MATERIALS AND METHODS

Cell Culture 3T3-L1 cells and HEK 293 cells, obtained from the Human Science Research Resources Bank (Osaka, Japan), were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum and in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum, respectively. For induction of adipose differentiation, 3T3-L1 cells were grown to confluence. The cells were then fed with differentiation medium (a 3: 1 mixture of DMEM and Ham’s F12 containing fetal bovine serum, 1.6 mM insulin, 0.0005% transferrin, 180mM adenine, 20pM triiodothyronine, 0.25 mM dexamethasone (DEX), and 500mM isobutylmethylxanthine (IBMX)). After 3 d, the cells were re-fed with fresh differentiation medium without IBMX and DEX and maintained over the following days.