Recently several groups reported that four orphan receptors

Recently, several groups reported that four orphan receptors, GPR40, GPR41, GPR43, and GPR120, can be activated by free fatty acids. Short-chain fatty acids are specific agonists for GPR41 and GPR43 . Medium and long-chain fatty acids can activate GPR40 and GPR120 . Our previous report showed that acetate and propionate regulate adipogenesis through GPR43 . GPR120 is abundantly expressed in the intestine and, following activation by long-chain fatty acids, mediates glucagon-like peptide-1 secretion. In addition, long-chain fatty acids stimulate the extracellular signal-regulated kinase (ERK) and PI3K-Akt pathways through GPR120 . The roles of functional fatty acids are gradually being elucidated; however, it is not known yet whether GPR120, GPR40 or both influence adipogenesis and adipocyte development. In the present study, we investigated the relationship between the GPRs for long-chain fatty acids and adipocyte differentiation and adipose tissue development. Materials and methods Animals. The mice were used C57BL/6J mice obtained from Charles River Japan. And then weighed and divided into two groups of six with approximately equal mean body weights. One group was fed the standard diet (Oriental Yeast, Chiba, Japan) containing 8.5% (w/w) fat, 43.7% carbohydrate, and 29.7% protein, with an energy content of 3.69kcal/g, and the other received a high-fat diet for 11 weeks (4–15 weeks of age). The high-fat diet was contained 41% fat, 36% carbohydrate, and 23% protein, with an energy content of 4.33kcal/g; its fat source was the same as that of the standard diet and it contained the same absolute amounts of protein and fiber as the standard diet. White adipose tissues and non-adipose tissues were rapidly frozen in liquid nitrogen, and stored at −80°C until RNA extraction. All experiments were conducted in accordance with the Shinshu University Guide for the Care and Use of Experimental Animals. Isolation of adipocytes and stromal-vascular (S-V) calcitonin receptor from adipose tissues. White adipose tissue from the perirenal and parametrial fat regions of 15-week-old female mice was separated from connective tissue and blood vessels. The adipose tissue was then divided into adipocyte and S-V cell fractions as previously described [9]. Culture of 3T3-L1 cells and induction of differentiation. 3T3-L1 cells were purchased from ATCC (Manassas, VA, USA). Culture of 3T3-L1 cells and induction of differentiation was performed as previously described [9]. In another set of experiments, cells were treated with troglitazone (5μM) for up to 7 days and all-trans retinoic acid (0.1μM) for up to 9 days during adipocyte differentiation. Oil red O staining. Cytoplasmic lipid droplets were stained with oil red O. Briefly, cells were rinsed three times in PBS and then fixed in 10% (v/v) formaldehyde for 10min. After being washed twice with PBS, cells were stained for 30min at 37°C in freshly diluted oil red O (Sigma Chemical Co., St. Louis, MO, USA) solution (six parts oil red O stock and four parts H2O; oil red O stock solution is 0.5% oil red O in isopropanol), followed by further washing with PBS. Total RNA extraction from tissues and cells. Total RNA was extracted from non-adipose tissues, confluent 3T3-L1 cells and differentiated adipocytes using Trizol Reagent (Gibco/Invitrogen). Total RNA was extracted from four adipose tissues, adipocytes, and S-V cells using RNeasy® Lipid Tissue Mini Kit (Qiagen KK, Tokyo, Japan). Total RNAs from human small intestine and pancreas were purchased from Cell Applications Inc. (San Diego, CA). Total RNAs from human cultured preadipocytes, differentiated adipocytes, and four subcutaneous adipose tissues were purchased from Zen-Bio Inc. (Research Triangle Park, NC). Semi-quantitative RT-PCR analysis. Semi-quantitative RT-PCR was performed as previously described [9] to measure the levels of mouse and human GPR120, GPR40, PPAR-γ2, leptin, and β-actin mRNAs. The gene-specific primers are shown in Table 1. PCR products were resolved on a 2% agarose gel. The DNA was visualized by ethidium bromide staining and analyzed using NIH image software. The mRNA levels of GPR120 were corrected using the transcription level of the β-actin gene as an internal standard. The data in Fig. 1, Fig. 3, Fig. 4 were expressed as fold differences (n-fold) over the appropriate control value, as described in the figure legends. The amplified cDNAs were subcloned into the pGEM-T easy vector (Promega, Madison, WI), and the sequences were confirmed using an ABI 3100® Genetic Analyzer (Applied Biosystems, Tokyo, Japan).