siRNA construction and transfection For small interfering RN

siRNA construction and transfection. For small interfering RNA (siRNA) experiments, we used Stealth™ select RNAi oligonucleotides targeted against GPR120 (Gibco/Invitrogen). The target sequence of siGPR120 used were; sense 5′-AAGUGGGUGCGAUUGACUUGGUCCA-3′ and antisense 5′-UGGACCAAGUCAAUCGCACCCACUU-3′. As a negative control, we used scrambled Stealth RNAi Negative Control Kit with Mediums GC (Gibco/Invitrogen). It does not match any mammalian sequences. siRNA transfection to 3T3-L1 VUF 11207 fumarate synthesis was conducted according to the modified manufacture’s instructions. GPR120 and control siRNAs were diluted to the final concentration of 1nM in Opti-MEM® I Reduced Serum Medium (Gibco/Invitrogen). Lipofectamine™ RNAiMAX (Gibco/Invitrogen) was diluted to 50 times with Opti-MEM® I Reduced Serum Medium and incubated for 15min at room temperature. After the 15-min incubation, the equal volumes of diluted siRNA and Lipofectamine™ RNAiMAX were mixed gently and incubated for 15min at room temperature to allow complexes to form. siRNA–lipofectamine complexes were diluted 4 times with Opti-MEM I® Reduced Serum Medium before transfection. Upon confluence, the 3T3-L1 cell medium was replaced by growth medium. When the cells reached confluence, the medium was replaced with one lacking antibiotics. Two days later, the cells were incubated with siRNA–lipofectamine complexes at 37°C for 4h before induced cell differentiation (see above). siRNA-mediated down-regulation of GPR120 expression was confirmed by RT-PCR analysis. Statistical analysis. The data in Fig. 1C are presented as means±SEM of six animals treated with the same protocol. The data in Fig. 3A and Fig. 4A are presented as means±SEM of three experiments with the same protocol. Differences in mean values in Fig. 3A were assessed statistically using Duncan’s multiple range test followed by a one-way analysis of variance. The data in Figs. 1C and 4A were analyzed using Student’s t-test.
Results The expressions of GPR120 and GPR40 mRNAs were determined in 15 tissues. In a subsequent experiment to validate our results, the expressions of leptin and PPAR-γ2 mRNAs were analyzed. As shown in Fig. 1A, although GPR120 mRNA was detected in all tested tissues, it was highly expressed in the pituitary, lung, small intestine, colon, and four different adipose tissues. GPR40 mRNA was not detected in the four adipose tissues. In order to identify which cell type was responsible for the high expression of GPR120 mRNA, adipocytes, and S-V cells were isolated from the four adipose tissues. The GPR120 transcript was present at a very low expression in the S-V cells but at a high expression in adipocytes (Fig. 1B). GPR40 mRNA was not detected in either adipocytes or S-V cells (Fig. 1B). To investigate the relationship between the level of GPR120 mRNA and nutritional status, four different WATs from animals subjected to a normal or a high fat diet were used for analysis of GPR120 and GPR40 mRNA levels (Fig. 1C). After 11 weeks on a high-fat diet, the body weight of mice was 30% greater than that of mice that had been fed a normal diet. In addition, the respective weights of these animals’ four different fat pads were approximately 2–3 times greater than were those in mice fed the normal diet. An increase in the amount of GPR120 mRNA was observed in mice fed the high fat diet. However, there were no differences in mRNA levels in perirenal adipose tissues of normal and high fat diet mice. There is no available information regarding GPR120 expression in human preadipocytes, differentiated adipocytes or adipose tissues. We therefore investigated expression of GPR120 and GPR40 in human adipose tissue and adipocytes. A high expression of GPR120 mRNA was found in human differentiated adipocytes but no expression could be detected in human preadipocytes (Fig. 2A). GPR40 mRNA was not detected in either preadipocytes or adipocytes. Similarly to mice, GPR120 mRNA was highly expressed in human adipose tissues but GPR40 was not detected (Fig. 2B). Human small intestine and pancreas tissues were used as positive controls for RT-PCR. As previously reported, GPR120 and GPR40 mRNA were present in both the intestine and pancreas.