Materials and Methods
Reagents RA (see chemical structure in Fig. 1A) and gentamicin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). C2C12 cells were from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). Bovine serum albumin (BSA) was obtained from Medical & Biological Laboratories (Nagoya, Japan). Fetal bovine serum was from Biological Industries (Beth Haemek, Israel). Horse serum was from Life Technologies (Gaithersburg, MA, USA). 3H-Palmitate was from Perkin Elmer Japan Co., Ltd. (Tokyo, Japan). FastStart Universal SYBR Green Master (ROX) was from Roche Applied Science (Indianapolis, IN, USA). Antibodies for β-actin, AMPKα, phospho-AMPKα (Thr-172), ACC, phospho-ACC (Ser-79), Akt, phospho-Akt (Thr-308) and acetyl-lysine were from Cell Signaling Technology (Beverly, MA, USA). Antibodies for CaMKK, PGC1α and SIRT1 were from Abcam Inc. (Cambridge, MA, USA).
RA treatment promotes fatty acid oxidation and glucose utilization in C2C12 myotubes.
(A) RA structure. (B) Effect of RA on Fatty acid oxidation. (C) Effect of RA on glucose utilization. Cells were treated with various concentrations of RA, 100 µM L-carnitine and 3H palmitate (200 µM cold palmitate and 185 kBq/mL 3H palmitate in each well) for 24 h. The culture media were collected for measurement of palmitate oxidation and glucose concentration. Values are mean ± SD (n = 4). Data are representative of multiple experiments. *P < 0.05; **P < 0.01 vs. control.
Cell culture and treatment C2C12 myoblasts were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 50 µg/mL gentamicin and 10% fetal bovine serum. To differentiate myoblasts into myotubes, cells were fed DMEM containing 2% horse serum for 4 days. Differentiated myotubes were serum-starved for 6 h in serum-free DMEM supplemented with 1% BSA. Subsequently, cells were incubated in serum-free DMEM supplemented with 1% BSA, 100 µM L-carnitine, RA and 3H-palmitate (mixture of 200 µM cold palmitate and 185 kBq/mL 3H-palmitate in each well) for 24 h. After incubation, the culture media were collected for measurement of palmitate oxidation and glucose concentration.
Measurement of palmitate oxidation and glucose concentration For measurement of palmitate oxidation, proteins bound to palmitate were removed by trichloroacetic acid precipitation, and the supernatant was loaded into a 96-well filter plate (Unifilter GF/B, GE Healthcare Japan, Tokyo, Japan) containing an equilibrated, activated charcoal slurry. The plate was centrifuged for 10 min at 1,000 × g. Following centrifugation, the charcoal-containing plate was discarded and the filtrate was counted using a scintillation counter (Plate Chameleon V, Turk, Finland). For measurement of glucose concentration, collected media were assayed for glucose concentration using a Glucose CII-test kit (Wako Pure Chemical Industries, Ltd.).
Quantitative real-time PCR Total RNA from C2C12 myotubes was isolated using Isogen reagent (Wako Pure Chemical Industries, Ltd.). To obtain cDNA, RNA (1 µg) was reverse transcribed using a PrimeScript RT Reagent Kit (Takara Bio Inc., Shiga, Japan). Quantitative real-time PCR was performed using an ABI 7500 Real-Time PCR System (Life Technologies) according to the manufacturer's protocol. Primer sequences used in PCR were: 36B4, 5′-ACTGGTCTAGGACCCGAGAAG-3′ and 5′-CTCCCACCTTGTCTCCAGTC-3′; CD36, 5′-TTGTACCTATACTGTGGCTAAATGAGA-3′ and 5′-CTTGTGTTTTGAACATTTCTGCTT-3′; long chain acyl-CoA dehydrogenase (LCAD), 5′-GCTTATGAATGTGTGCAACTCC-3′ and 5′-CCGAGCATCCACGTAAGC-3′; uncoupling protein 3 (UCP3), 5′-TACCCAACCTTGGCTAGACG-3′ and 5′-GTCCGAGGAGAGAGCTTGC-3′; SIRT1, 5′-CAGTGAGAAAATGCTGGCCTA-3′ and 5′-TTGGTGGTACAAACAGGTATTGA-3′; and glucose transporter 4 (GLUT4), 5′-CATGGCTGTCGCTGGTTTC-3′ and 5′-AAACCCATGCCGACAATGA-3′. Data analyses were performed using the 7500 System SDS software, version 1.3.1 (Life Technologies).
Western blot analysis Cells were lysed with M-PER mammalian protein extraction reagent (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail solutions (Wako Pure Chemical Industries, Ltd.). The protein concentration was determined using the BCA protein assay (Thermo Scientific). Equal amounts of protein (20 µg/lane) from each treated group mixed with SDS sample buffer (Wako Pure Chemical Industries, Ltd.) were loaded on a 10% or 12.5% SuperSep Ace gel (Wako Pure Chemical Industries, Ltd.). After electrophoresis, proteins were transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was first blocked in Tris-buffered saline with Tween 20 (TBST) with 4% skim milk or BSA for 1 h, followed by incubation with primary antibodies in TBST with 2% skim milk or BSA for 1 h at room temperature. Blots were washed three times with TBST and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The immunoreactive proteins were visualized using the ECL Plus Western Blotting Detection System (GE Healthcare Japan), and band intensities were quantified with GeneSys/GeneTools software using the GeneGnome-5 Chemiluminescent Imaging System (Syngene, Cambridge, UK).
Immunoprecipitation Immunoprecipitation of PGC1α was carried out using a Dynabeads Protein G Immunoprecipitation Kit (Veritas, Tokyo, Japan) according to the manufacturer's instructions. Briefly, anti-PGC1α antibody was immobilized on Dynabeads Protein G. Then, 500 µg of protein extract were incubated with the Dynabeads-antibody complex for 10 min at room temperature. The Dynabeads-antibody-antigen complex was magnetically isolated. After washing, the immunoprecipitate was eluted in elution buffer. Proteins were separated by SDS-PAGE, and Western blot analysis was performed using anti-PGC1α and anti-acetyl-lysine antibodies.
Statistical analysis Results are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Dunnett's post-hoc test using Ekuseru-Toukei 2012 (Social Survey Research Information Co. Ltd., Tokyo, Japan). Values of P < 0.05 were considered statistically significant.