Oleuropein and Hydroxytyrosol Inhibit Adipocyte Differentiation in 3T3-L1

1 Oleuropein and hydroxytyrosol inhibit adipocyte differentiation in 3T3-L1 2 cells 3 4 Riadh Driraa, Shu Chena and Kazuichi Sakamotoa* 5 6 aGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, 7 Ibaraki 305-8572, Japan 8 9 *Corresponding author. Graduate School of Life and Environmental Sciences, University of 10 Tsukuba, Tennodai 1-1-1, Tsukuba Ibaraki 305-8572, Japan. Tel.: +81 2985346761; fax: +81 11 298534676. 12 E-mail address: [email protected] 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 1 34 Abstract 35 Aims: 36 Oleuropein and hydroxytyrosol, which are antioxidant molecules found in olive leaves and oil, 37 have been reported to exert several biochemical and pharmacological effects. These 38 polyphenols are able to prevent low-density lipoprotein oxidation and protect cells against 39 several diseases. Here, we studied the effect of these compounds on adipocyte differentiation 40 in 3T3-L1. 41 Main Methods: 42 To perform this study, 3T3-L1 preadipocytes viability was analysed via Trypan blue and 43 MTT assays, and triglycerides were stained with Oil Red O. Adipogenesis releated genes 44 expression were checked by RT-PCR and qRT-PCR. Also, cells counting and flow cytometry 45 were used to analyse the mitotic cell cycle during the adipogenesis clonal expansion phase. 46 Results: 47 Oleuropein and hydroxytyrosol dose-dependently suppressed intracellular triglyceride 48 accumulation during adipocyte differentiation without effect on cell viability. PPARγ, 49 C/EBPα and SREBP-1c transcription factors and their downstream targets genes (GLUT4, 50 CD36 and FASN) were down-regulated after treatment by oleuropein and hydroxytyrosol. At 51 200 and 300 µmol/L oleuropein or 100 and 150 µmol/L hydroxytyrosol, the greatest effect on 52 the adipogenesis process was observed during the early stages of differentiation. Flow 53 cytometry revealed both polyphenols to inhibit the division of 3T3-L1 preadipocytes during 54 mitotic clonal expansion and cause cell cycle delay. Furthermore, oleuropein and its derivate 55 hydroxytyrosol decreased the transcriptional activity of SREBP-1c in a stable transfected 56 3T3-L1 cell line. 57 Significance: 58 These findings indicate that both compounds are able to prevent 3T3-L1 differentiation by 59 inhibition of the mitotic clonal expansion and downregulation of the adipogenesis related 60 genes. 61 62 Keywords: adipogenesis, polyphenols, olive leaves, SREBP-1c, cell cycle. 63 64 65 66 67 2 68 Introduction 69 Obesity, a complex disorder with multiple causes that include both genetic and environmental 70 factors, is a major health problem in both developed and developing countries. At the cell 71 biological level, obesity is characterised by an increase in the number and size of adipocytes 72 in adipose tissue, and leads to the development of type II diabetes mellitus, cardiovascular 73 disease and hyperlipidemia (Spiegelman and Flier 1996; Saltiel and Kahn 2001). Several 74 antiobesity mechanisms have thus far been proposed: reduction of energy and food intake, 75 decreased preadipocyte proliferation and differentiation, and increased lipolysis and fat 76 oxidation. Current studies on obesity focus on discovering food ingredients that have the 77 capability to suppress the proliferation and the differentiation of adipocytes in adipose tissues 78 (Lee et al. 2008; Kim et al. 2010; Yang et al. 2006; Maeda et al. 2006). 79 Phenolic compounds, which are secondary plant products, are consumed regularly as part of 80 the human diet and are associated with the prevention of some diseases. Mediterranean diets 81 are associated with lower mortality from cardiovascular disease and cancers (Trichopoulou et 82 al. 2003). Olive tree products are known to be the main source of healthy Mediterranean diet 83 ingredients due to their high phenolic content (Visioli et al. 2002). The phenolic compounds 84 in olive oil and leaves are a complex mixture of secoiridoid derivates that include 85 hydroxytyrosol, tyrosol, hydroxytyrosol acetate and other benzoic and cinnamic acid 86 derivatives (Mateos et al. 2001; Litridou et al. 1997). Oleuropein appears to be the principal 87 phenolic compound in olive oil and leaves (Fig. 1). Its concentration varies with cultivar and 88 climate and is several times higher in the olive leaf than the oil (Ryan et al. 2002). On 89 hydrolysis, oleuropein can produce other bioactive substances, including elenolic acid and 90 hydroxytyrosol (Fig. 1). Several in vitro and in vivo studies have shown that oleuropein and 91 its derivate hydroxytyrosol possess a wide range of biochemical and pharmacological 92 properties. In fact, oleuropein is able to inhibit hyperglycemia and oxidative stress induced in 93 diabetic rabbits, increase the resistance of LDLs to oxidation, inhibit cell proliferation and 94 induce cell apoptosis in MCF-7 cancer cells, and enhance osteoblastogenesis and inhibit 95 adipogenesis in stem cells derived from human bone marrow (Al-Azzawie and Alhamdani 96 2006; Han et al. 2009; Santiago-Mora et al. 2010). In addition, hydroxytyrosol, regarded as 97 the most potent antioxidant in olive leaves and oil phenolic fraction, provides in vitro 98 protection of human hepatoma cells (HepG2) against oxidative stress, inhibits the cell cycle 99 progression in HL60 and MCF-7 cells and reduces in vivo serum levels of total cholesterol, 100 triglycerides and LDL when administered to rats fed a cholesterol-rich diet (Han et al. 2009; 101 Fabiani et al. 2008; Goya et al. 2007; Gonzalez-Santiago et al. 2006). 3 102 To explore the possibility that oleuropein and hydroxytyrosol might inhibit in vitro adipocyte 103 differentiation, we carried out the following experiments to determine the effects of both 104 phenolic compounds on the adipogenesis and differentiation of 3T3-L1 cells. 105 106 107 Materials and methods 108 Materials. 3T3-L1 cells were provided from the Health Science Research Resources Bank 109 (HSRRB, Osaka, Japan). Oleuropein was purchased from Extrasynthese Company (Genay, 110 France). Hydroxytyrosol was obtained from Cayman Chemical Company (Michigan, USA). 111 Dulbecco’s modified Eagle’s medium (DMEM high-glucose), Dexamethasone, 3-iso-butyl-1- 112 methylxanthine, and Insulin were purchased from Sigma-Aldrich (Missouri, USA). 113 114 Cell culture. 3T3-L1 cells were cultured in DMEM medium containing 10% FBS at 37 °C 5 4 115 and 5% CO2. Cells were plated at a density of 3 × 10 cells in 60 mm culture dish, and 5 × 10 116 in 24 wells plate. After reaching the confluence, adipocyte’s differentiation was initiated 117 using the same medium containing 10 mg/L insulin, 0.5 mmol/L isobutylmethylxanthine, and 118 1 µmol/L dexamethasone for 2 days. The medium was then replaced with DMEM containing 119 5 mg/L insulin for more 2 days, and then changed to fresh medium every 2 days. 120 Hydroxytyrosol was diluted in ethanol, the final quantity of the solvent was 0.1% for control 121 and treated cells for all experiment. Oleuropein was dissolved in dDW water and directly 122 diluted in DMEM medium. 123 124 Oil red O staining and quantification. After differentiation, 3T3-L1 cells were washed 125 twice with phosphate buffered saline (PBS, pH 7.4), fixed with 4% paraformaldehyde 126 (Kantou Chemistry, Tokyo, Japan) at 4 °C for 1 h, and then stained with 3 g/L Oil red O (in 127 60% isopropanol) at room temperature for 10 min. Cells were washed exhaustively with 128 sterile water, and pictures were taken using a microscope (BioZero BZ-8000; Keyence, Osaka, 129 Japan). Moreover, differentiated adipocytes were stained with 0.3 g/L Oil Red O, the dye was 130 extracted with isopropanol, and the absorbance (OD. 420 nm) was measured by a Spectra 131 Max microplate reader (Spectra Max 190; Molecular Devices Corporation, CA, USA). 132 133 Triglyceride assay. 3T3-L1 cells were rinsed twice with PBS buffer and lysed in 50 mmol/L 134 Tris-HCL [pH: 6.8], 2% SDS and 6% β-mercapthoethanol. Total fat was extracted according 135 to Bligh and Dyer method (Bligh and Dyer 1959). The cell extract (400 µL) was incubated 4 136 with 1 mL methanol and 0.5 mL chloroform for 2 h, and then 0.5 mL chloroform and 0.5 mL 137 of sterile water were added, centrifuged briefly to collect chloroform phase. This extract was 138 dried for overnight, and was dissolved in 10% triton-isopropanol solution. According to a 139 manual of triglyceride E-test Wako (Wako, Osaka, Japan), the quantity of Triglyceride was 140 measured. The quantity of triglycerides (µmol) was normalized by total protein content. 141 142 Measurement of GPDH activity. 3T3-L1 cells were differentiated on 60 mm culture dish for 143 8 days in the presence of hydroxytyrosol (0-150 µmol/L) or oleuropein (0-300 µmol/L). The 144 cells were rinsed twice with PBS, and then scraped into 200 µL enzyme extract buffer 145 (Sucrose 280 mmol/L, Tris-HCl 5 mmol/L [pH 8.0], EDTA 1 mmol/L, and β- 146 mercapthoethanol 0.2%). Cells were sonicated and centrifuged at 15000×g at 4 °C for 10 min. 147 The supernatant was collected to measure glycerol-3-phosphate dehydrogenase activity. The 148 total protein quantity was quantified with protein assay kit (Bio-Rad laboratories, Inc., Tokyo, 149 Japan). 150 151 MTT assay. 3T3-L1 cells were harvested in 24-well plate. After reaching the confluence, the 152 culture medium was replaced by 500 µL containing hydroxytyrosol (0-200 µmol/L) or 153 oleuropein (0-400 µmol/L), and the cells were incubated for further 48 hours. The culture 154 medium was removed and replaced by 500 µL of fresh culture medium containing 10 % of 155 sterile filtered MTT (Sigma-Aldrich). After 3 hours, the insoluble formazan crystals were 156 dissolved in 500 µL/well isopropanol and absorbance was measured at 570nm against 630nm.

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