Caffeic Acid Phenethyl Ester Inhibits Differentiation to Adipocytes in 3T3-L1 Mouse Fibroblasts

Abstract

We investigated the inhibitory effect of caffeic acid phenethyl ester (CAPE) on the differentiation of 3T3-L1 mouse fibroblasts to adipocytes. 3T3-L1 cells were differentiated for adipocytes given high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 mM dexamethasone (DEX), 500 mM isobutylmethylxanthine (IBMX), and 5 mg/ml insulin for 7 days. After differentiation, cells were stained with Oil-Red-O to detect oil droplets in adipocytes. Additionally, the cells were lysed and measured for triglyceride contents. Total RNA was isolated from differentiated cells on day 0, 4 and 7. Then, RNA was analyzed using reverse transcription (RT)-polymerase chain reaction (PCR). CAPE dose-dependently suppressed oil droplet accumulation and reduced the droplet size. These findings showed that CAPE at concentrations of 25 to 50 mM could significantly inhibit triglyceride deposition (pϽ0.05). Treatment of 3T3-L1 with CAPE reduced the mRNA levels of peroxisome proliferator-activated receptor (PPAR) gamma and CCAAT/enhancer-binding protein (C/EBPalpha). Fatty acid synthase (Fas) and adipocyte-specific fatty acid binding protein (aP2) are known to be associated with lipid metabolism in adipocytes, and both Fas mRNA and aP2 mRNA were significantly suppressed by CAPE treatment. These findings suggested that CAPE suppresses 3T3-L1 differentiation to adipocytes through inhibition of PPARgamma, C/EBPalpha, Fas and aP2 expression.Key words caffeic acid phenethyl ester; adipocyte differentiation; fatty acid synthase; peroxisome proliferator-activated receptor gamma; CCAAT/enhancer-binding protein alpha; adipocyte-specific fatty acid binding protein 2 Biol. Pharm. Bull. 33(9) 1484-1488 (2010) © 2010 Pharmaceutical Society of Japan * To whom correspondence should be addressed. e-mail: manju129@mukogawa-u.ac.jp Oil-Red-O Staining After differentiation, cells were stained with Oil-Red-O to detect oil droplets in adipocytes. Cells were washed 3 times with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 2 min and then stained with 3.3 mg/ml Oil-Red-O in 75% isopropanol for 30 min. Cells were washed with PBS 3 times and observed under a microscope (Olympus CKX41, Japan). Stained oil droplets in the cells were dissolved containing 4% (v/v) Nonidet-P40 in isopropanol with gentle agitation for 5 min. Supernatant was measured with a spectrophotometer at 500 nm.Triglyceride Determination Differentiated cells were lysed with Lysis buffer (50 mM Tris, 0.15 M NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.1% Tween-20, pH 7.5 with HCl) and measured for triglyceride content on day 4 and 7. The triglyceride content in the cell lysates were quantified using the Triglyceride E test WAKO. The concentration was corrected using DNA as an internal standard. DNA was quantified using a Quant-iT TM dsDNA HS Assay kit. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) To detect mRNA expression levels, total RNA was extracted from differentiated cells on day 0, 4 and 7 with an RNeas...

Caffeic Acid Phenethyl Ester Suppresses the Production of Adipocytokines, Leptin, Tumor Necrosis Factor -Alpha and Resistin, during Differentiation to Adipocytes in 3T3-L1 Cells

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Caffeic acid phenethyl ester suppresses oxidative stress in 3T3-L1 adipocytes

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Caffeic Acid Phenethyl Ester Suppresses the Production of Pro-inflammatory Cytokines in Hypertrophic Adipocytes through Lipopolysaccharide-Stimulated Macrophages

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