FAS and SREBP-1c Inhibition via AMPK Activation in HepG2 Cells by Biovip, Tox-off, and Traphanoside GO1 from Glinus oppositifolius

Abstract

Glinus oppositifolius is an endemic herbaceous plant found in tropical Asian countries and is native in Vietnam. It is used in traditional folk medicine because of its flavor and antiseptic and laxative effects. In the current research, the effects of Tox-off, Biovip, and the purified compounds isolated from G. oppositifolius in the previous study were evaluated on the activation of adenosine 5′-monophosphate-activated protein kinase (AMPK)-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) in C2C12 myoblasts. In addition, the most potent active compounds, traphanoside-GO1 (TRA-GO1) and TRA-GO5 have validated the reduction of fatty acid synthase (FAS) and sterol regulatory element binding protein (SREBP)-1c in HepG2 cells. We found that Tox-off and Biovip significantly increased the phosphorylation of AMPK and ACC in C2C12 myoblasts. Furthermore, TRA-GO1 and TRA-GO5 significantly increased the AMPK activation and phosphorylation of its downstream substrate ACC in a concentration-dependent way compared to the dimethyl sulfoxide (DMSO) control. Besides, the protein level of FAS and SREBP-1c decreased by TRA-GO1 and TRA-GO5 in a concentration-dependent manner. Taken together, our results showed that the increased AMPK and ACC phosphorylation by active components of G. oppositifolius may activate the AMPK signaling pathways, which are useful for the anti-obesity and its related metabolic disorders.

INTRODUCTION

Glinus oppositifolius (GO) L. Aug. DC. (Molluginaceae) syn. Mollugo oppositifolia or Mollugo spergula, also known as “Rau đắng đất” in Vietnamese, is typically located in dry tropical regions. It is used as traditional medicine for the treatment of hepatitis, inflammation, fever, and wounds.¹⁾ Africans, Indians, and Filipinos use this bitter herb, which has a high content of iron and calcium, in daily meals.¹⁾ Recent studies have found that G. oppositifolius contains triterpenoid saponins, which are the main bioactive compounds,^2,3) as well as pectin polysaccharides,⁴⁾ flavonoids,⁵⁾ and steroids.⁶⁾ It has potential antioxidant,³⁾ hepatoprotective,⁴⁾ antiprotozoal,²⁾ and immunomodulating effects.⁷⁾

AMP-activated protein kinase (AMPK) plays a vital role in the maintenance of energy balance at the cellular and whole-body levels. It appears in the liver, skeletal muscle, brain, and fatty tissues. AMPK activation phosphorylates several cytoplasmic enzymes. The main substrates of AMPK are acetyl CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase.⁸⁾ HMG-CoA reductase is primarily responsible for the synthesis of ACC and HMG-CoA reductase, fatty acids, and cholesterol. ACC catalyzes the synthesis of malonyl-CoA from acetyl-CoA, which plays a major role in fatty acid synthase (FAS) in hepatocytes. AMPK activation (due to the phosphorylation of threonine molecule 172 on AMPK) inhibits ACC (due to phosphorylation of serin molecule 79 on ACC) reduces the concentration of malonyl-CoA in the cell and inhibits FAS.⁹⁾ Several studies have found that AMPK is the main protein kinase accountable for ACC inactivation, which decreases malonyl-CoA levels. It also increases the β-oxidation of fatty acids in organs like the liver and heart, and in skeletal muscle.^10–12)

The transcription factor, sterol regulatory element-binding protein 1 (SREBP-1), promotes FAS through increased expression of lipid biosynthetic genes, which plays a crucial role in liver metabolic disorders. AMPK phosphorylation inhibits SREBP-1c proteolysis and nuclear translocation, decreasing the expression of lipid pathogenic genes. Furthermore, its activation prevents fatty liver and hyperlipidemia, and accelerates atherosclerosis, partly through SREBP-1c phosphorylation.^13,14) Therefore, AMPK activators may be used for the treatment of metabolomic disorder diseases such as diabetes melitus, obesity, and liver failure.^15–19)

In the previous study, a new saponin, traphanoside GO1 along with eleven compounds were isolated from the aerial parts of G. oppositifolius.¹⁷⁾ However, the extracts and isolates have not been able to evaluate biological effects. To better understand the bioactive effects of GO on the anti-obesity and its related metabolic disorders, this study was carried out. Specifically, five characteristic substances (TRA-GO1–TRA-GO5) and two standardized extracts (Tox-off and Biovip) were examined for the effects on AMPK and ACC phosphorylation in C2C12 myoblasts. In addition, the effects of the most potential isolates (TRA-GO1 and TRA-GO5) were evaluated on SREBP-1c and FAS activation in HepG2 cells.

MATERIALS AND METHODS

Materials

The aerial parts of GO were provided by the Traphaco Joint Stock Company. The voucher specimen (GO-2021TJC) was deposited in the R&D Research Department of Traphaco Joint Stock Company (TJSC) and the Department of Analytical Chemistry and Standardization, National Institute of Medicinal Materials. The Biovip and Tox-off extracts were provided by the TJSC. The extracts containing TRA-GO1 (standardized for 2.83  ±  0.02% of Biovip extract and 2.24  ±  0.01% of Tox-off extract) obtained from the aerial parts of GO were evaluated for their effects on AMPK activation in C2C12 cells and FAS and SREBP-1c inhibition in human HepG2 hepatocytes.

Extraction and Isolation of Compounds from Tox-off Extract of GO

Tox-off extract (800 g) was suspended in 3.0 L water and separated sequentially using n-hexane, ethyl acetate, and n-butanol to obtain the corresponding extracts and a water layer.

The ethyl acetate extract (90.0 g) was separated by silica gel column chromatography (CC) by eluting with 100% dichloromethane, dichloromethane–methanol (9 : 1 to 7 : 3, v/v), and 100% methanol to obtain 10 fractions (E1–10). Fraction E6 (25.84 g) was subjected to silica gel CC and eluted using a gradient of dichloromethane–methanol (9 : 1 to 1 : 1, v/v), which obtained 10 sub-fractions (E6.1–6.10) and TRA-GO5 (50.0 mg). Fraction E6.4 (6.6 g) was applied to RP-C₁₈ CC and eluted with methanol–water (1 : 1 to 7 : 3, v/v) to obtain TRA-GO2 (110.0 mg). TRA-GO3 (80.0 mg) was obtained from fraction E6.6 (2.86 g) using an RP-C₁₈ CC and an eluent solvent system (methanol–water (1 : 1 to 3 : 2, v/v)). The isolation of fraction E6.8 (3.0 g) using silica gel CC and dichloromethane–methanol (9 : 1 to 1 : 1, v/v) provided E6.8.1–6.8.8. The isolation of fraction E6.8.5 (600.0 mg) using silica gel CC and dichloromethane–methanol (10 : 1, v/v) as the eluent system isolated TRA-GO1 (100.0 mg) and TRA-GO4 (10.0 mg).

The isolated compounds (TRA-GO1 to TRA-GO5) were identified respectively as traphanoside GO1 (TRA-GO1), spergulacin (TRA-GO2), spergulacin A (TRA-GO3), 3-O-(β-ᴅ-xylopyranosyl)-spergulagenin A (TRA-GO4), and vitexin (TRA-GO5) by comparing their NMR spectra with those of the reported compounds that we have published in the previous report.²⁰⁾

Qualitative Analysis of TRA-GO1 Content in Tox-off and Biovip Extracts Using HPLC-Diode Array Detector (DAD)

Reference Solution

TRA-GO1 was accurately weighed and dissolved in methanol in a volumetric flask to prepare a solution containing 1580 µg/mL as the reference solution. A series of concentrations of the reference solution (39.5–790 µg/mL) were used to construct a calibration curve.

Test Solution

In all, 200 mg powder and 100 mL 70% ethanol were accurately weighed and added to a round bottom flask, and maintained under reflux for 30 min. Cool, filter, and collect the filtrate in a separate flash, and weighed again. The loss of solvent was replenished with 70% ethanol and mixed. The filtrate was used as the test solution. Before injection, pass through a membrane filter of 0.45 µm or fine pore size, discarding a few ml of the filtrate.

HPLC-DAD analysis

A Shimadzu LC-30AD pump system (Shimadzu, Kyoto, Japan), equipped with a quaternary solvent system, an Autosampler SIL-30AC, and an SPD-M20A, was applied. Separation was performed using an Agilent C₁₈ column (250  ×  4.6 mm, 5 µm; Agilent, Santa Clara, CA, U.S.A.) at a temperature of 40 °C. The mobile phase consisted of acetonitrile and 0.1% phosphoric acid using a gradient elution of 0–10 min (28% can), 10–12 min (28–32% acetonitrile (ACN)), 12–20 min (32–36% ACN), 20–22.5 min (36–42% ACN), 22.5–24 min (42–100% ACN), and 24–27.5 min (100% ACN). The solvent flow rate was maintained at 0.8 mL/min. The column oven temperature was set at 40 °C. Detection was performed at a wavelength of 205 nm.

Calculation

The percentage of TRA-GO1 in the tested sample was calculated as:

  

where C is the concentration of the analyzed compound in the sample solution determined using the calibration curve (mg/mL), V is the volume of the sample solution (mL), m is the weight of the tested sample used to prepare the test solution (mg), and a is the moisture of the sample (%).

Cell Culture

The immortal C2C12 cell line is a subclone of the mouse myoblast cell line and HepG2 cells were isolated from human hepatocellular carcinoma, which were purchased from ATCC. These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 µg/mL streptomycin in a 5% CO₂ atmosphere at 37 °C. Cells have to be subcultured every three days.

Cell Viability Assay

C2C12 cells were grown in 96-well plates at an initially seeded cell density of 2  ×  10³ cells/well. The cells were incubated with extracts (Biovip and Tox-off at 1, 3, and 10 µg/mL) and compounds TRA-GO1–TRA-GO5 (1, 3, and 10 µM) for 24 h. The cytotoxicity of tested samples was determined by adding 40 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (1 mg/mL) to each well and incubating for 1–2 h. After that, 100 µL of dimethyl sulfoxide (DMSO) was used to dissolve formazan crystals and absorbance was measured at 550 nm using a Microplate Reader. The cell viability percentage was calculated based on the absorbance of the cells treated with the test sample compared with the absorbance of the untreated control cells.

Immunoblotting Analysis

C2C12 and HepG2 cells were incubated with samples (30 µg/mL for extracts and 3, 10, and 30 µM for isolated substances) for 2 h for pAMPK (#2531, Cell Signaling, Danvers, MA, U.S.A.), pACC (#366, Cell Signaling), and 6 h for FAS (#3189, Cell Signaling), SREBP-1c (#MBS8529268, MyBioSource, San Diego, CA, U.S.A.) testing. Cells were washed in ice-cold phosphate-buffered saline (PBS) and lysed in 100 µL lysis buffer [60 mM Tris–HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol] for 30 min. The cell lysate was boiled for 5 min (100 °C) and centrifuged for 30 min at 12000 rpm at 4 °C in a microcentrifuge. After that, their proteins were determined using the BCA protein assay kit (Pierce, Rockford, IL, U.S.A.), run on 10% SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto nitrocellulose membranes. These membranes were then incubated with primary antibodies (p-AMPK 1 : 1000, p-ACC 1 : 1000, FAS 1 : 100, SREBP-1c 1 : 1000, and β-actin 1 : 5000). The membranes were continuously incubated with secondary anti-mouse or anti-rabbit antibodies (1 : 4000). Finally, protein bands were detected using enhanced chemiluminescence (ECL) Western detection West Femto Kit (Thermo Scientific, Waltham, MA, U.S.A., Catalog: #34096) and the LI-COR machine (Lincoln, NE, U.S.A.) was used to capture and analyze images.

Statistical Analysis

Quantitative data are presented as the mean  ±  standard error of the mean (SEM) Data were analyzed by Student’s t-tests or one-way ANOVA for multiple comparisons with Šídák's multiple comparisons test using GraphPad Prism Software 5 (GraphPad Prism, San Diego, CA, U.S.A.). The difference was considered significant when p < 0.05.

RESULTS AND DISCUSSION

Qualitative Analysis of TRA-GO1 in Tox-off and Biovip Extracts

Figure 1 shows the high-performance liquid chromatograms of TRA-GO1, Tox-off, and Biovip extracts, as well as their combinations. The TRA-GO1 content (%, g/100 g) in Biovip and Tox-off extracts was 2.83 and 2.24%, respectively.

Fig. 1. High-Performance Liquid Chromatograms of TRA-GO1, Biovip and Tox-off Extracts

The retention time of TRA-GO1 peak appeared to be at 22.5 min. HPLC chromatogram of (A). TRA-GO1; (B). Biovip; (C). Tox-off; (D): combined HPLC chromatograms of TRA-GO1, Biovip, and Tox-off.

The Effects of Extracts and Purified Compounds on AMPK and ACC Phosphorylation in C2C12 Cells

The in vitro cytotoxic assay was set up to evaluate the activities of two extracts of G. oppositifolius (Biovip and Tox-off; 1, 3, 10, and 30 µg/mL) on C2C12 cell survival. The survival of C2C12 cells was 80–100% (Fig. 2) after 24 h incubation with Biovip and Tox-off extracts at a concentration of 30 µg/mL. Therefore, the effects of two extracts (Biovip and Tox-off) at 30 µg/mL on AMPK and ACC phosphorylation were evaluated in C2C12 cells. Figure 3 presents the effects of two extracts (Biovip and Tox-off) on phospho-ACC and phospho-AMPK protein expression in C2C12 cells after 2 h incubation. In addition, the phospho-ACC and phospho-AMPK levels were compared to those after control DMSO treatment. Biovip (30 µg/mL) treatment significantly increased the levels of ACC and AMPK phosphorylation in C2C12 cells compared to the Tox-off treatment. Previously, we isolated five compounds (TRA-GO1–TRA-GO5) from the aerial part of G. oppositifolius²⁰⁾ (Fig. 4).

Fig. 2. The Effects of Biovip, Tox-off, TRA-GO1–TRA-GO5 in Dose-Dependent on C2C12 Cells Viability for 24 h by Using MTT Assay

* p < 0.05; ** p < 0.01; compared to DMSO control group.

Fig. 3. Effects of AMPK and ACC Pathway Activation in C2C12 Cells by Biovip and Tox-off Extracts at 30 µg/mL after Incubation for 2 h

(A) The cells were then lysed and protein-electrophoresed with antibodies against phosphorylated-AMPK, phosphorylated-ACC, and β-actin. (B, C) Quantification of AMPK and ACC phosphorylation in (A) by ImageJ. * p < 0.05; ** p < 0.01; *** p < 0.001, compared to DMSO control group.

Fig. 4. Chemical Structures of Compounds TRA-GO1–TRA-GO5

The MTT assay was used again to determine the cytotoxic effects of these compounds in C2C12 cells. The cell survival was 80–100% after incubation with 10 µM these compounds for 24 h (Fig. 2). The bioactivity tests showed that a compound concentration of 10 µM showed considerable effects on the phosphorylation of AMPK and ACC in C2C12 cells. Therefore, the effects of compounds TRA-GO1–TRA-GO5 on the phosphorylation of AMPK and ACC proteins were examined at a concentration of 10 µM.

As shown in Fig. 5, TRA-GO1 and TRA-GO5 exhibited the greatest increase in the phosphorylation of AMPK and ACC proteins compared to the DMSO control; meanwhile, TRA-GO2 and TRA-GO3 showed a moderate activation effect and TRA-GO4 showed a negligible effect. TRA-GO5 is a well-known flavonoid, vitexin, which increases AMPK activation in 3T3-L1 adipocytes at 10–50 µM.²¹⁾ Vitexin was used as the positive control for the AMPK activator in previous in vivo and in vitro studies.^22–24) We recently identified TRA-GO1 as a saponin compound, named traphanoside GO1.²⁰⁾ TRA-GO1 is typically found in G. oppositifolius and is a marker for the qualitative analysis of Tox-off and Biovip extracts.

Fig. 5. The Effects of AMPK and ACC Pathway Activation in C2C12 Cells by Purified Compounds at 10 µM after Incubation for 2 h

(A) The cells were then lysed and protein-electrophoresed with antibodies against phosphorylated-AMPK, phosphorylated-ACC, and β-actin. (B, C) Quantification of AMPC and ACC phosphorylation in (A) by ImageJ. * p < 0.05; ** p < 0.01; *** p < 0.001, compared to DMSO control group.

The effects of different doses of TRA-GO1 and TRA-GO5 on AMPK and ACC activation were evaluated to determine the dose-response relationship. Incubation of C2C12 cells in 3, 10, and 30 µM TRA-GO1 and TRA-GO5 for 2 h confirmed concentration dependent effects on AMPK and ACC phosphorylation. TRA-GO1 and TRA-GO5 significantly increased the AMPK and ACC phosphorylation at 10 and 30 µM compared to the DMSO control in a concentration-dependent way (Fig. 6). These data suggest strong effects of TRA-GO1 and TRA-GO5 on the activation of AMPK and ACC in C2C12 cells.

Fig. 6. The Concentration-Dependent Activities of AMPK and ACC Pathway Activation in C2C12 Cells by Purified TRA-GO5 (A–C) and TRA-GO1 at 3, 10, and 30 µM (D–F) after Incubation for 2 h

The cells were then lysed and protein-electrophoresed with antibodies against phosphorylated-AMPK, phosphorylated-ACC, and β-actin (A, D). (B, C, E, F) Quantification of AMPK and ACC phosphorylation in (A, D) by ImageJ. * p < 0.05; ** p < 0.01; *** p < 0.001, compared to DMSO control group.

Two upstream kinases have been reported to activate AMPK in in vitro are the tumor suppressor The serine/threonine kinase (LKB1) and Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK).^25,26) The LKB1 has reported a novel signaling pathway linking cellular metabolism to growth control, cell polarity, and the tumor suppressor. Although AMPK is not the only substrate of LKB1 and LKB1 also phosphorylates the activation loop of 12 other kinases related to AMPK, also resulting in their activation.^27,28) However, only AMPKα1 and AMPKα2 of these LKB1-dependent kinases, are activated under low ATP conditions.²⁹⁾ One of the major downstream signaling pathways of AMPK is the mammalian target-of-rapamycin (mTOR) pathway. Like AMPK, the mTOR serine/threonine kinase plays key roles in cell growth, cell cycle, cell survival, autophagy, metabolism, and cell proliferation. mTOR forms two functional complexes, mTORC1, and mTORC2.³⁰⁾ mTORC1 exists as a multiprotein complex containing mTOR, Raptor, mLST8 (GβL), and PRAS40. mTORC1 is shown to be acutely sensitive to rapamycin and regulated by nutrients status and AMPK whereas mTORC2 is not.^31,32) The studies demonstrated that the mTOR and AMPK pathways are linked comes from the study of protein-binding eukaryotic promoter S6 kinase (S6K) and 4E-1 (4EBP1), which are upstream targets of mTOR. AMPK regulates the activities of both S6K and 4EBP1.^33,34) Moreover, the phosphorylation and activity of S6K and 4EBP1 are widely used as indicators for signaling via the mTOR pathway, suggesting convergence of the AMPK and mTOR signaling pathways. Thus, a further study of the signaling pathway targets for AMPK activation by TRA-GO1 and TRA-GO5 will aim to clarify the mechanism of those compounds.

Reduction of FAS and SREBP-1c Expression by Purified Compounds in HepG2 Cells

The sterol regulatory element-binding proteins (i.e., SREBP-1c) regulate lipid biosynthesis by modulating the genes related to FAS, including fatty acid synthase and ACC.³⁵⁾ A previous study showed that AMPK inhibits cleavage and transcriptional activation of SREBP via direct phosphorylation.²¹⁾ Therefore, inhibition of adipose tissue formation may be useful for the treatment and prevention of obesity and related disorders. To confirm the effects of TRA-GO1 and TRA-GO5 in dose-dependent on AMPK and ACC activation in HepG2 cells, 3, 10, and 30 µM TRA-GO1 and TRA-GO5 were incubated with HepG2 cells for 2 h. The AMPK and ACC phosphorylation were measured. As shown as Fig. 6, both TRA-GO1 and TRA-GO5 significantly increased the AMPK and ACC phosphorylation at 10 and 30 µM compared to the DMSO control (Fig. 7).

Fig. 7. The Concentration-Dependent Activities of AMPK and ACC Pathway Activation in HepG2 Cells by Purified TRA-GO5 (A–C) and TRA-GO1 at 3, 10, and 30 µM (D–F) after Incubation for 2 h

The cells were then lysed and protein-electrophoresed with antibodies against phosphorylated-AMPK, phosphorylated-ACC, and β-actin (A, D). (B, C, E, F) Quantification of AMPK and ACC phosphorylation in (A, D) by ImageJ. * p < 0.05; ** p < 0.01; *** p < 0.001, compared to DMSO control group.

Sekiya et al.³⁶⁾ showed that FAS and SREBP-1c are increased by high glucose levels and T0901317 in primary hepatocytes and adipocytes. To further investigate the effects of TRA-GO1 and TRA-GO5 on FAS and SREBP-1c levels in HepG2 cells, the cells were incubated with high levels of glucose (30 mM) and T0901317 (10 µM) for 12 h. Then the cells were stimulated with TRA-GO1 or TRA-GO5 at 3, 10, or 30 µM for 6 h. As shown in Fig. 8, FAS and SREBP-1c were significantly decreased in a concentration-dependent way by TRA-GO1 and TRA-GO5 at 10–30 µM compared to the control (30 mM glucose + T0901317 10 µM). Furthermore, 3 µM TRA-GO5 did not affect the FAS protein level but significantly decreased the SREBP-1c protein level (p < 0.05). By contrast, 3 µM TRA-GO5 did not affect the SREBP-1c protein level but significantly inhibited the FAS protein level (p < 0.001).

Fig. 8. The Dose-Dependent Effects of FAS and SREBP-1c Pathway Inhibition in HepG2 Cells by Purified Compounds TRA-GO5 (A–C) and TRA-GO1 at 3, 10, and 30 µM Concentrations (D–F)

The cells were first incubated with high glucose 30 mM and T0901317 for 12 h and stimulated with compounds for 6 h. The cells were then lysed and protein-electrophoresed with antibodies against FAS, SREBP-1c, and β-actin (A, D). (B, C, E, F) Quantification of FAS and SREBP-1c in (A, D) by ImageJ. * p < 0.05; ** p < 0.01; *** p < 0.001, compared to glucose 30 mM and T0901317 group.

In both C2C12 and HepG2 cell lines, we observed the effects of TRA-GO1 and TRA-GO5 on AMPK- and ACC-phosphorylation were not yet saturated, even at 30 µM. However, the effect of TRA-GO1 and TRA-GO5 on the expression of SREBP-1c was saturated at 3–30 µM. These data may indicate AMPK signaling pathway is one of the pathways that regulate FAS and SREBP-1c by TRA-GO1 and TRA-GO5.

In summary, this study showed that although Biovip prepared by a different extract method than Tox-off but Biovip possessed a high content of TRA-GO1 and significantly increased the levels of ACC and AMPK phosphorylation as compared to the Tox-off. In addition, Biovip extract has advantages over Tox-off extract, such as being less expensive and can be upgraded to industrial scale in case of future product development orientation. Therefore, further studies in animal models and pre-clinical trials might be focus on Biovip extract.

Taken together, our results demonstrate the effects of the purified compounds (TRA-GO1 and TRA-GO5) obtained from G. oppositifolius on SREBP-1c and FAS suppression in HepG2., suggesting that G. oppositifolius may be used for the prevention and treatment of obesity and related metabolic disorders via activation of the AMPK and other target signaling pathways.

Acknowledgments

This work was financially sponsored by Traphaco Joint Stock Company under Grant 01-2022/HĐNCKH-RĐĐ signed 20/4/2022 for “Research procedure to establish a reference compound and evaluate for the reduction of hepatitis, hepatic steatosis, and diabetes of Glinus oppositifolius extracts and its marker compounds”.

Conflict of Interest

The  authors  declare  no  conflict  of  interest.

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