In Vitro Cell.Dev.Biol.—Animal (2014) 50:851–857 DOI 10.1007/s11626-014-9789-3
Aspartame downregulates 3T3-L1 differentiation Muthuraman Pandurangan & Jeongeun Park & Eunjung Kim
Received: 25 November 2013 / Accepted: 9 June 2014 / Published online: 25 June 2014 / Editor: T. Okamoto # The Society for In Vitro Biology 2014
Abstract Aspartame is an artificial sweetener used as an alternate for sugar in several foods and beverages. Since aspartame is 200 times sweeter than traditional sugar, it can give the same level of sweetness with less substance, which leads to lower-calorie food intake. There are reports that consumption of aspartame-containing products can help obese people lose weight. However, the potential role of aspartame in obesity is not clear. The present study investigated whether aspartame suppresses 3T3-L1 differentiation, by downregulating phosphorylated peroxisome proliferator-activated receptor γ (p-PPARγ), peroxisome proliferator-activated receptor γ (PPARγ), fatty acid-binding protein 4 (FABP4), CCAA T/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1), which are critical for adipogenesis. The 3T3-L1 adipocytes were cultured and differentiated for 6 d in the absence and presence of 10 μg/ml of aspartame. Aspartame reduced lipid accumulation in differentiated adipocytes as evidenced by Oil Red O staining. qRTPCR analysis showed that the PPARγ, FABP4, and C/EBPα mRNA expression was significantly reduced in the aspartametreated adipocytes. Western blot analysis showed that the induction of p-PPARγ, PPARγ, SREBP1, and adipsin was markedly reduced in the aspartame-treated adipocytes. Taken together, these data suggest that aspartame may be a potent substance to alter adipocyte differentiation and control obesity.
Keywords Aspartame . 3T3-L1 . Differentiation . Lipid accumulation . Obesity M. Pandurangan : J. Park : E. Kim (*) Department of Food Science and Nutrition, Catholic University of Daegu, 13-13 Hayang-ro, Hayang-eup, Gyeongsan 712-702, South Korea e-mail: [email protected]
Introduction The prevalence of obesity has been increased considerably over recent years in most developed and industrialized countries. Obesity is a systemic disease that predisposes the impaired individual to several comorbidities and complications such as hypertension, hyperlipemia, type 2 diabetes, and arteriosclerosis, which affect overall health. An increase in obesity will also aggravate the morbidity of any concurrent disease (Hirotaka et al. 2013). Strategies to reverse the upward trend in obesity rates need to focus on both decreasing energy intake and increasing energy expenditure. The provision of low-energy-dense foods is one of the best ways of helping people to reduce their energy intake and lose weight. The application of intense sweeteners as a substitute for sucrose potentially offers one of the ways of helping people to reduce the energy density of their diet without affecting palatability (Haslam and James 2005). Aspartame is the most widely used artificial sweetener and is used in a wide variety of foods, beverages, and drugs. Aspartame was first approved by the US Food and Drug Administration for use in solid food in 1981 (FDA 1981). Later, it was extended to soft drinks (FDA 1983). The acceptable daily intake of aspartame is currently 50 mg/kg body weight (BW) in the USA and 40 mg/kg BW in the European Union for both children and adults. Aspartame is metabolized in the gastric tract to its three metabolites: aspartic acid, phenylalanine, and methanol. In vitro and in vivo tests have shown that aspartame is not genotoxic. DNA repair assay for the genotoxicity evaluation did not show any significant DNA-damaging properties of aspartame (Jeffrey and Williams 2000). It has been reported that the use of aspartame is associated with an increased feeling of hunger (Rogers et al. 1988; Blundell and Green 1996; Lavin et al. 1997; Appleton and Blundell 2007) and with the increasing prevalence of obesity
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(Popkin and Nielsen 2003; Fowler et al. 2008; Yang 2010). Recently, however, ample evidences showed that increased consumption of aspartame may facilitate weight control without affecting satiety (Canty and Chan 1991; Drewnowski et al. 1994; Renwick 1994; Blackburn et al. 1997; Raben et al. 2002; Van Wymelbeke et al. 2004; Phelan et al. 2009). De la Hunty et al. (2006) reported that aspartame was useful in reducing body weight and the maintenance of lower body weight after dieting. The meta-analyses of energy intake and weight loss demonstrated that the foods and drinks sweetened with aspartame instead of sucrose resulted in a significant reduction in energy intake and body weight (0.2 kg/wk) (De la Hunty et al. 2006). Raben and Richelson (2012) also reported that artificial sweeteners can be useful in maintaining reduced energy intake, body weight, decreased risk of type 2 diabetes, and cardiovascular disease compared to sucrose. Drewnowski (1999) reported that the application of intense sweeteners maintains sweetness with reduced energy density. This suggests that the substitution of calorie-rich sugar with low-calorie sweeteners may be an efficient method of weight control (Anton et al. 2010). Nevertheless, the effects of aspartame on the body weight and obesity are still inconclusive. In this study, we therefore investigated the anti-obesity effect of aspartame, focusing on 3T3-L1 preadipocyte differentiation. To our best knowledge, this is the first report showing that aspartame inhibits 3T3-L1 differentiation by downregulating adipogenic gene expression.
Materials and Methods Materials. 3T3-L1 preadipocytes were obtained from American Type Culture Collection. Dulbecco’s modified Eagle’s medium (DMEM), insulin, 3-isobutyl-1-methylxanthine, dexamethasone, and 3-(4, 5-dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical (St Louis, MO). Aspartame was purchased from Supelco (Bellefonte, PA). Fetal bovine serum (FBS), penicillin/streptomycin, and trypsin-EDTA were purchased from Gibco (Gaithersburg, MD). Antibodies for phosphorylated peroxisome proliferator-activated receptor γ (p-PPARγ) (S273) (Bioss Inc., Woburn, MA), peroxisome proliferatoractivated receptor γ (PPARγ), sterol regulatory elementbinding protein 1 (SREBP1), adipsin, and glyceraldehyde-3phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotech, Inc. (Santa Cruz, TX). Primers of PPARγ, fatty acid-binding protein 4 (FABP4), and CCAAT/enhancerbinding protein α (C/EBPα) were purchased from Macrogen (Seoul, South Korea). Reverse transcriptase and Taq DNA polymerase were purchased from Promega (Madison, WI). Cell culture. 3T3-L1 preadipocytes were incubated at a density of 8,000 cells/cm2 and grown in DMEM containing 10%
fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in 5% CO2. Confluent 3T3-L1 preadipocytes were induced to differentiate with a standard differentiation medium consisting of DMEM supplemented with 10% FBS, 250 nM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 5 μg/ml insulin, and antibiotics. Cultures were refed every 2 d to allow 90% of the cells to reach complete differentiation (Muthuraman and Ravikumar 2013). Cells with the differentiation medium were considered controls. Cells with the differentiation medium and aspartame were considered treatment. Both control and aspartame-treated 3T3-L1 cells were maintained in the differentiation medium for 6 d. MTT assay. The cytotoxicity of aspartame was determined by 3-(4, 5-dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) assay (Kim et al. 2008; Hu and Davies 2009). 3T3L1 preadipocytes were seeded at a seeding density of 1.5×104 cells/ml into 96-well microplates and allowed to adhere for 24 h and treated with aspartame for 6 d. Cells were labeled with MTT solution for 4 h, and the resulting formazan was solubilized in DMSO. The absorption was measured at 570 nm by plate reader. Measurement of lipid accumulation. 3T3-L1 adipocyte differentiation was determined by measuring cellular lipid accumulation. Lipid accumulation was determined by the Oil Red O staining method (Sen et al. 2001). 3T3-L1 adipocytes differentiated on six-well plates were fixed with 10% ice-cold formalin in phosphate-buffered saline (PBS) for 2 h and stained by 1.5 ml of working Oil Red O solution (stock, 0.5 g in 100 ml of isopropanol; working, 30 ml of stock solution in 20 ml of distilled water) for 10 min. The cells were rinsed with distilled water. The cell-retained reddish dye was eluted by isopropanol for 1 h and measured at 500 nm. A blank value was obtained by staining with the dye (without cells). qRT-PCR. Total RNA was isolated from control and aspartame-treated adipocytes. RNA was reversibly transcribed with reverse transcriptase and oligo-(dT) primer. Messenger RNA (mRNA) expression of PPARγ (forward primer: 5′CCACCGTTGACTTCTCCA-3′, reverse primer: 5′-AGGC TCCACTTTGATTGC-3′), FABP4 (forward primer: 5′AGATGAAGGTGCTCTGGT-3′, reverse primer: 5′-CTCA TAAACTCTGGTGGC-3′), C/EBPα (forward primer: 5′GCGGCAAAGCCAAGAAGTCC-3′, reverse primer: 5′GCGGCTCAGTTGTTCCACCC-3′), and GAPDH (forward primer: 5′-CACCCTCAAGATTGTCAGC-3′, reverse primer: 5′-TAAGTCCCTCCACGATGC-3′) were determined by qRT-PCR (Li et al. 2010). The reaction was carried out in 10 μl using SYBR Green Master Mix (Invitrogen, Eugene, OR) according to the manufacturers’ instructions. Relative ratios were calculated based on the 2−△△CT method (Pfaffl
ASPARTAME ON 3T3-L1
853
2001). Relative quantification is based on the expression levels of PPARγ, FABP4, and C/EBPα (target gene) versus GAPDH (reference gene). Expressions of PPARγ, FABP4, and C/EBPα mRNA are presented as fold. PCR was monitored using the Mini Opticon Real Time PCR System (BioRad, Philadelphia, PA). Western blot analysis. Control and aspartame-treated 3T3-L1 adipocytes were washed three times with ice-cold PBS and lysed with 10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1% NP40, 50 mM NaF, 2 mM EDTA (pH 8.0), 1 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. Equal amounts of lysate protein samples were run on SDS polyacrylamide gel and then transferred onto a PVDF membrane. Nonspecific binding was blocked by soaking the membrane in Trisbuffered saline-Tween (TBST) buffer that contained 5% nonfat dry milk for 1 h. The membrane was probed overnight with an antibody against p-PPARγ, PPARγ, SREBP1, and adipsin. After washing with TBST buffer, the membrane was incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG. The protein levels of p-PPARγ, PPARγ, SREBP1, and adipsin were determined by using an enhanced chemiluminescence kit (Bioscience Technology, Pohang, South Korea). Statistical analysis. All the values are expressed as means± SEM. Statistical analysis was performed using SPSS version 16.0 (Statistical Package). Student’s t test and one-way ANOVA test were performed to determine the differences between control and treatments. P
Aspartame downregulates 3T3-L1 differentiation Muthuraman Pandurangan & Jeongeun Park & Eunjung Kim
Received: 25 November 2013 / Accepted: 9 June 2014 / Published online: 25 June 2014 / Editor: T. Okamoto # The Society for In Vitro Biology 2014
Abstract Aspartame is an artificial sweetener used as an alternate for sugar in several foods and beverages. Since aspartame is 200 times sweeter than traditional sugar, it can give the same level of sweetness with less substance, which leads to lower-calorie food intake. There are reports that consumption of aspartame-containing products can help obese people lose weight. However, the potential role of aspartame in obesity is not clear. The present study investigated whether aspartame suppresses 3T3-L1 differentiation, by downregulating phosphorylated peroxisome proliferator-activated receptor γ (p-PPARγ), peroxisome proliferator-activated receptor γ (PPARγ), fatty acid-binding protein 4 (FABP4), CCAA T/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1), which are critical for adipogenesis. The 3T3-L1 adipocytes were cultured and differentiated for 6 d in the absence and presence of 10 μg/ml of aspartame. Aspartame reduced lipid accumulation in differentiated adipocytes as evidenced by Oil Red O staining. qRTPCR analysis showed that the PPARγ, FABP4, and C/EBPα mRNA expression was significantly reduced in the aspartametreated adipocytes. Western blot analysis showed that the induction of p-PPARγ, PPARγ, SREBP1, and adipsin was markedly reduced in the aspartame-treated adipocytes. Taken together, these data suggest that aspartame may be a potent substance to alter adipocyte differentiation and control obesity.
Keywords Aspartame . 3T3-L1 . Differentiation . Lipid accumulation . Obesity M. Pandurangan : J. Park : E. Kim (*) Department of Food Science and Nutrition, Catholic University of Daegu, 13-13 Hayang-ro, Hayang-eup, Gyeongsan 712-702, South Korea e-mail: [email protected]
Introduction The prevalence of obesity has been increased considerably over recent years in most developed and industrialized countries. Obesity is a systemic disease that predisposes the impaired individual to several comorbidities and complications such as hypertension, hyperlipemia, type 2 diabetes, and arteriosclerosis, which affect overall health. An increase in obesity will also aggravate the morbidity of any concurrent disease (Hirotaka et al. 2013). Strategies to reverse the upward trend in obesity rates need to focus on both decreasing energy intake and increasing energy expenditure. The provision of low-energy-dense foods is one of the best ways of helping people to reduce their energy intake and lose weight. The application of intense sweeteners as a substitute for sucrose potentially offers one of the ways of helping people to reduce the energy density of their diet without affecting palatability (Haslam and James 2005). Aspartame is the most widely used artificial sweetener and is used in a wide variety of foods, beverages, and drugs. Aspartame was first approved by the US Food and Drug Administration for use in solid food in 1981 (FDA 1981). Later, it was extended to soft drinks (FDA 1983). The acceptable daily intake of aspartame is currently 50 mg/kg body weight (BW) in the USA and 40 mg/kg BW in the European Union for both children and adults. Aspartame is metabolized in the gastric tract to its three metabolites: aspartic acid, phenylalanine, and methanol. In vitro and in vivo tests have shown that aspartame is not genotoxic. DNA repair assay for the genotoxicity evaluation did not show any significant DNA-damaging properties of aspartame (Jeffrey and Williams 2000). It has been reported that the use of aspartame is associated with an increased feeling of hunger (Rogers et al. 1988; Blundell and Green 1996; Lavin et al. 1997; Appleton and Blundell 2007) and with the increasing prevalence of obesity
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(Popkin and Nielsen 2003; Fowler et al. 2008; Yang 2010). Recently, however, ample evidences showed that increased consumption of aspartame may facilitate weight control without affecting satiety (Canty and Chan 1991; Drewnowski et al. 1994; Renwick 1994; Blackburn et al. 1997; Raben et al. 2002; Van Wymelbeke et al. 2004; Phelan et al. 2009). De la Hunty et al. (2006) reported that aspartame was useful in reducing body weight and the maintenance of lower body weight after dieting. The meta-analyses of energy intake and weight loss demonstrated that the foods and drinks sweetened with aspartame instead of sucrose resulted in a significant reduction in energy intake and body weight (0.2 kg/wk) (De la Hunty et al. 2006). Raben and Richelson (2012) also reported that artificial sweeteners can be useful in maintaining reduced energy intake, body weight, decreased risk of type 2 diabetes, and cardiovascular disease compared to sucrose. Drewnowski (1999) reported that the application of intense sweeteners maintains sweetness with reduced energy density. This suggests that the substitution of calorie-rich sugar with low-calorie sweeteners may be an efficient method of weight control (Anton et al. 2010). Nevertheless, the effects of aspartame on the body weight and obesity are still inconclusive. In this study, we therefore investigated the anti-obesity effect of aspartame, focusing on 3T3-L1 preadipocyte differentiation. To our best knowledge, this is the first report showing that aspartame inhibits 3T3-L1 differentiation by downregulating adipogenic gene expression.
Materials and Methods Materials. 3T3-L1 preadipocytes were obtained from American Type Culture Collection. Dulbecco’s modified Eagle’s medium (DMEM), insulin, 3-isobutyl-1-methylxanthine, dexamethasone, and 3-(4, 5-dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical (St Louis, MO). Aspartame was purchased from Supelco (Bellefonte, PA). Fetal bovine serum (FBS), penicillin/streptomycin, and trypsin-EDTA were purchased from Gibco (Gaithersburg, MD). Antibodies for phosphorylated peroxisome proliferator-activated receptor γ (p-PPARγ) (S273) (Bioss Inc., Woburn, MA), peroxisome proliferatoractivated receptor γ (PPARγ), sterol regulatory elementbinding protein 1 (SREBP1), adipsin, and glyceraldehyde-3phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotech, Inc. (Santa Cruz, TX). Primers of PPARγ, fatty acid-binding protein 4 (FABP4), and CCAAT/enhancerbinding protein α (C/EBPα) were purchased from Macrogen (Seoul, South Korea). Reverse transcriptase and Taq DNA polymerase were purchased from Promega (Madison, WI). Cell culture. 3T3-L1 preadipocytes were incubated at a density of 8,000 cells/cm2 and grown in DMEM containing 10%
fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in 5% CO2. Confluent 3T3-L1 preadipocytes were induced to differentiate with a standard differentiation medium consisting of DMEM supplemented with 10% FBS, 250 nM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 5 μg/ml insulin, and antibiotics. Cultures were refed every 2 d to allow 90% of the cells to reach complete differentiation (Muthuraman and Ravikumar 2013). Cells with the differentiation medium were considered controls. Cells with the differentiation medium and aspartame were considered treatment. Both control and aspartame-treated 3T3-L1 cells were maintained in the differentiation medium for 6 d. MTT assay. The cytotoxicity of aspartame was determined by 3-(4, 5-dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) assay (Kim et al. 2008; Hu and Davies 2009). 3T3L1 preadipocytes were seeded at a seeding density of 1.5×104 cells/ml into 96-well microplates and allowed to adhere for 24 h and treated with aspartame for 6 d. Cells were labeled with MTT solution for 4 h, and the resulting formazan was solubilized in DMSO. The absorption was measured at 570 nm by plate reader. Measurement of lipid accumulation. 3T3-L1 adipocyte differentiation was determined by measuring cellular lipid accumulation. Lipid accumulation was determined by the Oil Red O staining method (Sen et al. 2001). 3T3-L1 adipocytes differentiated on six-well plates were fixed with 10% ice-cold formalin in phosphate-buffered saline (PBS) for 2 h and stained by 1.5 ml of working Oil Red O solution (stock, 0.5 g in 100 ml of isopropanol; working, 30 ml of stock solution in 20 ml of distilled water) for 10 min. The cells were rinsed with distilled water. The cell-retained reddish dye was eluted by isopropanol for 1 h and measured at 500 nm. A blank value was obtained by staining with the dye (without cells). qRT-PCR. Total RNA was isolated from control and aspartame-treated adipocytes. RNA was reversibly transcribed with reverse transcriptase and oligo-(dT) primer. Messenger RNA (mRNA) expression of PPARγ (forward primer: 5′CCACCGTTGACTTCTCCA-3′, reverse primer: 5′-AGGC TCCACTTTGATTGC-3′), FABP4 (forward primer: 5′AGATGAAGGTGCTCTGGT-3′, reverse primer: 5′-CTCA TAAACTCTGGTGGC-3′), C/EBPα (forward primer: 5′GCGGCAAAGCCAAGAAGTCC-3′, reverse primer: 5′GCGGCTCAGTTGTTCCACCC-3′), and GAPDH (forward primer: 5′-CACCCTCAAGATTGTCAGC-3′, reverse primer: 5′-TAAGTCCCTCCACGATGC-3′) were determined by qRT-PCR (Li et al. 2010). The reaction was carried out in 10 μl using SYBR Green Master Mix (Invitrogen, Eugene, OR) according to the manufacturers’ instructions. Relative ratios were calculated based on the 2−△△CT method (Pfaffl
ASPARTAME ON 3T3-L1
853
2001). Relative quantification is based on the expression levels of PPARγ, FABP4, and C/EBPα (target gene) versus GAPDH (reference gene). Expressions of PPARγ, FABP4, and C/EBPα mRNA are presented as fold. PCR was monitored using the Mini Opticon Real Time PCR System (BioRad, Philadelphia, PA). Western blot analysis. Control and aspartame-treated 3T3-L1 adipocytes were washed three times with ice-cold PBS and lysed with 10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1% NP40, 50 mM NaF, 2 mM EDTA (pH 8.0), 1 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. Equal amounts of lysate protein samples were run on SDS polyacrylamide gel and then transferred onto a PVDF membrane. Nonspecific binding was blocked by soaking the membrane in Trisbuffered saline-Tween (TBST) buffer that contained 5% nonfat dry milk for 1 h. The membrane was probed overnight with an antibody against p-PPARγ, PPARγ, SREBP1, and adipsin. After washing with TBST buffer, the membrane was incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG. The protein levels of p-PPARγ, PPARγ, SREBP1, and adipsin were determined by using an enhanced chemiluminescence kit (Bioscience Technology, Pohang, South Korea). Statistical analysis. All the values are expressed as means± SEM. Statistical analysis was performed using SPSS version 16.0 (Statistical Package). Student’s t test and one-way ANOVA test were performed to determine the differences between control and treatments. P
