2019 Volume 25 Issue 6 Pages 817-826
Resveratrol and its dimer ε-viniferin are polyphenolic compounds in red wine. Although various beneficial functions of resveratrol have been identified, those of ε-viniferin remain unclear. The present study therefore compared the effects of ε-viniferin and resveratrol on the differentiation of 3T3-L1 preadipocytes. We found that ε-viniferin more effectively suppressed intracellular lipid accumulation than resveratrol. ε-Viniferin, but not resveratrol, reduced protein expression of PPARγ and fatty acid synthase that contribute to lipogenesis, and increased expression of adipose triglyceride lipase, a lipolytic enzyme. Adiponectin protein expression, which plays a critical role in preventing type 2 diabetes and metabolic syndrome, was enhanced only by ε-viniferin. In addition, only ε-viniferin raised SIRT1 expression with increased phosphorylation of AMPK. These findings suggest that ε-viniferin is more effective than resveratrol in promoting favorable adipocyte differentiation with enhanced adiponectin expression and decreased lipid accumulation. Therefore, ε-viniferin may be a candidate phytochemical in red wine for preventing obesity.
Obesity has become a global public health concern because it is associated with the development of various diseases including type 2 diabetes, coronary heart disease, and hypertension (Bouchard et al., 2010; Heber et al., 2010; Tsai et al., 2011). Obesity originates from an energy imbalance due to excess caloric intake relative to energy expenditure, and it is characterized by adipocyte hypertrophy, an increase in adipocyte size, and hyperplasia, an increase in adipocyte number (Siriwardhana et al., 2013). Thus, maintaining a normal state of adipose is critical to prevent the development of obesity and its related diseases.
Adipocyte differentiation, known as adipogenesis, is a complex process that is accompanied by coordinated changes in morphology, hormone sensitivity, and gene expression. Peroxisome proliferator activator receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) are two key regulators of adipocyte differentiation that orchestrate the expression of adipogenic and lipogenic genes (Gregoire et al., 1998; Li et al., 2016; Lowell, 1999;). Fatty acid synthase (FAS) and adipose triglyceride lipase (ATGL) play central roles in lipogenesis and lipolysis, respectively (Gregoire et al., 1998; Li et al., 2016; Lowell, 1999; Nakamura et al., 2014). The activation of AMP-activated protein kinase (AMPK) leads to suppressed PPARγ and C/EBPα expression (Jiang et al., 2012; Picard et al., 2004). The NAD+-dependent protein deacetylase SIRT1, a critical factor that affects obesity and lifespan, often functions coordinately with AMPK and inhibits PPARγ activity (Jiang et al., 2012; Picard et al., 2004).
The primary role of adipose tissue is to store energy in the form of triglycerides when energy intake is sufficient and to release energy in the form of free fatty acids during starvation. Adipose tissue also acts as an endocrine organ that generates various biologically active molecules called adipocytokines. Among them, adiponectin, which is abundantly expressed in adipocytes, is regarded as beneficial because it efficiently reduces insulin resistance, exerts anti-inflammatory effects, and regulates lipid metabolism (Un and Myung-Sook, 2014). Therefore, impaired adipocyte differentiation with decreased adiponectin expression is thought to contribute to insulin resistance, type 2 diabetes and other metabolic disorders (Geffken et al., 2001; Kadowaki et al., 2006; Pischon et al., 2003).
Numerous studies have shown the role of dietary polyphenols in the prevention of obesity and obesity-related chronic diseases (Aguirre et al., 2014; Li et al., 2016). Resveratrol, a natural polyphenolic compound in grapes and red wine, is probably the most widely studied among them. The results of many cellular and animal studies have shown that resveratrol exerts anti-obesity effects by reducing the viability and proliferation of preadipocytes, suppressing adipocyte differentiation with decreased triglyceride accumulation, inhibiting lipogenesis, and/or stimulating lipolysis and fatty acid β-oxidation (Aguirre et al., 2014; Carpéné et al., 2015; Cho et al., 2012; Kang et al., 2012; Kwon et al., 2012; Li et al., 2016; Shan et al., 2013). In contrast, less is understood about its dimer ε-viniferin, although its content in grapes and red wine is comparable to or greater than that of resveratrol, depending on the type of red wine and the stage of the noble rot on grapes (Mazauric et al., 2005; Tran et al., 2008). Many reports have described ε-viniferin function over the past decade (Guschlbauer et al., 2013; Karaki et al., 2016; Mi et al., 2007; Nivelle et al., 2018; Yu et al., 2017; Zghonda et al., 2012) and most of them suggest that ε-viniferin is more effective than resveratrol. Mi et al. (2007) demonstrated that ε-viniferin induces the vasorelaxation of isolated thoracic aortas more potently than resveratrol. We also showed that ε-viniferin facilitated the repair of wounded vascular endothelial cells more effectively than resveratrol, and improved blood pressure and cardiac hypertrophy in spontaneously hypertensive rats (Zghonda et al., 2012).
However, only two reports have described the effects of ε-viniferin on adipose tissue (Hsu et al., 2007; Ohara et al., 2015). Ohara et al. (2015) reported that only ε-viniferin at the same dose as resveratrol prevented diet-induced obesity in mice, and decreased triglyceride accumulation and PPARγ expression more effectively than resveratrol during 3T3-L1 adipogenesis. Lu et al. (2017) also revealed that ε-viniferin suppressed diet-induced obesity in mice more effectively than resveratrol and reduced hydroxymethylglutaryl-CoA reductase activity, a key enzyme for catalyzing the rate-limiting step of cholesterol, more effectively than resveratrol at high concentrations (80, 120, and 160 µM) in 3T3-L1 preadipocytes. However, these studies did not apparently compare the effects of ε-viniferin and resveratrol on important regulators of adipogenesis in 3T3-L1 preadipocytes such as FAS, ATGL, AMPK, and SIRT1. In particular, the expression of adiponectin that increases during adipocyte differentiation has not been described, even though a reduction in such expression appears to result in insulin resistance, type 2 diabetes and metabolic syndrome (Geffken et al., 2001; Kadowaki et al., 2006; Pischon et al., 2003).
The aims of the present study were to examine the effects of resveratrol and ε-viniferin on 3T3-L1 preadipocyte differentiation, focusing on the expression of adiponectin and regulators of differentiation and lipid accumulation, and to compare the effects of the two compounds. We used these compounds at concentrations that avoid inducing apoptosis when evaluating their effects on preadipocyte differentiation.
Chemical reagents and antibodies Trans-resveratrol was purchased from Wako Pure Chemical Industries (Osaka, Japan). We respectively obtained 3T3-L1 preadipocytes and newborn calf serum (CS) from the American Type Culture Collection (Manassas, VA, USA) and Gibco (Gaithersburg, MD, USA). Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium with high glucose (DMEM), 3-isobutyl-1-methylxanthine (IBMX), a mixture of penicillin and streptomycin, insulin, dexamethasone (DEX), Oil Red O, and Triton X-100 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies for PPARγD69), adiponectin, ATGL, FAS, and anti-rabbit and -mouse secondary antibodies coupled to horseradish peroxidase were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-fibroblast growth factor 21 (FGF21) antibody was purchased from Sigma Chemical Co. Anti-SIRT1 (H-300) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
Cell culture and manipulations The 3T3-L1 mouse embryo fibroblasts that can be induced to differentiate into an adipocyte-like phenotype were maintained in DMEM containing high glucose (4500 mg/L) supplemented with 10% CS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified 5% CO2 atmosphere, and sub-cultured when they reached approximately 70% confluence. When the 3T3-L1 preadipocytes reached 100% confluence, the medium was changed to DMEM containing 10% FBS, 1 mM DEX, 0.5 mM IBMX, and 1 µg/mL insulin (≥27 IU/mg) to initiate adipogenic differentiation. After 2 days of initiation, the medium was replaced with DMEM containing 1 µg/mL insulin for 10 days. Resveratrol and ε-viniferin were applied throughout the period of differentiation.
Cell viability Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assays using CellTiter 96 AQueous One Solution (Promega, Madison, WI, USA). The 3T3-L1 preadipocytes (6 × 104) were seeded in 96-well plates, cultured for 12–16 h, and then incubated with or without resveratrol and ε-viniferin for 24 h. The cells were incubated with 20 µL of MTS solution for 4 h at 37 °C and absorbance was measured at 490 nm to determine cell viability.
Oil Red O staining and lipid quantitation Cells differentiated into adipocytes during a period of 10 days were washed three times with PBS (−) and fixed in 10% neutral formalin for 30 min at room temperature. After three rinses with distilled water, the adipocytes were incubated with 60% isopropanol for 5 min, and stained with fresh 0.5% Oil Red O in isopropanol diluted 3:2 (v/v) with distilled water for 20 min. Stained adipocytes were washed three times and then stained with hematoxylin for 60 s. After three washes with distilled water, the cells were assessed by microscopy and images were collected on an Olympus microscope (Tokyo, Japan). Oil Red O stain was dissolved in DMSO and optical density was quantified at 520 nm.
Triglyceride content After differentiation for 10 days, the adipocytes were washed three times with PBS (-) and scraped into cell lysis buffer for 1 min on ice. Total triglyceride contents of the cells were determined using LabAssay™ triglyceride kits (Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. Triglyceride contents were normalized by protein content and expressed as a percentage of the value for differentiated cells in the absence of the compounds.
Western blot analysis On day 10 after initiating differentiation, adipocytes were washed three times with cold PBS and scraped into lysis buffer (50 mM HEPES pH7.5, 50 mM NaCl, 1 mM EDTA, 50% glycerol, 100 mM NaF, 10 mM sodium pyrophosphate, 1% Triton X-100, 1 mM NaVO4, 1 mM PMSF,10 µg/mL antipain, 10 µg/mL leupeptin, 10 µg/mL aprotinin). The cell lysates were centrifuged at 14,000 rpm for 15 min and the supernatant was collected. Cell lysates containing equal quantities of proteins (35 µg) were separated by electrophoresis on 12% polyacrylamide gels and proteins were transferred to polyvinylidene difluoride (PVDF) membranes using a Trans-Blot® Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% BSA in TBS-T (150 mM NaCl, 0.05% Tween 20, and 10 mM Tris-HCl (pH 7.4) for 60 min at room temperature and then incubated with appropriately diluted primary antibodies overnight at 4 °C. The primary antibodies were then detected using horseradish peroxidase-conjugated goat anti-mouse or donkey anti-rabbit secondary antibodies (1/5,000) for 60 min at room temperature. Immunocomplexes were visualized using the Chemi-Lumi One L kit according to the manufacturer's instructions (Nacalai Tesque Ltd., Kyoto, Japan).
Statistical analysis Data are presented as means ± standard deviation and were statistically analyzed using Dunnett for Figs. 2–5 and Games-Howell for Fig. 6. Values with p < 0.05 were considered statistically significant.
Chemical structures of resveratrol (A) and ε-viniferin (B).
Effect of resveratrol and ε-viniferin on cell viability of 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were treated with different concentrations (0, 5, 10, 20 and 30 µM) of resveratrol (A) and ε-viniferin (B) for 24 h, and the cell viability was assessed by MTS assay. Data are presented as means ± SD (n=3). *P < 0.05 vs. Control (0 µM), **P < 0.01 vs. Control (0 µM).
Effect of resveratrol and ε-viniferin on lipid accumulation during differentiation of 3T3-L1 preadipocytes. 3T3-L1 cells were treated with different concentrations (0, 5, 10 and 15 µM) of resveratrol and ε-viniferin (A–C) or 50 µM resveratrol (D, E) for 10 days. Intracellular lipid accumulation was visualized with Oil Red O staining (A, D) and subjected to spectrometric quantification after dissolved in DMSO (B, C, E). Intracellular triacylglycerol contents were measured as described in Materials and Methods. The 100% indicates triglyceride content at a concentration of 25.33 mg/mg protein. D: differentiated, U: undifferentiated. Data are presented as means ± SD (n=3). *P µ 0.05 vs. D, **P µ 0.01 vs. D.
Effect of resveratrol and ε-viniferin on protein expression of adiponectin, PPARγ, FAS, and ATGL during differentiation of 3T3-L1 cells. 3T3-L1 cells were treated with different concentrations (0, 5, 10 and 15 µM) of resveratrol and ε-viniferin for 10 days, and protein expression of adiponectin (A), PPARγ (B), FAS (C), and ATGL (D) was detected by western blotting. D: differentiated, U: undifferentiated. Data are presented as means ± SD (n=3). *P < 0.05 vs. D.
Effect of resveratrol and ε-viniferin on SIRT1, p-AMPK, and FGF-21 protein expression during differentiation of 3T3-L1 cells. 3T3-L1 cells were treated with different concentrations (0, 5, 10 and 15 µM) of resveratrol and ε-viniferin for 10 days and protein expression of SIRT1 (A), p-AMPK (B), and FGF-21 (C) was measured by western blotting. D: differentiated, U: undifferentiated. Data are presented as means ± SD. (n=3). *P < 0.05 vs. D, **P < 0.01 vs. D.
Effect of SIRT1 inhibitor on lipid accumulation during 3T3-L1 preadipocyte differentiation in the pesence and absence of resveratrol and ε-viniferin. 3T3-L1 cells were treated with and without 15 µM resveratrol or ε-viniferin in the presence and absence of 10 µM Ex527 for 10 days. Intracellular lipid droplets were stained with Oil Red O and subjected to spectrometric quantification after dissolved in DMSO. D: differentiated, U: undifferentiated. Data are presented as means ± SD (n=3). *P > 0.05.
Resveratrol, but not ε-viniferin, reduces cell viability of 3T3-L1 preadipocyte cells Figure 1A and B show the structural formulae of resveratrol and ε-viniferin, respectively. Resveratrol is thought to suppress lipid accumulation by decreasing cell viability and reducing adipogenesis (Rayalam et al., 2008). Therefore, we investigated whether resveratrol and ε-viniferin would affect the viability of 3T3-L1 preadipocytes under our experimental conditions. The preadipocytes were cultured with various concentrations (5 to 30 µM) of these compounds for 24 h. Resveratrol at 20 and 30 µM significantly decreased cell viability whereas ε-viniferin did not exert detectable effects at 5 to 30 µM (Fig. 2). Thus, we used 5, 10, and 15 µM resveratrol and ε-viniferin for subsequent experiments to determine their effects on adipocyte differentiation.
ε-Viniferin suppresses intracellular lipid accumulation more effectively than resveratrol during 3T3-L1 preadipocyte differentiation We assessed whether resveratrol and ε-viniferin promote or suppress total lipid accumulation in 3T3-L1 preadipocytes to determine their effects on adipocyte differentiation. The preadipocytes were incubated with these compounds from days 0 to 10 of differentiation. Accumulated lipid was detected on day 10. The results of Oil Red O staining indicated that 10 and 15 µM ε-viniferin significantly decreased intracellular oil droplets by 22% and 27%, respectively, whereas resveratrol exerted the significant decrease at only 15 µM (Fig. 3A and B). Similarly, ε-viniferin at all of these concentrations significantly reduced intracellular triglyceride accumulation whereas 10 and 15 µM, but not 5 µM resveratrol significantly reduced the accumulation (Fig. 3C). These data suggested that ε-viniferin suppresses adipocyte differentiation more effectively than resveratrol in terms of lipid accumulation.
Many reports indicated that high concentrations of resveratrol reduce lipid accumulation during 3T3-L1 preadipocyte differentiation (Aguirre et al., 2014; Hwang et al., 2013; Kwon et al., 2012; Lee et al., 2013; Rayalam et al., 2008). Consistent with the findings of these studies, 50 µM resveratrol decreased lipid accumulation by 32% (Fig. 3D and E). This reduction may be due to apoptosis induced by high concentrations of resveratrol.
Effect of resveratrol and ε-viniferin on protein expression of adiponectin, PPARγ, FAS, and ATGL during 3T3-L1 cell preadipocyte differentiation Adiponectin plays a critical role in preventing insulin resistance, type 2 diabetes and metabolic syndrome (Geffken et al., 2001; Kadowaki et al., 2006; Pischon et al., 2003). Therefore, understanding the effects of resveratrol and ε-viniferin on adiponectin expression is extremely important. Figure 4A shows that 15 µM ε-viniferin significantly enhanced adiponectin protein expression, whereas 15 µM resveratrol did not.
We further compared the effects of the two compounds on the protein expression of PPARγ, FAS, and ATGL because PPARγ is a key regulator of adipocyte differentiation, and FAS and ATGL are critically involved in lipogenesis and lipolysis, respectively, in adipose tissue. We found that ε-viniferin significantly reduced PPARγ and FAS expression, and increased ATGL expression (Fig. 4B–D). These results agree well with the finding that ε-viniferin suppressed lipid accumulation during adipocyte differentiation (Fig. 3A–C). Therefore, the ε-viniferin-dependent reduction in lipid accumulation may be associated with both decreased FAS expression and increased ATGL expression. In contrast, resveratrol at 5 to 15 µM did not exert any effects.
Together, these findings suggest that ε-viniferin, but not resveratrol, shows favorable effects with reduced lipid accumulation accompanied by enhanced adiponectin expression during adipocyte differentiation.
Expression of SIRT1, p-AMPK and FGF21 is increased by ε-viniferin, but not resveratrol A number of reports have indicated that resveratrol activates AMPK (Hwang et al., 2008; Shang et al., 2008; Tauriainen et al., 2011; Zang et al., 2006), a major metabolism-sensing protein, and derives several of its beneficial effects by targeting SIRT-1 (Ajmo et al., 2008; Barger et al., 2008; Howitz et al., 2003; Tauriainen et al., 2011), which is activated by AMPK. There are also reports demonstrating that polyphenols including resveratrol suppress adipocyte differentiation and obesity (Aguirre et al., 2014; Li et al., 2016). Therefore, we investigated whether these compounds increase the expression of SIRT1 and of phosphorylated AMPK (p-AMPK), an active form of AMPK, during adipocyte differentiation. Fig. 5A and B show that 15 µM ε-viniferin significantly elevated the expression of these proteins, whereas 15 µM resveratrol did not. These findings indicate that ε-viniferin function is mediated by increased p-AMPK and SIRT1 expression.
Although mainly acting as a hepatic endocrine regulator in glucose and lipid metabolism, FGF21 is also expressed in adipocytes (Ohta et al., 2014). Several studies have suggested that FGF21 causes weight loss in mice with diet-induced obesity (Ohta et al., 2014; Véniant et al., 2015). Therefore, we compared the effects of ε-viniferin and resveratrol on FGF21 expression. Figure 5C shows that 15 µM ε-viniferin increased FGF21 protein expression, which might be associated with its anti-obesity function.
Effect of SIRT1 inhibitor EX527 on ε-viniferin-induced suppression of intracellular lipid accumulation during adipocyte differentiation To determine whether SIRT1 mediates the inhibitory effect of ε-viniferin on lipid accumulation, we differentiated 3T3-L1 preadipocytes in the presence or absence of the SIRT1 inhibitor EX527. Lipid accumulation was suppressed by 15 µM ε-viniferin and this suppression was completely blocked by EX527, which is consistent with the result that ε-viniferin elevated SIRT1 expression (Fig. 5A). However, EX527 slightly but significantly increased lipid accumulation in the absence of ε-viniferin, suggesting that SIRT1 is also involved in basal lipid accumulation during adipocyte differentiation.
The present study compared the effects of resveratrol and its dimer ε-viniferin at 5 to 15 µM on the adipogenesis of 3T3-L1 preadipocytes, a condition in which these compounds do not reduce cell viability. Our main findings are as follows. First, ε-viniferin promoted favorable adipocyte differentiation with enhanced adiponectin expression and decreased triglyceride accumulation, whereas resveratrol did not. Second, ε-viniferin reduced the expression of PPARγ and FAS, and increased the expression of SIRT1, p-AMPK and ATGL, suggesting that these proteins may coordinately contribute to inducing favorable adipocyte differentiation.
Impaired adipogenesis in addition to obesity is implicated in insulin resistance (Rosen et al., 2006). This is because adipogenesis contributes to increasing the numbers of insulin sensitive adipocytes accompanied by increased generation of adiponectin, which plays a critical role in preventing insulin-resistant type 2 diabetes. Therefore, whether adiponectin expression is affected by the suppression of adipogenesis induced by various phytochemicals should be determined. Hsu et al. (2007) investigated the inhibitory effect of 15 phenolic acids and six flavonoids on adipocyte differentiation. Most of the investigated compounds suppressed adipogenesis, and among them, o-coumaric acid and rutin remarkably reduced lipid accumulation and increased adiponectin expression. We found that ε-viniferin lowered lipid accumulation and up-regulated adiponectin. Thus, these compounds are considered to promote favorable adipocyte differentiation.
In contrast to a number of reports indicating that phytochemicals exert anti-adipogenic effects, only three reports have shown the phytochemical-dependent promotion of adipogenesis (Han et al., 2017; Hassan et al., 2007; Li et al., 2016). The phytochemicals in these three reports are phloretin in apples, 4-hydroxyderricin in Angelica keiskei, and 4-methoxychalcone, all of which belong to the chalcone class of flavonoids. These three compounds stimulate the adipogenesis of 3T3-L1 preadipocytes by enhancing the expression of PPARγ and increasing that of adiponectin and some other proteins involved in adipocyte differentiation. Anti-diabetic thiazolidinedione derivatives (TZD) are PPARγ activators that improve insulin sensitivity and lower the blood glucose levels associated in type 2 diabetes (Staels et al., 2005). These agents promote adipocyte differentiation by increasing the uptake of glucose and free fatty acids, triglyceride storage, and the generation of adiponectin via PPARγ activation. Therefore, these chalcone derivatives might function as PPARγ ligands. Some phytochemicals inhibit and others promote adipogenesis although both types of compounds can increase adiponectin expression. These findings suggest that although PPARγ is critically involved in adipogenesis with increased lipid accumulation, adiponectin expression may be independent of PPARγ activation during preadipocyte differentiation. In fact, there are reports indicating that the activation of SIRT1 or AMPK raised adiponectin expression during preadipocyte differentiation (Li et al., 2011; Wang et al., 2017) whereas these two molecules also inhibited PPARγ activity (Jiang et al., 2012; Picard et al., 2004). We cannot exclude the possibility that elevated adiponectin expression by ε-viniferin is caused by suppressed adiopogenesis with decreased lipid accumulation. However, based on our data together with the past reports, we believe that ε-viniferin-dependent elevation of adiponectin expression is induced by activation of the SIRT1/AMPK pathway, which lowers PPARγ expression leading to lipid accumulation. Relationships between chemical structures and their effects on adipose tissue need to be clarified so that dietary compounds can be effectively applied to prevent and improve obesity followed by type 2 diabetes and its related diseases.
He et al. (2013) showed that ursolic acid lowers lipid accumulation during 3T3-L1 preadipocyte differentiation accompanied by AMPK activation, increases SIRT1 expression, and decreases PPARγ and FAS expression. These data on protein expression are in good agreement with our findings. However, the SIRT1 inhibitor nicotinamide did not reverse this anti-adipogenic effect of ursolic acid. In contrast, EX527, also a SIRT1 inhibitor, blocked lipid accumulation in the presence of ε-viniferin in the present study. Our findings are consistent with those reported by Pichard et al. (2004), who found that SIRT1 suppressed the adipogenesis of 3T3-L1 preadipocytes with reduced expression of PPARγ and that this effect was reversed by the expression of SIRT1 RNAi. Bai et al. (2008) also indicated that resveratrol at high concentrations (50 to 100 µM) suppressed pig preadipocyte differentiation with increased SIRT1 expression and reduced PPARγ expression, and that SIRT1 might modulate the differentiation of these cells based on the effects of nicotinamide. The involvement of SIRT1 in adipocyte differentiation might differ depending on the nature of phytochemicals. Ono et al. (2011) reported that apigenin suppresses lipid accumulation during 3T3-L1 differentiation with decreased PPARγ expression and AMPK activation. However, ATGL expression did not change in response to 4 days of incubation with apigenin. Adipose triglyceride lipase promotes the catabolism of stored triglycerides in adipose tissues (Zimmermann et al., 2004). There are many reports demonstrating that various phytochemicals including resveratrol upregulate ATGL expression accompanied by reduced lipid accumulation (Aguirre et al., 2014). As far as we know, the present study is the first to suggest that the increased expression of ATGL, a lipolytic enzyme, and the decreased expression of FAS, a lipogenic enzyme, are involved in the reduction of lipid accumulation induced by resveratrol and its oligomers during adipocyte differentiation.
There is a discrepancy between the suppressive effect of resveratrol on lipid accumulation and resveratrol-dependent expression change in proteins such as PPARγ, FAS, and ATGL. That is, low concentrations of resveratrol slightly but significantly suppressed lipid accumulation without changing the expression of these proteins that are closely related to adipogenesis. There are reports indicating that high concentrations of resveratrol reduced lipid accumulation by suppressing PPARγ and FAS expression or by decreasing ATGL expression (Aguirre et al., 2014; Bai et al., 2008; Lasa et al., 2012; Lee et al., 2013). We speculate that low concentrations of resveratrol are too weak to constantly change the expression of these proteins. Actually, the effect of resveratrol was weaker than that of ε-viniferin as shown in Fig. 3A, B, and C. However, we cannot exclude the possibility that low concentrations of resveratrol suppress lipid accumulation through a pathway independent of PPARγ, FAS, and ATGL because many other factors including C/EBPα are complicatedly involved in adipocyte differentiation.
In summary, we found that low concentrations of ε-viniferin promote favorable adipocyte differentiation with enhanced adiponectin expression and decrease triglyceride accumulation, whereas resveratrol at low concentrations does not stimulate adiponectin expression in spite of slightly suppressing lipid accumulation Therefore, ε-viniferin may prevent and improve obesity and its related disorders more effectively than resveratrol.
Acknowledgements We thank N. Foster for help in preparing the manuscript.
peroxisome proliferator activator receptor γ
C/EBPαCCAAT/enhancer binding protein α
FASfatty acid synthase
ATGLadipose triglyceride lipase
AMPKAMP-activated protein kinase
FGF21fibroblast growth factor 21
