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Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2022 Apr 4;60(3):1015–1025. doi: 10.1007/s13197-022-05436-x

Resveratrol butyrate esters inhibit lipid biosynthesis in 3T3-L1 cells by AMP-activated protein kinase phosphorylation

Ming-Kuei Shih 1, Shu-Ling Hsieh 2, Yu-Wen Huang 2, Anil Kumar Patel 2,3, Cheng-di Dong 2, Chih-Yao Hou 2,✉
PMCID: PMC9998790  PMID: 36908355

Abstract

Resveratrol butyrate esters (RBEs), which are novel resveratrol-synthesized derivatives, exhibit increased biological activity. This study elucidated the effect of RBEs on fat metabolism and their anti-obesity characteristics. Their molecular mechanism was investigated in the 3T3-L1 murine preadipocyte cells and adipocytes. RBE doses of < 2 μM did not induce a significant change in the viability of 3T3-L1 adipocytes. After RBEs treatment, intracellular lipid droplet accumulation in 3T3-L1 adipocytes was stimulated by methylisobutylxanthine, dexamethasone, and insulin-containing medium. However, a significant dose-dependent reduction in intracellular lipid levels was observed. The mRNA levels of two adipogenic transcription factors (peroxisome proliferator-activated receptor [PPAR] and CCAAT/enhancer-binding proteins [C/EBP]) and lipogenic proteins (fatty acid-binding protein 4 [FABP4] and fatty acid synthase [FAS]) were significantly attenuated by RBE treatment in both MDI-stimulated and differentiated 3T3-L1 adipocytes. Moreover, the phosphorylation level of adenosine monophosphate-activated protein kinase (AMPK) also dramatically increased in the MDI + RBE-treated group compared to that in the MDI + vehicle-treated group. Collectively, our study provides strong evidence that RBEs inhibit adipogenesis by regulating adipogenic protein expression and increasing the p-AMPK/AMPK ratio. Future studies will be conducted on animal models to validate the application of RBEs as a functional food ingredient in improving human health.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-022-05436-x.

Keywords: Resveratrol butyrate ester; Lipogenesis; Isobutyl-methylxanthine, dexamethasone, and insulin; 3T3-L1 adipocyte; Adenosine monophosphate kinase

Introduction

Obesity is a multifaceted chronic condition resulting in detrimental health conditions such as diabetes mellitus type 2 (DM2), coronary thrombosis, malignancy and osteoarthritis (Kopelman 14). Differentiation of 3T3-L1 cells is an efficient and convenient method to obtain adipocyte-like cells for experimental studies. It has also been used to evaluate the cellular mechanisms of bioactive compounds with potential anti-lipogenic herbs (Guru et al. 9). Adipogenesis is the transformation of preadipocytes into adipocytes. The CCAAT/enhancer-binding protein (C/EBP) gene family and peroxisome proliferator-activated receptor (PPAR) are two transcription factors that mutually enhance the expression of the other and maintain the differentiated state (Rosen et al. 24). Additionally, adenosine monophosphate-activated protein kinase (AMPK), a critical enzyme in energy metabolism, can function as a cellular sensor in adipocytes by regulating various lipid metabolism-related variables (Hardie 10). Recent studies have shown that AMPK can limit adipogenesis and suppress the expression of sterol regulatory element-binding protein (SREBP)-1c, PPAR, and fatty acid synthase (FAS) in adipocytes (Day et al. 5; Herzig andShaw 11).

Resveratrol (RSV) inhibits PPARγ and C/EBPαβ induction in adipocytes (Floyd et al. 7). RSV may activate AMPK in differentiated 3T3-L1 adipocytes (Bruckbauer and Zemel 2) or proliferating preadipocytes (Mitterberger andZwerschke 19); however, little is known about its adverse effects, especially in preadipocytes undergoing differentiation. RSV is limited by its low bioavailability in vivo, and some reports have demonstrated that esterification may increase its bioactivity (Intagliata et al. 12). We reported that the esterification of RSV with butyrate produced RSV, resveratrol butyrate esters (RBE) mono-, RBE di-, and RBE tri-esters with enhanced H2O2-scavenging activity than RSV (Tain et al. 27). In our previous reports (Tain et al. 26, 27; Liao et al. 16; Shih et al. 25), RBEs esterified with butyric acid and resveratrol reduced lipid oxidation (Tain et al. 27) and fat accumulation (Tain et al. 26). In addition, RBEs can reduce obesity in female Sprague Dawley (SD) rats (Shih et al. 25). Since RBEs can significantly alter the gut microbiota (reduced Firmicutes/Bacteroidetes (F/B) ratio) in female SD rat offspring, we speculate that bisphenol A (BPA) affects the slow body fat metabolism of female offspring and causes obesity. RBEs adjusted the gut microbiota of the female offspring group to normalize the lipid metabolism. Further studies are required to explore the related physiological and metabolic pathways. Liao et al. (16) reported that RBEs also reduce liver damage in male SD rats (Liao et al. 16). Observations of antioxidant gene mRNA levels, antioxidant enzyme activities, angiogenesis, and immunohistochemical (IHC) staining of nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) distribution in liver tissue sections indicated that RBEs enhanced the expression of all selected genes and effectively attenuated extramedullary hematopoiesis and mononuclear cell infiltration. RBEs reduced fat accumulation in HepG2 cells by regulating acetyl-CoA carboxylase (ACC) and SREBP-1c, and this effect was more potent than that of RSV at the same concentration (Tain et al. 26). These results suggest that RBEs may serve as potential anti-fat accumulation agents in functional food ingredients, additives and supplements to improve health. However, the mechanism of action in adipocytes remains unclear. Thus, this study aimed to elucidate the influence of RBEs on fat metabolism, their anti-obesity effects, and the molecular mechanisms that govern the biological activity of RBEs.

Materials and methods

Chemicals and reagents

Dulbecco’s modified Eagle’s high-glucose medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and trypsin were purchased from Gibco (Waltham, MA USA A). Sodium bicarbonate and phosphate-buffered saline (PBS) were purchased from Uni-onward (Taipei, Taiwan). 3-Isobutyl-methylxanthine (IBMX), dexamethasone (DEX), insulin, 3-(4,5-Dimethyazol-2-yl)-2,5-Diphenyltetrazolium bromide (MTT), Oil Red O, formalin, Triton X-100, TRIzol reagent, chloroform, sodium dodecyl sulfate (SDS), ammonium persulfate (APS), glycerol, β-mercaptoethanol, and Tween-20 were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Isopropanol and Tris-base were purchased from J. T. Baker (Phillipsburg, New Jersey, USA). Commercial total triglycerides (TR212) kits were purchased from RANDOX (Crumlin, County Antrim, UK). Chemiluminescence Kit was purchased from GE Healthcare (Los Altos, CA, USA). Glycine, Tris–HCl, and tetramethylethylenediamine (TEMED) were purchased from Amresco (Boise, ID, USA). Acrylamide was purchased from Bio-Rad Laboratories, Inc. (Hercules, California, USA). Oligo dT, M-MLV reverse transcriptase, and RNase were purchased from Promega (Madison, Wisconsin, USA). Primer and 2X SYBR Frist MM were purchased from Topgen (Kaohsiung, Taiwan). AMPKα antibody, phospho-AMPKα (Thr172), and β-actin were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Synthesis of resveratrol butyrate ester

RBE was synthesized according to our previously study (Tain et al. 27). The novel RBEs, including RSV (approximately 17.1%), RBE monoester (47.1%), RBE diester (35.0%) and RBE tri-ester (less than 1%), were produced by the esterification of RSV and butyric acid(Tain et al. 27).

Cell culture and adipocyte differentiation

The 3T3-L1 cell line is a well-characterized and reliable model for studying the conversion of preadipocytes into adipocytes (Guru et al. 9). 3T3-L1 preadipocytes from the Bioresource Collection and Research Center (Hsinchu, Taiwan) were cultivated at 37 °C in a humidified environment of 5% CO2 in DMEM with FBS (10%), penicillin/streptomycin (1%, 100 U/mL), and sodium bicarbonate (3.7 g/L). 3T3-L1 cells were cultured for 24 h (0 days) on 3 cm plates (5 × 104 cells) and subsequently treated for 10 days with various RBE dosages. On days 2–4, the cells were supplemented with MDI (containing 3-isobutyl-methylxanthine (0.5 mM), dexamethasone (1 mM), and insulin (10 g/mL)). The cells were grown with only insulin-containing (10 g/mL) medium during days 5–8 after MDI stimulation (day 4). After an additional four days of incubation in the aforementioned condition (day 8), the cells were grown in DMEM for two days. By day 10, complete differentiation had occurred.

Cell proliferation assay

The dose selection for RSV and RBEs was based on previous studies with modifications (Tain et al. 2020). 3T3-L1 preadipocytes were incubated for 24 h on 3 cm plates (1 × 105 cells) and subsequently treated for 72 h with 1, 2, 5 and 10 μM RBEs. The cell proliferation experiment was performed by pooling the cultivated cells, washing them twice with PBS and incubating them for 3 min at 37 °C and 5% CO2 in a trypsin-containing solution. The cells were then placed in a DMEM medium and centrifuged for 3 min at 201 × g and 25 °C in a 1.5 mL Eppendorf tube. The supernatant was then removed and mixed with PBS at 4 °C. Preadipocyte 3T3-L1 cells were suspended in 20 μL of PBS with 1 μL of solution-13 (containing 286 μM 4′,6-diamidino-2-phenylindole [DAPI] and 80 μM acridine orange stains). A NucleoCounter® NC-3000™ fluorescence image cytometer (Chemometec, Allerod, Denmark) was employed for analysis using an 8-chamber NC-Slides A8™.

Cell cycle assay

3T3-L1 preadipocytes were incubated for 24 h on 3 cm plates (1 × 105 cells) and subsequently treated for 72 h with 1, 2, 5 and 10 M RBEs. The cell cycle experiment was performed by pulling cultivated cells, washing them twice with PBS and incubating for 3 min at 37 °C and 5% CO2 in a trypsin-containing solution. The cells were then placed in DMEM and centrifuged for 3 min at 201 × g and 25 °C in a 1.5 mL tube. Cells were counted and thoroughly resuspended to a final count of approximately 1 × 106–2 × 106 cells in 0.5 ml PBS. Cell suspensions were transferred in ice-cold ethanol (70%)-containing tubes, vortexed vigorously, and then maintained in the fixative for at least 2 h. The ethanol-suspended cells were centrifuged for 5 min at 201 × g. The ethanol was thoroughly decanted. The cell pellet was then suspended in 5 ml PBS, incubated for 50 s, and then centrifuged for 5 min at 201 × g. The cell pellet was resuspended in 0.5 mL solution-3 (1 µg/ml DAPI, 0.1% Triton X-100 in PBS) and incubated for 5 min at 37 °C. The supernatant was then removed and mixed with PBS at 4 °C. 3T3-L1 preadipocytes were suspended in 20 μL of PBS with 1 μL of solution-3. A NucleoCounter® NC-3000™ fluorescence image cytometer (Chemometec, Allerod, Denmark) was employed for analysis using an 8-chamber NC-Slides A8™.

Lipid accumulation assay

Oil red O staining assay

During the differentiation process, cells were stained every 2 days with Oil Red O in 60% isopropanol. The cells were gently rinsed twice with 1 mL PBS before incubation in formalin (10%) for 10 min. After staining in Oil Red O for 30 min, the solution was withdrawn and the plates were washed once with water. A microscope (Olympus, Tokyo, Japan) was used to examine the stained lipid droplets. After 15 min, isopropanol was added, and the mixture was shaken for 15 min. The optical density (OD) was measured at 510 nm. Inhibition was reported as the percentage of viable cells compared to that in control.

Triglyceride deposition assay

The cells were washed twice in PBS to determine the number of cellular triglycerides (TG), then scraped into a 1.5 mL tube with 70 µL of 0.5% Triton X-100 in PBS and stirred for 30 s using a sonicator (Qsonica, Connecticut, USA) (frequency: 20 kHz; power rating: 125 watts; and amplitude: 80%). The TG content of the cell samples was evaluated using an assay kit (Triglycerides assay kit TR212, Randox) and the protein concentration was assessed using the Lowry assay (Pierce™ Modified Lowry Protein Assay Kit, 23,240, Thermo). The findings are expressed in milligrams of triglycerides per milligram of cellular protein. Staining was performed using Harris hematoxylin and eosin. Stained slides were examined under a microscope (Olympus, Tokyo, Japan).

Western blot analysis

The cells were washed twice with PBS, and 70 μL of PBS (containing 10 mmol/L potassium phosphate, 150 mmol/L potassium chloride, and 1 mM PMSF, pH 7.4) was added. The cells were then scraped into a 1.5 mL tube and stirred for 30 s using a sonicator (Qsonica, Connecticut, USA) (frequency: 20 kHz; power rating: 125 watts; and amplitude: 80%). Protein concentrations of the cell homogenates were determined using the Lowry assay. Subsequently, 10–20 μg of total protein from each cell sample was loaded onto a 10% SDS gel, resolved by 10% SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes. These membranes were then incubated with anti-AMPK (1:1000) and anti-pAMPK (1:1000) antibodies at 24 °C for 12 h. They were subsequently incubated with a peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibody (1:10,000) at 37 °C for 1 h. PVDF signals were visualized using an enhanced chemiluminescence detection kit (GE Healthcare). The blots were treated with an enhanced chemiluminescence reagent for densitometry analysis and exposed using a ChemiDoc XRS + system (Bio-Rad Laboratories, Inc.).

mRNA expression

Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions. The total RNA was quantified using an Epoch microvolume spectrophotometer (Agilent, Santa Clara, USA). RNase and Moloney murine leukemia virus (M-MLV) reverse transcriptase were used to transcribe poly (A) + RNA using oligo-dT primers for first-strand complementary DNA (cDNA) synthesis by reverse transcription (RT). SYBR Green was used for qPCR analysis. Real-time PCR was used to measure the relative gene expression (LightCycler® 96 Real-Time PCR System, Roche Life Science, Basel, Switzerland). For all experiments, the PCR cycling parameters were denaturation at 95 °C for 120 s, primer annealing and elongation continued for 40 cycles sequentially at 95 °C for 5 s, then 60 °C for 30 s. The PCR cycle was as follows: denaturing at 95 °C for 10 s, cooling to 65 °C for 60 s, and then heating to 97 °C. Specific primers were used to amplify the cDNA. The threshold cycle (Ct) values of the target genes were standardized to the Ct values of β-actin. Fold changes in gene expression were calculated using the comparative Ct technique. The control group for the gene-specific primers was β-actin, and the target genes were PPAR, C/EBP, fatty acid-binding protein 4 (FABP4) and FAS (Table. S1).

Statistical analysis

All experiments were repeated in quadruplicate, and the data are presented as the mean ± SD. Data were analyzed by the SPSS statistical analysis software for Windows, version 20.0 (SPSS, Inc., Chicago, Illinois, USA). Tukey’s test was used to evaluate the significance of the differences between the two mean values. A p-value of < 0.05 was considered statistically significant.

Results

Effect of RBEs on cell proliferation in 3T3-L1 preadipocytes

the viability of 3T3-L1 adipocytes was assessed following exposure to 1, 2, 5 and 10 μM RSV and RBEs to assess the cell proliferative ability of these compounds. All the treated groups maintained a constant level of viability. After 48 h of culture, the samples showed decreased cell proliferation at higher concentrations. The results showed that the RSV at 1, 2, 5 and 10 μM significantly reduced the total cell number after 72 h (Fig. 1A), with 10 μM demonstrating the most significant reduction, reaching 25% and 28% at 48 h and 72 h, respectively. The RBEs at 2, 5, and 10 μM considerably reduced the total cell number after 48 and 72 h (Fig. 1B), with 10 μM registering the most significant reduction, reaching 28% and 32% at 48 h and 72 h, respectively.

Fig. 1.

Fig. 1

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Effect of RSV or RBEs on cell proliferation in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were treated or non-treated with 1, 2, 5, 10 μM RSV A or RBEs B for 72 h. The significance of difference in cell proliferation was evaluated by Duncan’s test. Data are the means ± SD (n = 4). Different letters (a–d) indicate significant differences among the different concentrations of the RSV or RBEs group (p < 0.05)

Effect of RE or RBEs on cell cycle in 3T3-L1 preadipocytes

To prove the effect of RSV or RBEs on mitosis, the cell cycle was analyzed using an NC-3000 cytometer. The MTT assay results suggested that high RBE concentrations might affect the growth of 3T3-L1 preadipocytes(Fig. 1).

DAPI was used to stain the nucleic acids to observe the amount of DNA in the cells. In normal cells, there are two sets of chromosomes (2 N) in the G0/G1 phase of the cell cycle, four sets of chromosomes (4 N) in the G2/M phase, and an S phase between the aforementioned phases. When cells undergo apoptosis, DNA is fragmented, and chromosomes are reduced to less than 2 N. Therefore, this study utilized DAPI staining to label the DNA in cells. Using flow cytometry, the percentage of apoptosis and cell cycle changes was analyzed and the peak appeared before the G0/G1 phase, which was Sub-G1. The percentage of apoptotic cells and alterations in the cell phase was confirmed.

The amount of 3T3-L1 preadipocyte DNA in the G0/G1 phase was higher than that in the control group (p < 0.05). In the S phase and G2/M phase, the amount of DNA was similar or lower (p < 0.05) (Fig. 2). This indicates that 1, 2, 5 and 10 μM RSV (Fig. 2A, B) as well as RBEs (Fig. 2C, D) blocked the cell cycle, especially at the G0/G1 transition at 24 h and 48 h. The data obtained at the G0/G1 phase showed that the DNA amounts were comparable (with approximately 5% difference) under different treatment conditions, except for that at 10 μM RBE treatment (24 h), which registered approximately 13% higher difference in the DNA amount compared to that in control (Fig. 2C).

Fig. 2.

Fig. 2

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Effect of RSV or RBEs on cell cycle in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were treated with 1, 2, 5, 10 μM RSV for 24 (A), 48 h(B) and 1, 2, 5, 10 μM RBEs for 24 (C) and 48 h(D). The significance of difference in cell cycle was evaluated by Duncan’s test. Data are the means ± SD (n = 4). Different letters (a–c) indicate significant differences among the different concentrations of the RSV group (p < 0.05)

Effect of RBEs on lipid accumulation in 3T3-L1 adipocytes

Adipocyte differentiation involves whole-cell changes and is triggered by coordinated signaling by growth factors, cytokines, and hormones. To investigate the inhibitory effect of the samples on lipid accumulation, intracellular lipid droplets were measured by Oil Red O staining of MDI-stimulated 3T3-L1 adipocytes treated with RSV or RBEs for 10 days. The extent of Oil Red O staining increased with the increased accumulation of numerous large intracellular droplets in the MDI-stimulated mature 3T3-L1 adipocytes. Cell lipid accumulation levels were measured by staining with Oil Red O and were observed under a microscope (200 ×). The images are representative of the results from the final day.

The cells were incubated for 0–10 days with 1, 2, 5 and 10 μM RSV or RBEs. A change in adipocyte differentiation was observed after 10 days (Fig. 3A, Fig. 4A). In Oil Red O-stained cells, RSV at 5 and 10 μM significantly reduced intracellular lipid content on days 6, 8 and 10 (p < 0.05) compared to that in the positive control (MDI + vehicle treatment) (Fig. 3B). RBEs at 2, 5 and 10 μM significantly reduced intracellular lipid content on days 6, 8, 10 (p < 0.05) (Fig. 4B). Upon treatment with 10 μM RSV, the ability to inhibit MDI-induced fat accumulation was 7%, 8% and 30% on days 6, 8 and 10, respectively. Upon treatment with 10 μM RBEs, the ability to inhibit MDI-induced fat accumulation was 10%, 16% and 32%, respectively. TG accumulation assays suggested that 5 and 10 μM RSV significantly reduced intracellular TG content on days 8 and 10 (p < 0.05) compared to that in the positive control (MDI + vehicle treatment) (Fig. 3C). RBEs (2, 5 and 10 μM) significantly reduced intracellular TG content on days 4, 6, 8 and 10 (p < 0.05), respectively (Fig. 4C). Upon treatment with 10 μM RSV, the TG levels that reduced MDI-induced fat accumulation were 0.38 mg, 0.43 mg, and 0.55 mg on days 6, 8 and 10, respectively, and that upon treatment with 10 μM RBEs were 0.53 mg, 0.54 mg, and 0.63 mg, respectively.

Fig. 3.

Fig. 3

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Effect of RSV on lipid accumulation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated in MDI medium containing 1, 2, 5, 10 μM RSV for 10 days. Cells were collecting every two days. A Cell lipid accumulation levels were measured by staining with Oil Red O and observed under the microscope (200)-picture only shows the final day. B 3T3-L1 adipocytes relative amount of control lipid accumulation (%). C Calculation followed by TG concentration (mg TG/mg protein) = mg TG/mg protein. Control: undifferentiated cells. The significance of difference in lipid accumulation evaluated by Turkey’s test. Data are the means ± SD (n = 4). Different letters (a–d) indicate significant differences among the different concentrations of the RSV group (p < 0.05)

Fig. 4.

Fig. 4

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Effect of RBEs on lipid accumulation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated in MDI medium containing 1, 2, 5, 10 μM RBEs for 10 days. Cells were collecting every two days. A Cell lipid accumulation levels were measured by staining with Oil Red O and observed under the microscope (200)-picture only shows the final day. B 3T3-L1 adipocytes relative amount of control lipid accumulation (%). C Calculation followed by TG concentration (mg TG/mg protein) = mg TG/mg protein. Control: undifferentiated cells. The significance of difference in lipid accumulation evaluated by Turkey’s test. Data are the means ± SD (n = 4). Different letters (a–d) indicate significant differences among the different concentrations of the RBEs group (p < 0.05)

Overall, the lipid accumulation test results showed that from days 6–10, the positive control group (MDI + vehicle treatment) showed enhanced Oil red O staining (Fig. 3B, Fig. 4B). The TG content (Fig. 3C, Fig. 4C) was significantly higher than that in the control group (p < 0.05). However, lipid accumulation significantly decreased in a dose-dependent manner after combined treatment with RSV or RBEs. Notably, there was no difference compared to the lipid accumulation levels in the control group (p > 0.05). Collectively, these results indicate that RBEs suppress the MDI-stimulated increased accumulation of lipid droplets in the 3T3-L1 adipocytes.

Effect of RBEs on mRNA expression of lipid metabolism-related genes in 3T3-L1 adipocytes

To examine the inhibitory effects of the samples on the expression of adipogenic and lipogenic factors, the mRNA levels of two transcription factors (PPAR and C/EBP) and two lipogenic proteins (FABP4 and FAS) were evaluated in MDI-stimulated 3T3-L1 adipocytes after exposure to RSV or RBEs for 10 days. The mRNA expression of the two transcription factors was higher in the MDI + vehicle-treated group than in the untreated group, with a significant dose-dependent decrease observed in the MDI + RSV-treated group (Fig. 5A) and MDI + RBE-treated groups (Fig. 5B). A similar response was observed in the mRNA expression of the two lipogenic protein-encoding genes, although the decrease in FAS mRNA expression was higher than that of FABP4 mRNA (Fig. 5A, Fig. 5B). These results suggest that RSV and RBEs suppressed the increased mRNA expression of adipogenic transcription factors and lipogenic protein-encoding genes induced when cultured in an MDI medium. In summary, both RSV and RBEs significantly regulated 3T3-L1 adipocyte lipid metabolism by reducing adipogenesis- and lipogenesis-regulating molecules.

Fig. 5.

Fig. 5

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Effect of RSV or RBEs on mRNA expression of genes related to lipid metabolism in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated in MDI medium containing 1, 2, 5, 10 μM RSV A or RBEs B for 10 days. The significance of difference in mRNA expression of genes related to lipid metabolism was evaluated by Turkey’s test. Data are the means ± SD (n = 4). Different letters (a–c) indicate significant differences among the different concentrations of the RSV or RBEs group (p < 0.05)

Effect of RBEs on protein expression of lipid metabolism in 3T3-L1 adipocytes

AMPK expression is regulated by the AMP/adenosine triphosphate (ATP) ratio and is a significant regulator of intracellular energy metabolism. Therefore, it is known as an energy sensor. To investigate whether the activation of AMPK was implicated in RSV or RBE-mediated suppression of adipocyte differentiation, the extent of AMPK phosphorylation was determined. 3T3-L1 adipocytes were incubated in an MDI medium containing 1, 2, or 5 μM RSV or RBEs for 10 days. The protein expression of AMPK, phosphorylated AMPK (p-AMPK), and the relative protein levels of RSV or RBEs were measured by western blot analysis on day 10 (Fig. 6A). The expression levels of p-AMPK and AMPK in MDI + RSV-treated 3T3-L1 adipocytes (Fig. 6B) and MDI + RBE-treated 3T3-L1 adipocytes (Fig. 6C) were measured. As shown in Fig. 6B, the p-AMPK/AMPK ratio in 1, 2, 5, and 10 μM RSV were significantly reduced compared to those in control. When 3T3-L1 adipocytes were treated with RBEs, the p-AMPK/AMPK ratio was significantly increased (Fig. 6C). In addition, enhancement of p-AMPK by culturing in MDI medium dramatically increased in the MDI + RBE-treated group compared to that in the MDI + vehicle-treated group (p < 0.05) (Fig. 6C). Compared to the positive control group (MDI + vehicle treatment), the ratios at 10 μM (RSV and BREs) were increased by approximately 8% and 136%, respectively. These results suggest that RBE administration promotes AMPK activity, enhances lipid catabolism, and decreases lipid synthase. These results were consistent with our previous studies (Tain et al. 2020; Shih et al. 2021). RBEs can reduce lipid accumulation more potently than RSV (Tain et al. 2020) and reduce hyperlipidemia and obesity induced by BPA in offspring rats (Shih et al. 2021).

Fig. 6.

Fig. 6

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Effect of RSV or RBEs on protein expression of lipid metabolism in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated in MDI medium containing 1, 2, 5, 10 μM RSV or RBEs for 10 days. The protein expression of AMPK and p-AMPK A and relative protein levels of RSV B and RBEs C were measured by western blot analysis on 10 day. The significance of difference protein expression of lipid metabolism was evaluated by Turkey’s test. Data are the means ± SD (n = 4). Different letters (a–c) indicate significant differences among the different concentrations of the RSV or RBEs group (p < 0.05)

Discussion

Various compounds with lipolytic and anti-lipogenic activities have been isolated as potential drug candidates from herbal medicines and natural products used to prevent body fat accumulation. To date, many studies have reported the anti-obesity effects of several natural compounds such as RSV (Chang et al. 3) and zeaxanthin (Liu et al. 17). In this study, we obtained scientific evidence supporting the role of RBEs in the inhibition of lipogenesis and stimulation of lipolysis in 3T3-L1 cells and adipocytes. The results indicate that RBEs prevent lipid accumulation in MDI-stimulated 3T3-L1 cells by regulating adipogenic transcription factors and increasing the p-AMPK/AMPK ratio. The biological activity of RBEs is more potent than that of RSV.

During the induction of mitotic clonal expansion (MCE), the number of adipocytes in the early stages of adipogenesis increased with the cell cycle progression (Patel andLane 22). Several anti-obesity compounds may modulate cell cycle progression in MDI-stimulated 3T3-L1 cells. The reduction of G0/G1-blocking populations in MDI-stimulated cells recovered after treatment with dioscin (Poudel et al. 23) and ramalin (Kim et al. 13). Furthermore, cell cycle progression is mediated by the MAPK signaling pathway during adipogenesis (Tang et al. 2003). High levels of phosphorylated p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) were significantly reduced in MDI-stimulated adipocytes after exposure to ramalin (Kim et al. 13) and dioscin (Poudel et al. 23). In contrast, the inhibition of ERK and p38 phosphorylation has been observed in dioscin-treated adipocytes (Poudel et al. 23). In the present study, we observed an RSV and RBE-mediated inhibition of G0/G1 arrest and the S phase of cell cycle progression in 3T3-L1 adipocytes. The results of this study were consistent with those of previous studies, especially because the G2/M phase was not different from that of the control group at 48 h after RBEs treatment (p > 0.05). This may involve G2/M phase arrest to reduce preadipocyte division, activate apoptosis-promoting proteins, induce mitochondrial membrane potential imbalance to promote apoptosis, and induce apoptosis related to endoplasmic reticulum (ER) stress mechanisms (Chen et al. 4). As RBEs are obtained from RSV-derived complexes, future studies are required to verify the actual effect of RBE treatment on the cell cycle.

Biological events leading to obesity include changes in adipocyte number, which may occur through a complex interplay between the proliferation and differentiation of preadipocytes and changes in lipid metabolism in mature adipocytes. It has previously been noted that RSV inhibits both preadipocyte differentiation (Bai et al. 1) and lipid accumulation in mature adipocytes (Lasa et al. 15). The present study investigated the potential effects of RSV metabolites on adipogenesis and triacylglycerol metabolism in mature adipocytes. The results showed that the TG content of the positive control group (MDI + vehicle treatment) was significantly higher than that of the negative control group. In contrast, RSV and RBE treatment significantly reduced lipid accumulation, and the reduction was similar to that in the negative control group (p > 0.05) (Fig. 3C, Fig. 4C). The results concerning RBEs are in accordance with those reported in previous studies, which observed no effect with 10 µM or 12.5 µM of this polyphenol and a significant reduction in triacylglycerol content with 20 µM or 25 µM (Lasa et al. 15).

Two adipogenesis transcription factors (PPARg and C/EBPα) and adipogenesis proteins (FABPs and FAS) play critical roles in adipogenesis, which is the differentiation of fibroblast-like preadipocytes into mature lipid-accumulating, insulin-responsive adipocytes cells (Nagai et al. 21). Notably, in the intermediate stage of adipocyte differentiation, the expression of PPAR and C/EBP is enhanced (Tang and Lane 28) The expression of PPARγ and C/EBPα is significantly enhanced during the intermediate stages of adipocyte differentiation (Tang andLane 1999). Upregulated PPAR and C/EBP induce the transcription of FABPs and FAS genes, which are associated with developing and maintaining an advanced adipocyte phenotype (desá et al. 6). Therefore, previous studies have used them as distinct markers to identify novel compounds with anti-obesity activity. This study clearly showed that the expression levels of adipogenic transcription factors and lipogenic proteins were significantly increased in MDI-stimulated 3T3-L1 cells. These levels recovered after treatment with RSV and RBEs in a dose-dependent manner. FABPs have been proposed to actively facilitate the transport of fatty acids to specific organelles in the cell for lipid oxidation in the mitochondria or peroxisomes, lipid-mediated transcriptional regulation in the nucleus, signaling, trafficking, and membrane synthesis in the endoplasmic reticulum (ER), regulation of enzyme activity, and storage as lipid droplets in the cytoplasm (Furuhashi andHotamisligil 8). FABPs are also involved in converting fatty acids to eicosanoids and stabilizing leukotriene (Dickinson Zimmer et al. 30). However, the mRNA levels of adipogenic transcription factors and lipogenic protein-encoding genes were significantly increased in MDI-stimulated 3T3-L1 cells. These levels recovered after treatment with several single compounds isolated from natural products, including RSV (Chang et al. 3) and eupatilin (Kim et al. 13). Similar to previous studies, the present study noted decreased mRNA levels of two adipogenic transcription factors and two lipogenic protein-encoding genes in MDI-stimulated 3T3-L1 cells following RSV or RBE treatment. These results provide scientific evidence of the molecular mechanism underlying the anti-adipogenic effect of RBEs on the differentiation and lipid accumulation of 3T3-L1 adipocytes.

The AMPK cascade is an important therapeutic target in obesity and type 2 diabetes mellitus (Luo et al. 18). AMPK, whose expression is regulated by the AMP/ATP ratio, is regarded as an energy sensor as it significantly regulates intracellular energy metabolism. According to these findings, AMPK may limit adipogenesis and suppress SREBP-1c, PPARγ, and FAS expression in adipocytes (Day et al. 5; Herzig andShaw 11). AMPK activation is essential for inhibiting 3T3-L1 adipocyte lipogenesis by phytochemicals (Moon et al. 20). To determine whether RBEs inhibited adipocyte differentiation by activating AMPK, the level of AMPK phosphorylation was determined. This study indicated that the MDI + RBE-treated group had significantly increased p-AMPK levels when cultured in MDI medium compared to that in the MDI + vehicle-treated group (p < 0.05). The level of AMPK phosphorylation was significantly elevated after RBE treatment. Collectively, these results show that RBEs can inhibit lipogenesis and differentiation of 3T3-L1 adipocytes via AMPK activation. Therefore, they may be potential therapeutic candidates for preventing and treating obesity and obesity-related diseases.

Conclusion

The results indicate that RBEs significantly regulate 3T3-L1 adipocyte lipid metabolism by reducing the mRNA levels of adipogenesis- and lipogenesis-regulating molecules such as PPAR, C/EBP, FABP4, and FAS. Furthermore, the study confirmed that, compared to RSV, RBEs significantly promoted the p-AMPK/AMPK ratio to enhance lipid catabolism, thereby reducing lipid synthesis. Considering that the above data was derived from cell lines, the lipolytic ability of RBEs and their potential therapeutic use in obesity treatments should be further verified in animal models.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 13 KB) (13.4KB, docx)

Acknowledgements

The authors would like to thank all the individuals who volunteered for this study.

Abbreviations

RBEs

Resveratrol butyrate esters

RSV

Resveratrol

FBS

Fetal bovine serum

MDI

Methyl-isobutyl-xanthine, dexamethasone, insulin

AMPK

AMP-activated protein kinase

DM2

Diabetes mellitus type 2

C/EBP

CCAAT/enhancer-binding protein

PPAR

Peroxisome proliferator-activated receptor

BPA

Bisphenol A

ACC

Acetyl CoA carboxylase

SREBP-1

Sterol regulatory element-binding protein-1

DMEM

Dulbecco’s modified eagle medium

PBS

Phosphate buffered saline

IBMX

3-Isobutyl-methylxanthine

DEX

Dexamethasone

MTT

3-(4,5-Dimethyazol-2-yl)-2,5-Diphenyltetrazolium bromide

SDS

Sodium dodecyl sulfate

APS

Ammonium persulfate

DAPI

4′,6-Diamidino-2-Phenylindole

TG

Triglyceride

Authors’ contributions

M.-K.S., Y.-W.H. and C.-Y.H.; Data curation, M.-K.S. and S.-L.H.; Funding acquisition, Y.-W.H. and C.-Y.H.; Investigation, Y.-W.H., S.-L.H. and C.-Y.H.; Methodology, C.-Y.H. and S.-L.H.; Project administration, C.-Y.H.; Resources, M.-K.S. and C.-Y.H.; Software, Y.-W.H. and S.-L.H.; Supervision, M.-K.S. and C.-Y.H.; Validation, M.-K.S., S.-L.H. and Y.-W.H.; Visualization, Y.-W.H. and S.-L.H.; Writing—Original draft, C.-Y.H., C.-D.D. and A.-K.P.; Writing—Review & editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Science and Technology, Republic of China (Grant No. 110-2320-B-992 -001-MY3 and 110-2622-E-992-012-).

Declarations

Conflicts of interest

The authors declare no conflict of interest.

Consent for publication

All authors have read and agreed to the published version of the manuscript.

Footnotes

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Contributor Information

Ming-Kuei Shih, Email: mkshih@mail.nkuht.edu.tw.

Shu-Ling Hsieh, Email: slhsieh@nkust.edu.tw.

Yu-Wen Huang, Email: may2377234@gmail.com.

Anil Kumar Patel, Email: anilkpatel22@gmail.com.

Cheng-di Dong, Email: cddong@nkust.edu.tw.

Chih-Yao Hou, Email: chihyaohou@gmail.com.

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