T L cells can be induced to

3T3-L1 cells can be induced to differentiate into adipocytes by the appropriate hormonal treatment and they offer a useful in vitro model system for adipogenesis. GPR120 mRNA was detected in differentiated mature adipocytes but not in confluent preadipocytes (Fig. 3A). The level of GPR120 mRNA increased during late stage adipocyte differentiation, whereas GPR40 mRNA could not be detected (Fig. 3A). To investigate the effects of altering PPAR-γ2 expression on that of GPR120, 3T3-L1 cells were treated during differentiation with troglitazone, synthetic ligands of PPAR-γ2, or retinoic acid. Troglitazone stimulated expression of PPAR-γ2 during differentiation and increased the expression of GPR120 mRNA at the same time (Fig. 3B). Retinoic melatonin receptor agonist inhibited the expressions of PPAR-γ2 and of GPR120 mRNAs (Fig. 3C). We next examined how the down-regulation of GPR120 using GPR120 siRNA treatment. Adipogenic genes such as PPAR-γ2 and aP2 mRNAs were dramatically down-regulated in 3T3-L1 adipocytes transfected with GPR120 siRNA compared with those that received control siRNA (Fig. 4A). In addition, the number of cells stained by oil red O was reduced in GPR120 siRNA-transfected cells (Fig. 4B).
Discussion In the present study, we have demonstrated that GPR120, but not GPR40, was highly expressed in adipose tissues. This suggests that GPR120 may be involved with the development of adipose tissues and differentiation of adipocytes. GPR120 and GPR40 are activated by long-chain fatty acids with similar characteristics although the homology of the two receptors is low. Linolenic acid is more selective for GPR120 whereas 5,8,11-eicosatriynoic acid (C20H28O2) is more active on GPR40. In addition, it has been reported that these GPRs activate similar G proteins: GPR120 activates Gαq in STC-1 cells [8], [10] and GPR40 activates Gαq in β cells [11], [12]. We could not detect GPR40 mRNA in adipose tissues or differentiated adipocytes derived from 3T3-L1 cells, even though GPR40 mRNA was present at a high expression in the spleen. High fat diets are widely used in nutritional experiments as a good strategy to induce overweight conditions and fat deposition in animals [13]. It is known that the control of energy balance in adipose tissues is regulated by a variety of proteins related to lipid metabolism. As with PPAR-γ2, differential expression of GPR120 in response to changes in nutritional status appears to be an essential feature of the biological function of this receptor. When GPR120 expression was knocked down using siRNA, the proportion of cells staining positively with oil red O for lipid droplets decreased, and expression of PPAR-γ2 further declined during adipogenesis. These results suggest that nutritional regulation exerts on expression of GPR120 in adipose tissues, and GPR120 may function as a regulator of adipocyte development and differentiation. The level of GPR120 mRNA increased during adipocyte differentiation in 3T3-L1 cells but not in confluent 3T3-L1 cells. In addition, GPR120 mRNA was not detected in S-V cells but was found at high expression in mature adipocytes. One of the important findings of this study was that the expression of GPR120 mRNA was influenced by up- and down-regulation of PPAR-γ2. The transcription network, involving the C/EBP family and PPAR-γ2, that controls adipocyte differentiation has been thoroughly investigated [14]. In addition to regulation of adipogenesis, this network simultaneously controls expression of adipocyte-specific genes such as aP2 and leptin [4]. Although troglitazone is known to induce adipose differentiation via PPAR activation, the suppressive effect of agonist to retinoic acid receptor was found by the PPAR and retinoic acid receptor heterodimer [14]. Therefore, it is not unreasonable to speculate that troglitazone increases GPR120 mRNA and RA decreases. However, adipocyte differentiation is not likely to be responsible for the increased GPR120 mRNA in 3T3-L1 adipocytes. Up-regulation of GPR120 mRNA may be essential for modulation of the adipogenic process in mature adipocytes. Numerous studies have shown that fatty acids influence cell metabolism in many tissues. Long-chain fatty acids regulate leptin secretion in isolated rat adipocytes [15], although the mechanism of action remains unknown. Long-chain fatty acids also mediate cell survival via GPR120 and GPR40 [10], [16]. Fatty acid metabolism in adipose and other tissues is implicated in the development of obesity and insulin resistance. Increased plasma fatty acid concentration, which is a hallmark of obesity and insulin resistance, can be caused by increased lipolysis and fatty acid release from adipose tissue, increased hepatic VLDL triglyceride synthesis and secretion, and reduced peripheral fatty acid uptake [17]. These suggested the possibility that fatty acids functionally activate adipocyte through GPR120 as its receptor even though there are no direct evidences on acts of long-chain fatty acids.