Oul, Korea) were maintained in Dulbecco's modified eagle's medium (DMEM)/high glucose (Hyclone, UT, USA) with

Oul, Korea) were maintained in Dulbecco’s modified eagle’s medium (DMEM)/high glucose (Hyclone, UT, USA) with 10 newborn calf serum (GibcoTM, Life Technologies, NY, USA), one hundred units/ml penicillin, and one hundred g/ml streptomycin at 37 in five CO2 incubator. To induce differentiation, one hundred confluent 3T3-L1 pre-adipocytes (day 0) have been stimulated for two days with MDI media [0.5 mM 3-isobutyl-1-methylxanthine (Sigma Aldrich, MO, USA), 1 M dexamethasone (Sigma Aldrich), and 5 g/ml insulin (GibcoTM) in DMEM/10 fetal bovine serum (FBS, Hyclone)] [21]. On day 3, the MDI media was replaced with differentiation media (5 g/ml insulin in DMEM/10 FBS). On day six, the differentiation media was replaced with development media (DMEM/ ten FBS). The cell culture media was changed each 2 day.Table 1. The primer sequences of adipogenic genes Gene aP2 C/EBP FAS LPL PPAR MIG/CXCL9, Human (HEK293, His) SREBP-1c -actin Primer Sequence Forward 5′-TCA CCT GGA AGA CAG CTC CT-3′ 5′-GAA CAG CAA CGA GTA CCG GGT-3′ 5′-GCT TTG CTG CCG TGT CCT TCT-3′ 5′-AGT AGA CTG GTT GTA TCG GG-3′ 5′-CCA GAG CAT GGT GCC TTC GCT-3′ 5′-CGC TAC CGG TCT TCT ATC ATT G-3′ 5′-AGG CTG TGC TGT CCC TGT AT-3′ Reverse 5′-AAT CCC CAT TTA CGC TGA TG-3′ 5′-GCC ATG GCC TTG ACC AAG GAG-3′ 5′-TCT AGC CCT CCC GTA CAC TCA-3′ 5′-AGC GTC ATC AGG AGA AAG G-3′ 5′-CAG CAA CCA TTG GGT CAG CTC-3′ 5′-TTG CTT TTG TGT GCA CTT CG-3′ 5′-ACC CAA GAA GGA AGG CTG GA-3’Byulchorong Min et al.-actin, and presented as fold changes relative to controls (no arctiin therapy). Western blot evaluation 3T3-L1 cells were collected and suspended in lysis buffer containing 25 mM Tris-HCl (pH 7.6), 1 NP-40, 1 sodium deoxycholate, 150 mM NaCl, 1 SDS, 1 mM phenylmethanesulfonylfluoride, protease inhibitor tablet (Roche, IN, USA) and phosphatase inhibitor tablet (Roche). Total protein concentrations in lysates were measured by utilizing BCA protein assay kit (Pierce, IL, USA). 20 g protein homogenates were mixed with two ?loading buffer [25 mM Tris-HCl (pH six.8), 1 SDS, 30 glycerol, 10 2-mercaptoethanol (Sigma Aldrich), protease inhibitor tablet (Roche) and phosphatase inhibitor tablet (Roche), 0.7 bromophenol blue (Sigma Aldrich)], heated at 95 for five min, and separated on 10 SDS polyacrylamide gel. Proteins had been then transferred onto PVDF membrane (Millipore, Billerica, MA, USA). After blocking for two hours at area temperature with 5 non-fat skim milk (DIFCO, Paris, France) in TBS-T [Tris-buffered saline containing 0.01 Tween 20 (Sigma Aldrich)], the membranes have been incubated overnight at 4 with the following major antibodies: PPAR, C/EBP, phosphoAMPK, AMPK, phospho-acetyl CoA carboxylase (p-ACC) (Cell Signaling, Boston, MA, USA) and -actin (Santa Cruz Biotechnology, CA, USA). After washing five times with TBS-T, the membranes had been additional reacted with goat anti-rabbit IgG (H + L)-HRP conjugate (BioRad) or goat anti-mouse IgG (H + L)-HRP conjugate (BioRad) at space temperature for 90 min. Bands have been TM visualized by enhanced chemiluminescence (Clarity western ECL substrate, BioRad) and chemiluminescence imager instrument (CLINX Science Instrument, China). For quantification, the densities of each band were determined by a gel analysis software program (CLINX Science Instrument). Animals and diet regime Male C57BL/6J mice (SLC Inc., VIP Protein Biological Activity Hamamatsu, Japan) were housed in a space with controlled temperature (21 23 ), humidity (55 60 ) and lighting (12-h light/dark cycle). Soon after acclimation for 1 week, animals were divided, by weightmatching, into three groups (HF, HF + AC, and CON). HF and HF + AC.