cell biochemistry and function Cell Biochem Funct 2015; 33: 220–225. Published online 23 April 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cbf.3107
Euphorbiasteroid, a component of Euphorbia lathyris L., inhibits adipogenesis of 3T3-L1 cells via activation of AMP-activated protein kinase Su-Jin Park1, Jae Ho Park1, Anna Han2, Munkhtugs Davaatseren3, Hyun Jin Kim4, Myung-Sunny Kim1, Haeng Jeon Hur1*, Mi-Jeong Sung1, Jin-Taek Hwang1, Hye Jeong Yang1 and Dae Young Kwon1 1
Korea Food Research Institute, Division of Nutrition And Metabolism Research, Seongnam-si, Korea The University of Tennessee-Knoxville, Department of Nutrition, College of Education, Health & Human Sciences, Knoxville, TN, USA 3 Konkuk University, Department of Bioresources and Food Science, College of Life and Environmental Sciences, Seoul, Gwangjin-Gu, Korea 4 Gyeongsang National University, Department of Food Science & Technology, Jinju, Korea 2
The purpose of this study is to investigate the effects of euphorbiasteroid, a component of Euphorbia lathyris L., on adipogenesis of 3T3-L1 pre-adipocytes and its underlying mechanisms. Euphorbiasteroid decreased differentiation of 3T3-L1 cells via reduction of intracellular triglyceride (TG) accumulation at concentrations of 25 and 50 μM. In addition, euphorbiasteroid altered the key regulator proteins of adipogenesis in the early stage of adipocyte differentiation by increasing the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase. Subsequently, levels of adipogenic proteins, including fatty acid synthase, peroxisome proliferator-activated receptor-γ and CCAAT/enhancer-binding protein α, were decreased by euphorbiasteroid treatment at the late stage of adipocyte differentiation. The anti-adipogenic effect of euphorbiasteroid may be derived from inhibition of early stage of adipocyte differentiation. Taken together, euphorbiasteroid inhibits adipogenesis of 3T3-L1 cells through activation of the AMPK pathway. Therefore, euphorbiasteroid and its source plant, E. lathyris L., could possibly be one of the fascinating anti-obesity agent. Copyright © 2015 John Wiley & Sons, Ltd. key words—euphorbiasteroid; obesity; adipogenesis; AMPK; 3T3-L1
INTRODUCTION Obesity is one of the critical health problems because it is associated with numerous serious diseases such as diabetes, hyperlipidemia, fatty liver diseases, hypertension, coronary heart disease and cancer.1 An abnormal increase in white adipose tissue (WAT), characterized by increases in cell size and number of adipocytes, is a major phenotype of obesity.2 Adipocytes play a key role in energy metabolism because they store excess energy in the form of triglycerides (TGs) and release energy in the form of glycerol and fatty acids.3 Pre-adipocytes differentiate into mature adipocytes because of excessive calorie intake and other various factors such as stimulation of insulin and glucocorticoids.4,5 Adipocyte differentiation can be divided into two main stages: mesenchymal stem cells differentiate into preadipocytes and then undergo terminal differentiation to mature adipocytes, which is accompanied by adipogenic proteins and/or gene expression changes, including CCAAT/enhancer binding proteins (C/EBPs), sterol regulatory element-binding
*Correspondence to: Haeng Jeon Hur, Korea Food Research Institute, Division of Nutrition And Metabolism Research, Seongnam-si, Korea. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
protein-1c (SREBP-1c) and peroxisome proliferator-activated receptor-γ (PPAR-γ).2,6 After differentiation, adipocytes regulate lipid metabolism through lipogenic proteins such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) and lipolytic enzymes such as hormone-sensitive lipase.3 AMP-activated protein kinase (AMPK) is a sensory protein that controls energy metabolism through regulation of the energy status.7 AMPK stimulates energy-producing processes such as cellular energy uptake, fatty acid oxidation, glycolysis and ketogenesis and inhibits energyconsuming processes such as lipogenesis, protein synthesis and gluconeogenesis.8 In adipocytes, activation of AMPK reversibly phosphorylates and inactivates ACC to inhibit the lipogenic pathway and increase adenosine triphosphate (ATP) production.9 Therefore, many studies have focused on the AMPK signalling pathway that appears to have an important role in preventing obesity and related metabolic diseases.10 In many scientific studies, natural compounds, such as resveratrol, epigallocatechin-3-gallate, quercetin, berberine, curcumin and ginsenosides, which activate the AMPK signalling pathway, are showing beneficial effects to inhibit adipogenesis in vitro and/or prevent the development of obesity induced by high fat diet in vivo.11–16 Received 16 July 2014 Revised 23 March 2015 Accepted 23 March 2015
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anti-adipogenic effect of euphorbiasteroid Euphorbia lathyris L. (E. lathyris L.), also known as caper spurge, is the biggest species of spurge family that have been widely used as traditional medicinal plant in Asia, especially in China.17 Recent studies reported that E. lathyris L. and its biologically active components including lathyrane, esculetin, ingenol derivatives and euphorbiasteroid (is a tricyclic diperpene, not a steroid), have shown beneficial effects on several disorders such as reverses of multi-drug resistance, inhibits tyrosinase and human immunodeficiency virus and act as anti-cancer drugs.18–21 However, the effects of E. lathyris L. and its biologically active components on obesity and their underlying mechanisms have not been studied yet. The present study was undertaken to investigate the potential anti-adipogenic activities of euphorbiasteroid on obesity by determining its effects on adipocyte differentiation and adipogenic protein expression.
Western blot Cytosolic proteins were extracted with a radioimmunoprecipitation assay (RIPA) buffer containing a SigmaFAST™ Protease Inhibitor Cocktail tablet (Sigma-Aldrich) and phosphatase inhibitors (2.5 mM sodium fluoride, 50 μM sodium orthovanadate, 500 μM sodium pyrophosphate and 500 μM β-glycerophosphate). Isolated proteins were quantified using the Bradford-Reagent (Biosesang, Seoul, Korea). Then, 20 to 50 μg proteins were separated on 12.5% acrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were then transferred to nitrocellulose membranes at 100 V for 1 h. All antibodies were used according to the manufacturer’s instructions, and protein bands were detected using an EZ-Western chemiluminescent detection kit (Daeil Lab., Seoul, Korea). Real time polymerase chain reaction (PCR)
MATERIALS AND METHODS Chemicals Euphorbiasteroid was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Bovine insulin, isobutylmethylxanthine and dexamethasone and all other chemicals and reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). Cell culture The murine pre-adipocyte cell line 3T3-L1 (American Type Culture Collection, Manassas, VA, USA) was maintained in culture medium (CM), Dulbecco’s modified Eagle’s medium (DMEM), containing 4.5 g/L D-glucose (Welgene, Daegu, Korea) supplemented with 10% foetal bovine serum (FBS) (Welgene) and penicillin/streptomycin (Welgene) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. For the differentiation to adipocytes, 3T3-L1 cells were grown in 96-well plates for 2 days to full confluency. At first (S1)-stage, CM were replaced with adipogenesis induction medium (MDI: 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone and 10 μg/ml bovine insulin in CM) and maintained for 2 days. At second (S2)-stage, MDI media were replaced with adipogenesis medium (AM: 10 μg/ml bovine insulin in CM) and maintained for 2 days. For the last (S3)-stage, AM medium was replaced with CM and maintained for 2 days. 3T3-L1 cells were treated with or without euphorbiasteroid at each differentiation stage. Oil red O staining For Oil red O (ORO) staining, differentiated 3T3-L1 cells were fixed with 4% para-formaldehyde for 1 h. After washing three times with phosphate-buffered saline (PBS), cells were stained with ORO solution (0.5% ORO in propylene glycol) for 1 h. After washing once with PBS, cells were washed three times with double-distilled water, dried in air and solubilized with isopropyl alcohol. Absorbance was measured using an enzyme-linked immunosorbent assay reader (Molecular Devices, CA, USA) at 550 nm. Copyright © 2015 John Wiley & Sons, Ltd.
Total RNA was isolated from cells using TRIzol reagent (Gibco, Grand Island, NY, USA). Total RNA (1 μg) was then reverse-transcribed into cDNA using AccuPower® PCR PreMix (Bioneer, Daejeon, Korea). The amplification reactions were performed on a Roche Light-Cycler® 480 system (Roche Diagnostics GmbH, Mannheim, Germany) using the following thermal cycling conditions: an initial activation step at 95 °C for 3 min, followed by 45 cycles of denaturation for 10 s at 95 °C, annealing for 15 s at 55 °C and extension for 20 s at 72 °C. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene, and the fold change in expression of each target mRNA relative to GAPDH was calculated based on the comparative 2-ΔΔct expression method. Primers were as follows: PPAR-γ: forward, 5’-CCC TGG CAA AGC ATT TGT AT-3’; reverse, 5’-ACT GGC ACC CTT GAA AAA TG-3’; C/EBP-α: forward, 5’-AGG TGC TGG AGT TGA CCA GT-3’; reverse, 5’-CAG CCT AGA GAT CCA GCG AC-3’; and GAPDH: forward, 5’-CAT GGC CTT CCG TGT TCC TA-3’; reverse 5’-GCG GCA CGT CAG ATC CA-3’. Statistical analysis Data are presented as mean ± standard deviation (SD). Statistical differences between the groups were assessed using Analysis of variance (one-way ANOVA followed by Tukey’s honest significant difference test). A value of P < 0.001 was considered statistically significant.
RESULTS Effects of euphorbiasteroid on differentiation of 3T3-L1 pre-adipocytes Adipogenesis is characterized by the formation of intracellular lipid droplets and expression of an endocrine cell-like phenotype. To evaluate the effects of euphorbiasteroid on adipogenesis, we measured TG content in differentiated 3T3-L1 cells treated with euphorbiasteroid. At concentrations of 25 μM and 50 μM, euphorbiasteroid significantly Cell Biochem Funct 2015; 33: 220–225.
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decreased TG accumulation in 3T3-L1 cells to the level of non-differentiation group (ND) (Figure 1). These data indicate that euphorbiasteroid could exert anti-adipogenic activity through inhibition or delay of adipogenesis in 3T3-L1 cells. Many anti-obesity agents inhibit adipogenesis by targeting genes involved in early adipogenesis such as phosphoinositide 3-kinase (PI-3K) pathway proteins and AMPK, however, some anti-obesity agents target adipogenesis in the late phase such as adipocyte fatty acid binding protein 2, FAS and ACC.22 To investigate whether euphorbiasteroid represents antiadipogenic activity during differentiation, we quantified the TG content in 3T3-L1 cells treated with or without euphorbiasteroid at each differentiation stages (S1, S2, S3). Treatment with euphorbiasteroid at the S1-stage inhibited
ET AL.
TG accumulation in differentiated 3T3-L1 cells and its anti-adipogenic activity was followed for S2-stage and S3stage. However, euphorbiasteroid treatment had no effect on intracellular TGs in differentiated 3T3-L1 cells at the S2-stage and S3-stage. On the basis of these results, we propose that euphorbiasteroid interferes with the progression of adipogenesis at the early stages of adipocyte differentiation (Figure 2). Effects of euphorbiasteroid on the expression of FAS, C/EBPs, PPAR-γ and SREBP-1c in 3T3-L1 cells during adipogenesis In order to examine the effect of euphorbiasteroid on adipogenic proteins, we measured the expression levels of FAS, C/EBPs, PPAR-γ and SREBP-1c in the cytosolic fraction of 3T3-L1 cells. Euphorbiasteroid decreased FAS levels in differentiated 3T3-L1 cells at 12.5 and 25 μM. Because FAS is a key regulator of lipid synthesis in adipocytes, its decrease led to a reduction in the TG level in 3T3-L1 cells. Next, we determined the effects of euphorbiasteroid on the expression of C/EBPs and PPAR-γ, upstream regulators of FAS, at the S2-stage or S3-stage of differentiation. We found that C/EBPs, PPAR-γ and SREBP-1c levels were decreased by euphorbiasteroid treatment in a dose-dependent manner (Figure 3). In addition, euphorbiasteriod treatment significantly decreased the C/EBPα and PPAR-γ gene expression in 3T3-L1 cells (Figure 4). These results are indicating that the underlying mechanism of the anti-adipogenic effects of euphorbiasteroid is related to the inhibition of signals involved in the initial stage of adipogenesis or activation of anti-adipogenic proteins and gene expressions. Effects of euphorbiasteroid on phosphorylation of Akt and ERK 1/2 in 3T3-L1 adipocytes The insulin signalling pathway is involved in the early stage of adipogenesis through activation of PI-3K and mitogenactivated protein kinases including extracellular signalregulated kinase (ERK) 1/2. However, euphorbiasteroid treatment did not significantly affect phosphorylation of ERK 1/2 and Protein Kinase B (Akt) (Figure 5). Effects of euphorbiasteroid on phosphorylation of AMPK and ACC in 3T3-L1 adipocytes
Figure 1. Effects of euphorbiasteroid on triglyceride accumulation in differentiating 3T3-L1 cells. 3T3-L1 cells were treated with euphorbiasteroid at concentrations of 6.25, 12.5, 25 and 50 μM for 2 days in adipogenesis induction medium (AI: 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone and 10 μg/ml bovine insulin in culture medium (CM)), 2 days in adipogenesis medium (AM: CM with 10 μg/ml bovine insulin) and 2 days in CM sequentially during differentiation. Intracellular triglycerides (TGs) were stained with Oil red O (ORO) solution. (A) Intracellular TGs levels were measured and quantified as fold change compared with the control. (B) Photomicrograph of 3T3-L1 cells stained with ORO solution (magnification, 200×). Data are expressed as mean ± SD (n = 3).*, P < 0.001 versus control. ND refers to non-differentiated group Copyright © 2015 John Wiley & Sons, Ltd.
Previous studies reported that activation of energy-sensing proteins, including AMPK, inhibits adipogenesis. AMPK is a protein kinase that plays a central role in regulating cellular metabolism and energy balance in adipose tissue. Thus, we determined the effects of euphorbiasteroid on phosphorylation of AMPK. Euphorbiasteroid increases phosphorylation of AMPK in the first stage of differentiation. In addition, euphorbiasteroid induce phosphorylation of ACC, a substrate of AMPK, which led to inhibition of lipid synthesis pathways (Figure 6). Cell Biochem Funct 2015; 33: 220–225.
anti-adipogenic effect of euphorbiasteroid
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Figure 2. Effects of euphorbiasteroid on differentiation at each stage in differentiating 3T3-L1 cells. 3T3-L1 cells were treated with or without euphorbiasteroid at concentrations of 12.5 and 25 μM in each differentiation medium. After differentiation, triglycerides in matured 3T3-L1 cells were stained with Oil red O solution. Data are expressed as mean ± SD (n = 3). ***, P < 0.001 versus control. ND refers to non-differentiated group
DISCUSSION
Figure 3. Effects of euphorbiasteroid on the expression of fatty acid synthase (FAS), CCAAT/enhancer binding proteins (C/EBPs), peroxisome proliferator-activated receptor-γ (PPAR-γ) and sterol regulatory elementbinding protein-1c (SREBP-1c) in differentiating 3T3-L1 cells. 3T3-L1 cells were treated with euphorbiasteroid at concentrations of 12.5 and 25 μM in differentiation medium for 4 days. Cytosolic proteins were extracted and used to determine the levels of proteins related to adipogenesis. ND defines non-differentiated group
Taken together, euphorbiasteroid inhibits adipogenesis of 3T3-L1 cells through activation of the AMPK pathway. Copyright © 2015 John Wiley & Sons, Ltd.
A range of evidence obtained from in vivo and human studies supports the idea that long-term consumption of natural anti-obesity agents prevents or lowers the occurrence of obesity.23 Anti-obesity agents inhibit weight gain and/or reduce body fat through various mechanisms such as reduction of de novo fat synthesis, expenditure of stored energy, loss of appetite, disturbance of fat digestion and absorption and stimulation of calorie restriction-like effects. De novo fat synthesis is mainly occurring in adipocytes, a major population of white adipose tissue.24–26 Adipogenesis has been used for a long time to identify biomarkers for the development of anti-obesity agents. By using in vitro tests, numerous natural products were identified as anti-adipogenic agents and many of them were also effective in diet-induced and/or genetically obese animal models.27,28 In this study, we demonstrated that euphorbiasteroid suppresses adipogenic differentiation of 3T3-L1 cells, mainly at the early stage, and stimulates the AMPK signalling pathway. Previous studies reported that AMPK activators such as 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), A-769662, RSVA314 and RSVA405 inhibit adipogenesis and decrease the levels of major genes involved in adipogenesis, including PPAR-γ, C/EPBα and FAS. In addition, AICAR is effective to restore metabolic alterations in mice in which obesity was induced by feeding of high-fat diets.29,30 These findings indicate that activation of AMPK is sufficient to terminate adipogenesis in pre-adipocytes. Thus, we are suggesting that the anti-adipogenic effects of euphorbiasteroid could Cell Biochem Funct 2015; 33: 220–225.
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ET AL.
Figure 6. Effects of euphorbiasteroid on phosphorylation of AMPactivated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in 3T3-L1 cells. 3T3-L1 cells were treated with euphorbiasteroid at concentrations of 12.5 and 25 μM in differentiation medium for 2 days. Cytosolic proteins were extracted and used to determine the phosphorylation of AMPK and ACC. ND defines non-differentiated group
Figure 4. Effects of euphorbiasteroid on the gene expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancerbinding protein α (C/EBP-α) in differentiating 3T3-L1 cells. 3T3-L1 cells were treated with euphorbiasteroid at concentrations of 25 μM in differentiation medium for 2 or 4 days. mRNA was extracted and used to determine the levels of PPAR-γ and C/EBP-α gene expressions. ND defines non-differentiated group. Data are expressed as mean ± SD (n = 3). ***, P < 0.001 versus adipogenesis induction medium (MDI)
In addition, activation of the AMPK pathway suppresses the development of obesity through lowering enzymes related to gluconeogenesis and energy storage and stimulating mitochondrial biogenesis in liver and glucose uptake in muscle and other organs. These research data support the hypothesis that AMPK activation stimulated by euphorbiasteroid contributes to the prevention of obesity. Taken together, euphorbiasteroid inhibits adipogenesis of 3T3-L1 cells through activation of the AMPK pathway. Therefore, euphorbiasteroid and its source plant, E. lathyris L., could possibly be used as an anti-obesity agent. However, further studies, including animal studies, are required.
ACKNOWLEDGEMENTS This work was supported by research grants from the Korea Food Research Institute, Republic of Korea and 2014 KU Brain Pool of Konkuk University, Seoul, Korea.
REFERENCES
Figure 5. Effects of euphorbiasteroid on phosphorylation of Protein Kinase B (Akt) and extracellular signal-regulated kinase (ERK) 1/2 in 3T3-L1 cells. 3T3-L1 cells were treated with euphorbiasteroid at concentrations of 12.5 and 25 μM in differentiation medium for 30 min. Cytosolic proteins were extracted and used to determine the phosphorylation of Akt and ERK 1/2. ND defines non-differentiated group
possibly be attributed to activation of the AMPK pathway, by decreasing the level of FAS and its up-regulators, including C/EBPs, PPAR-γ and SREBP-1c, without involving insulin signalling pathway. Copyright © 2015 John Wiley & Sons, Ltd.
1. Kopelman PG. Obesity as a medical problem. Nature 2000; 404: 635–643. 2. Cristancho AG, Lazar MA. Forming functional fat: a growing understanding of adipocyte differentiation. Nat Rev Mol Cell Biol 2011; 12: 722–734. 3. Rosen ED, Spiegelman BM. Adipocytes as regulators of energy balance and glucose homeostasis. Nature 2006; 444: 847–853. 4. Hauner H, Schmid P, Pfeiffer EF. Glucocorticoids and insulin promote the differentiation of human adipocyte precursor cells into fat cells. J Clin Endocrinol Metab 1987; 64: 832–835. 5. Fontana L, Klein S. Aging, adiposity, and calorie restriction. JAMA 2007; 297: 986–994. 6. Rosen ED, Hsu C-H, Wang X, et al. C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway. Genes Dev 2002; 16: 22–26. 7. Kahn BB, Alquier T, Carling D, Hardie DG. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 2005; 1: 15–25. Cell Biochem Funct 2015; 33: 220–225.
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21. Choi JS, Kang NS, Min YK, Kim SH. Euphorbiasteroid reverses P-glycoprotein-mediated multi-drug resistance in human sarcoma cell line MES-SA/Dx5. Phytother Res 2010; 24: 1042–1046. 22. Davaatseren M, Hur HJ, Yang HJ, et al. Taraxacum official (dandelion) leaf extract alleviates high-fat diet-induced nonalcoholic fatty liver. Food Chem Toxicol 2013; 58: 30–36. 23. Barone GB, Kinzel LS, Pettee GK, Chang Y-F, Kuller LH. Short- and long-term eating habit modification predicts weight change in overweight, postmenopausal women: results from the WOMAN study. J Acad Nutr Diet 2012; 112: 1347–1355, 1355.e1–2. 24. Cho KJ, Moon HE, Moini H, Packer L, Yoon DY, Chung AS. Alphalipoic acid inhibits adipocyte differentiation by regulating proadipogenic transcription factors via mitogen-activated protein kinase pathways. J Biol Chem 2003; 278: 34823–34833. 25. Kumar SG, Rahman MA, Lee SH, Hwang HS, Kim HA, Yun JW. Plasma proteome analysis for anti-obesity and anti-diabetic potentials of chitosan oligosaccharides in ob/ob mice. Proteomics 2009; 9: 2149–2162. 26. Lodhi IJ, Yin L, Jensen-Urstad AP, et al. Inhibiting adipose tissue lipogenesis reprograms thermogenesis and PPARγ activation to decrease diet-induced obesity. Cell Metab 2012; 16: 189–201. 27. Yang JY, Della-Fera MA, Rayalam S, et al. Enhanced inhibition of adipogenesis and induction of apoptosis in 3 T3-L1 adipocytes with combinations of resveratrol and quercetin. Life Sci 2008; 82: 1032–1039. 28. Rosen ED, MacDougald OA. Adipocyte differentiation from the inside out. Nat Rev Mol Cell Biol 2006; 7: 885–896. 29. Vingtdeux V, Chandakkar P, Zhao H, Davies P, Marambaud P. Smallmolecule activators of AMP-activated protein kinase (AMPK), RSVA314 and RSVA405, inhibit adipogenesis. Mol Med 2011; 17: 1022–1030. 30. do Ha T1, Trung TN, Phuong TT, Yim N, Chen QC, Bae K. The selected flavonol glycoside derived from Sophorae Flos improves glucose uptake and inhibits adipocyte differentiation via activation AMPK in 3 T3-L1 cells. Bioorg Med Chem Lett 2010; 20: 6076–6081.
SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at publisher’s web site.
Cell Biochem Funct 2015; 33: 220–225.
Euphorbiasteroid, a component of Euphorbia lathyris L., inhibits adipogenesis of 3T3-L1 cells via activation of AMP-activated protein kinase Su-Jin Park1, Jae Ho Park1, Anna Han2, Munkhtugs Davaatseren3, Hyun Jin Kim4, Myung-Sunny Kim1, Haeng Jeon Hur1*, Mi-Jeong Sung1, Jin-Taek Hwang1, Hye Jeong Yang1 and Dae Young Kwon1 1
Korea Food Research Institute, Division of Nutrition And Metabolism Research, Seongnam-si, Korea The University of Tennessee-Knoxville, Department of Nutrition, College of Education, Health & Human Sciences, Knoxville, TN, USA 3 Konkuk University, Department of Bioresources and Food Science, College of Life and Environmental Sciences, Seoul, Gwangjin-Gu, Korea 4 Gyeongsang National University, Department of Food Science & Technology, Jinju, Korea 2
The purpose of this study is to investigate the effects of euphorbiasteroid, a component of Euphorbia lathyris L., on adipogenesis of 3T3-L1 pre-adipocytes and its underlying mechanisms. Euphorbiasteroid decreased differentiation of 3T3-L1 cells via reduction of intracellular triglyceride (TG) accumulation at concentrations of 25 and 50 μM. In addition, euphorbiasteroid altered the key regulator proteins of adipogenesis in the early stage of adipocyte differentiation by increasing the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase. Subsequently, levels of adipogenic proteins, including fatty acid synthase, peroxisome proliferator-activated receptor-γ and CCAAT/enhancer-binding protein α, were decreased by euphorbiasteroid treatment at the late stage of adipocyte differentiation. The anti-adipogenic effect of euphorbiasteroid may be derived from inhibition of early stage of adipocyte differentiation. Taken together, euphorbiasteroid inhibits adipogenesis of 3T3-L1 cells through activation of the AMPK pathway. Therefore, euphorbiasteroid and its source plant, E. lathyris L., could possibly be one of the fascinating anti-obesity agent. Copyright © 2015 John Wiley & Sons, Ltd. key words—euphorbiasteroid; obesity; adipogenesis; AMPK; 3T3-L1
INTRODUCTION Obesity is one of the critical health problems because it is associated with numerous serious diseases such as diabetes, hyperlipidemia, fatty liver diseases, hypertension, coronary heart disease and cancer.1 An abnormal increase in white adipose tissue (WAT), characterized by increases in cell size and number of adipocytes, is a major phenotype of obesity.2 Adipocytes play a key role in energy metabolism because they store excess energy in the form of triglycerides (TGs) and release energy in the form of glycerol and fatty acids.3 Pre-adipocytes differentiate into mature adipocytes because of excessive calorie intake and other various factors such as stimulation of insulin and glucocorticoids.4,5 Adipocyte differentiation can be divided into two main stages: mesenchymal stem cells differentiate into preadipocytes and then undergo terminal differentiation to mature adipocytes, which is accompanied by adipogenic proteins and/or gene expression changes, including CCAAT/enhancer binding proteins (C/EBPs), sterol regulatory element-binding
*Correspondence to: Haeng Jeon Hur, Korea Food Research Institute, Division of Nutrition And Metabolism Research, Seongnam-si, Korea. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
protein-1c (SREBP-1c) and peroxisome proliferator-activated receptor-γ (PPAR-γ).2,6 After differentiation, adipocytes regulate lipid metabolism through lipogenic proteins such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) and lipolytic enzymes such as hormone-sensitive lipase.3 AMP-activated protein kinase (AMPK) is a sensory protein that controls energy metabolism through regulation of the energy status.7 AMPK stimulates energy-producing processes such as cellular energy uptake, fatty acid oxidation, glycolysis and ketogenesis and inhibits energyconsuming processes such as lipogenesis, protein synthesis and gluconeogenesis.8 In adipocytes, activation of AMPK reversibly phosphorylates and inactivates ACC to inhibit the lipogenic pathway and increase adenosine triphosphate (ATP) production.9 Therefore, many studies have focused on the AMPK signalling pathway that appears to have an important role in preventing obesity and related metabolic diseases.10 In many scientific studies, natural compounds, such as resveratrol, epigallocatechin-3-gallate, quercetin, berberine, curcumin and ginsenosides, which activate the AMPK signalling pathway, are showing beneficial effects to inhibit adipogenesis in vitro and/or prevent the development of obesity induced by high fat diet in vivo.11–16 Received 16 July 2014 Revised 23 March 2015 Accepted 23 March 2015
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anti-adipogenic effect of euphorbiasteroid Euphorbia lathyris L. (E. lathyris L.), also known as caper spurge, is the biggest species of spurge family that have been widely used as traditional medicinal plant in Asia, especially in China.17 Recent studies reported that E. lathyris L. and its biologically active components including lathyrane, esculetin, ingenol derivatives and euphorbiasteroid (is a tricyclic diperpene, not a steroid), have shown beneficial effects on several disorders such as reverses of multi-drug resistance, inhibits tyrosinase and human immunodeficiency virus and act as anti-cancer drugs.18–21 However, the effects of E. lathyris L. and its biologically active components on obesity and their underlying mechanisms have not been studied yet. The present study was undertaken to investigate the potential anti-adipogenic activities of euphorbiasteroid on obesity by determining its effects on adipocyte differentiation and adipogenic protein expression.
Western blot Cytosolic proteins were extracted with a radioimmunoprecipitation assay (RIPA) buffer containing a SigmaFAST™ Protease Inhibitor Cocktail tablet (Sigma-Aldrich) and phosphatase inhibitors (2.5 mM sodium fluoride, 50 μM sodium orthovanadate, 500 μM sodium pyrophosphate and 500 μM β-glycerophosphate). Isolated proteins were quantified using the Bradford-Reagent (Biosesang, Seoul, Korea). Then, 20 to 50 μg proteins were separated on 12.5% acrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were then transferred to nitrocellulose membranes at 100 V for 1 h. All antibodies were used according to the manufacturer’s instructions, and protein bands were detected using an EZ-Western chemiluminescent detection kit (Daeil Lab., Seoul, Korea). Real time polymerase chain reaction (PCR)
MATERIALS AND METHODS Chemicals Euphorbiasteroid was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Bovine insulin, isobutylmethylxanthine and dexamethasone and all other chemicals and reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). Cell culture The murine pre-adipocyte cell line 3T3-L1 (American Type Culture Collection, Manassas, VA, USA) was maintained in culture medium (CM), Dulbecco’s modified Eagle’s medium (DMEM), containing 4.5 g/L D-glucose (Welgene, Daegu, Korea) supplemented with 10% foetal bovine serum (FBS) (Welgene) and penicillin/streptomycin (Welgene) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. For the differentiation to adipocytes, 3T3-L1 cells were grown in 96-well plates for 2 days to full confluency. At first (S1)-stage, CM were replaced with adipogenesis induction medium (MDI: 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone and 10 μg/ml bovine insulin in CM) and maintained for 2 days. At second (S2)-stage, MDI media were replaced with adipogenesis medium (AM: 10 μg/ml bovine insulin in CM) and maintained for 2 days. For the last (S3)-stage, AM medium was replaced with CM and maintained for 2 days. 3T3-L1 cells were treated with or without euphorbiasteroid at each differentiation stage. Oil red O staining For Oil red O (ORO) staining, differentiated 3T3-L1 cells were fixed with 4% para-formaldehyde for 1 h. After washing three times with phosphate-buffered saline (PBS), cells were stained with ORO solution (0.5% ORO in propylene glycol) for 1 h. After washing once with PBS, cells were washed three times with double-distilled water, dried in air and solubilized with isopropyl alcohol. Absorbance was measured using an enzyme-linked immunosorbent assay reader (Molecular Devices, CA, USA) at 550 nm. Copyright © 2015 John Wiley & Sons, Ltd.
Total RNA was isolated from cells using TRIzol reagent (Gibco, Grand Island, NY, USA). Total RNA (1 μg) was then reverse-transcribed into cDNA using AccuPower® PCR PreMix (Bioneer, Daejeon, Korea). The amplification reactions were performed on a Roche Light-Cycler® 480 system (Roche Diagnostics GmbH, Mannheim, Germany) using the following thermal cycling conditions: an initial activation step at 95 °C for 3 min, followed by 45 cycles of denaturation for 10 s at 95 °C, annealing for 15 s at 55 °C and extension for 20 s at 72 °C. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene, and the fold change in expression of each target mRNA relative to GAPDH was calculated based on the comparative 2-ΔΔct expression method. Primers were as follows: PPAR-γ: forward, 5’-CCC TGG CAA AGC ATT TGT AT-3’; reverse, 5’-ACT GGC ACC CTT GAA AAA TG-3’; C/EBP-α: forward, 5’-AGG TGC TGG AGT TGA CCA GT-3’; reverse, 5’-CAG CCT AGA GAT CCA GCG AC-3’; and GAPDH: forward, 5’-CAT GGC CTT CCG TGT TCC TA-3’; reverse 5’-GCG GCA CGT CAG ATC CA-3’. Statistical analysis Data are presented as mean ± standard deviation (SD). Statistical differences between the groups were assessed using Analysis of variance (one-way ANOVA followed by Tukey’s honest significant difference test). A value of P < 0.001 was considered statistically significant.
RESULTS Effects of euphorbiasteroid on differentiation of 3T3-L1 pre-adipocytes Adipogenesis is characterized by the formation of intracellular lipid droplets and expression of an endocrine cell-like phenotype. To evaluate the effects of euphorbiasteroid on adipogenesis, we measured TG content in differentiated 3T3-L1 cells treated with euphorbiasteroid. At concentrations of 25 μM and 50 μM, euphorbiasteroid significantly Cell Biochem Funct 2015; 33: 220–225.
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decreased TG accumulation in 3T3-L1 cells to the level of non-differentiation group (ND) (Figure 1). These data indicate that euphorbiasteroid could exert anti-adipogenic activity through inhibition or delay of adipogenesis in 3T3-L1 cells. Many anti-obesity agents inhibit adipogenesis by targeting genes involved in early adipogenesis such as phosphoinositide 3-kinase (PI-3K) pathway proteins and AMPK, however, some anti-obesity agents target adipogenesis in the late phase such as adipocyte fatty acid binding protein 2, FAS and ACC.22 To investigate whether euphorbiasteroid represents antiadipogenic activity during differentiation, we quantified the TG content in 3T3-L1 cells treated with or without euphorbiasteroid at each differentiation stages (S1, S2, S3). Treatment with euphorbiasteroid at the S1-stage inhibited
ET AL.
TG accumulation in differentiated 3T3-L1 cells and its anti-adipogenic activity was followed for S2-stage and S3stage. However, euphorbiasteroid treatment had no effect on intracellular TGs in differentiated 3T3-L1 cells at the S2-stage and S3-stage. On the basis of these results, we propose that euphorbiasteroid interferes with the progression of adipogenesis at the early stages of adipocyte differentiation (Figure 2). Effects of euphorbiasteroid on the expression of FAS, C/EBPs, PPAR-γ and SREBP-1c in 3T3-L1 cells during adipogenesis In order to examine the effect of euphorbiasteroid on adipogenic proteins, we measured the expression levels of FAS, C/EBPs, PPAR-γ and SREBP-1c in the cytosolic fraction of 3T3-L1 cells. Euphorbiasteroid decreased FAS levels in differentiated 3T3-L1 cells at 12.5 and 25 μM. Because FAS is a key regulator of lipid synthesis in adipocytes, its decrease led to a reduction in the TG level in 3T3-L1 cells. Next, we determined the effects of euphorbiasteroid on the expression of C/EBPs and PPAR-γ, upstream regulators of FAS, at the S2-stage or S3-stage of differentiation. We found that C/EBPs, PPAR-γ and SREBP-1c levels were decreased by euphorbiasteroid treatment in a dose-dependent manner (Figure 3). In addition, euphorbiasteriod treatment significantly decreased the C/EBPα and PPAR-γ gene expression in 3T3-L1 cells (Figure 4). These results are indicating that the underlying mechanism of the anti-adipogenic effects of euphorbiasteroid is related to the inhibition of signals involved in the initial stage of adipogenesis or activation of anti-adipogenic proteins and gene expressions. Effects of euphorbiasteroid on phosphorylation of Akt and ERK 1/2 in 3T3-L1 adipocytes The insulin signalling pathway is involved in the early stage of adipogenesis through activation of PI-3K and mitogenactivated protein kinases including extracellular signalregulated kinase (ERK) 1/2. However, euphorbiasteroid treatment did not significantly affect phosphorylation of ERK 1/2 and Protein Kinase B (Akt) (Figure 5). Effects of euphorbiasteroid on phosphorylation of AMPK and ACC in 3T3-L1 adipocytes
Figure 1. Effects of euphorbiasteroid on triglyceride accumulation in differentiating 3T3-L1 cells. 3T3-L1 cells were treated with euphorbiasteroid at concentrations of 6.25, 12.5, 25 and 50 μM for 2 days in adipogenesis induction medium (AI: 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone and 10 μg/ml bovine insulin in culture medium (CM)), 2 days in adipogenesis medium (AM: CM with 10 μg/ml bovine insulin) and 2 days in CM sequentially during differentiation. Intracellular triglycerides (TGs) were stained with Oil red O (ORO) solution. (A) Intracellular TGs levels were measured and quantified as fold change compared with the control. (B) Photomicrograph of 3T3-L1 cells stained with ORO solution (magnification, 200×). Data are expressed as mean ± SD (n = 3).*, P < 0.001 versus control. ND refers to non-differentiated group Copyright © 2015 John Wiley & Sons, Ltd.
Previous studies reported that activation of energy-sensing proteins, including AMPK, inhibits adipogenesis. AMPK is a protein kinase that plays a central role in regulating cellular metabolism and energy balance in adipose tissue. Thus, we determined the effects of euphorbiasteroid on phosphorylation of AMPK. Euphorbiasteroid increases phosphorylation of AMPK in the first stage of differentiation. In addition, euphorbiasteroid induce phosphorylation of ACC, a substrate of AMPK, which led to inhibition of lipid synthesis pathways (Figure 6). Cell Biochem Funct 2015; 33: 220–225.
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Figure 2. Effects of euphorbiasteroid on differentiation at each stage in differentiating 3T3-L1 cells. 3T3-L1 cells were treated with or without euphorbiasteroid at concentrations of 12.5 and 25 μM in each differentiation medium. After differentiation, triglycerides in matured 3T3-L1 cells were stained with Oil red O solution. Data are expressed as mean ± SD (n = 3). ***, P < 0.001 versus control. ND refers to non-differentiated group
DISCUSSION
Figure 3. Effects of euphorbiasteroid on the expression of fatty acid synthase (FAS), CCAAT/enhancer binding proteins (C/EBPs), peroxisome proliferator-activated receptor-γ (PPAR-γ) and sterol regulatory elementbinding protein-1c (SREBP-1c) in differentiating 3T3-L1 cells. 3T3-L1 cells were treated with euphorbiasteroid at concentrations of 12.5 and 25 μM in differentiation medium for 4 days. Cytosolic proteins were extracted and used to determine the levels of proteins related to adipogenesis. ND defines non-differentiated group
Taken together, euphorbiasteroid inhibits adipogenesis of 3T3-L1 cells through activation of the AMPK pathway. Copyright © 2015 John Wiley & Sons, Ltd.
A range of evidence obtained from in vivo and human studies supports the idea that long-term consumption of natural anti-obesity agents prevents or lowers the occurrence of obesity.23 Anti-obesity agents inhibit weight gain and/or reduce body fat through various mechanisms such as reduction of de novo fat synthesis, expenditure of stored energy, loss of appetite, disturbance of fat digestion and absorption and stimulation of calorie restriction-like effects. De novo fat synthesis is mainly occurring in adipocytes, a major population of white adipose tissue.24–26 Adipogenesis has been used for a long time to identify biomarkers for the development of anti-obesity agents. By using in vitro tests, numerous natural products were identified as anti-adipogenic agents and many of them were also effective in diet-induced and/or genetically obese animal models.27,28 In this study, we demonstrated that euphorbiasteroid suppresses adipogenic differentiation of 3T3-L1 cells, mainly at the early stage, and stimulates the AMPK signalling pathway. Previous studies reported that AMPK activators such as 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), A-769662, RSVA314 and RSVA405 inhibit adipogenesis and decrease the levels of major genes involved in adipogenesis, including PPAR-γ, C/EPBα and FAS. In addition, AICAR is effective to restore metabolic alterations in mice in which obesity was induced by feeding of high-fat diets.29,30 These findings indicate that activation of AMPK is sufficient to terminate adipogenesis in pre-adipocytes. Thus, we are suggesting that the anti-adipogenic effects of euphorbiasteroid could Cell Biochem Funct 2015; 33: 220–225.
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ET AL.
Figure 6. Effects of euphorbiasteroid on phosphorylation of AMPactivated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in 3T3-L1 cells. 3T3-L1 cells were treated with euphorbiasteroid at concentrations of 12.5 and 25 μM in differentiation medium for 2 days. Cytosolic proteins were extracted and used to determine the phosphorylation of AMPK and ACC. ND defines non-differentiated group
Figure 4. Effects of euphorbiasteroid on the gene expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancerbinding protein α (C/EBP-α) in differentiating 3T3-L1 cells. 3T3-L1 cells were treated with euphorbiasteroid at concentrations of 25 μM in differentiation medium for 2 or 4 days. mRNA was extracted and used to determine the levels of PPAR-γ and C/EBP-α gene expressions. ND defines non-differentiated group. Data are expressed as mean ± SD (n = 3). ***, P < 0.001 versus adipogenesis induction medium (MDI)
In addition, activation of the AMPK pathway suppresses the development of obesity through lowering enzymes related to gluconeogenesis and energy storage and stimulating mitochondrial biogenesis in liver and glucose uptake in muscle and other organs. These research data support the hypothesis that AMPK activation stimulated by euphorbiasteroid contributes to the prevention of obesity. Taken together, euphorbiasteroid inhibits adipogenesis of 3T3-L1 cells through activation of the AMPK pathway. Therefore, euphorbiasteroid and its source plant, E. lathyris L., could possibly be used as an anti-obesity agent. However, further studies, including animal studies, are required.
ACKNOWLEDGEMENTS This work was supported by research grants from the Korea Food Research Institute, Republic of Korea and 2014 KU Brain Pool of Konkuk University, Seoul, Korea.
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Figure 5. Effects of euphorbiasteroid on phosphorylation of Protein Kinase B (Akt) and extracellular signal-regulated kinase (ERK) 1/2 in 3T3-L1 cells. 3T3-L1 cells were treated with euphorbiasteroid at concentrations of 12.5 and 25 μM in differentiation medium for 30 min. Cytosolic proteins were extracted and used to determine the phosphorylation of Akt and ERK 1/2. ND defines non-differentiated group
possibly be attributed to activation of the AMPK pathway, by decreasing the level of FAS and its up-regulators, including C/EBPs, PPAR-γ and SREBP-1c, without involving insulin signalling pathway. Copyright © 2015 John Wiley & Sons, Ltd.
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Cell Biochem Funct 2015; 33: 220–225.
