinase, including LKB1 and CaMKKβ [7]. In addition, the active form of AMPK prevents the phosphatases from accessing T172 of the catalytic subunit [31]. An in silico previous study reported that kaemferide is a promising AMPK activator [8]. Naringenin has shown an anti-adipogenic effect by binding to the AMP binding sites of the γ-subunit as the agonists of AMPK [9]. In the present study, molecular docking analysis showed that the flavonoids activate AMPK directly as positive modulators by interacting with the R groups of the CBS domains in the γ-subunit of AMPK.
In summary, the present study indicated that the flavonoids reduced the lipid accumulation and TG content in PA-treated HepG2 cells by increasing the AMPK and ACC phosphorylation levels while reducing the SREBP-2 and HMGCR expression levels (Fig. 8). In addition, the flavonoid-mediated AMPK activation increased the glucose uptake and the phosphorylation of GSK3β, thereby enhancing glycogen synthesis. A future study will examine the expression of genes involved in the esterification of fatty acid in PA-treated HepG2 cells. The overall results suggest that citrus flavonoids have anti-adipogenic effects on PA-treated HepG2 cells via the AMPK signaling pathway. These findings offer a valuable clue for elucidating the mechanism of the anti-adipogenic effect of these flavonoids for the treatment of obesity.