FGF21 Activates Preferentially Adipose FGFR1-KLB Figure 5. Differential activation of FGFRs and downstream MAPKs by FGF19 and FGF21. (A) Tyrosine phosphorylation of FGFR and activation of ERK1/2 in engineered T-Rex 293 cells [4]. Expression of FGFR1 and FGFR4 was induced with 30 ng/ml Tet overnight. Cells were stimulated with 100 ng/ml FGF21, FGF19 and FGF1 in presence of 1 mg/ml heparin, cell lysates in 16SDS sample buffer were used for immunoblot analyses using antibodies as indicated. The identity of FGFR1 and FGFR4 was pre-determined by their respective antibodies (not showed). (B) Responses of adipocytes and hepatocyte-like cells to the stimulation of FGF19 and FGF21. Mature 3T3-L1 adipocytes and HR4 hepatoma cells after overnight serum-starvation were treated by FGF21, FGF19 and FGF1 at the concentrations as indicated, and cell lysates were used for immunoblot analyses for MAPK activation as described above. The average relative activation level of Erk1/2 for each cell type under different stimulation condition is expressed as the percentage to the peak activation of Erk1/2 treated with FGF1 after normalized as ratio of pErk1/2 to total Erk1/2. doi:10.1371/journal.pone.0033870.g005 that no additional cross-linked complexes were detected with labeled FGF1 in the co-expression cells. This may explain why FGF21/19 cannot compete with FGF1 binding even in presence of KLB. On the other hand, the fact that FGF1 cannot compete with FGF19 may indicate a FGFR-independent binding site of FGF19/21 on KLB. Our results imply that KLB functions as a key regulator of FGF19 and FGF21 not only by promoting their high- affinity binding to and subsequent activation of FGFRs, but also by determining their tissue-specific activity where KLB and FGFRs are specifically co-expressed [17,21]. The differences in formation of the active canonic and non- canonic complexes may underlie their downstream functional divergence [1,4,64,65]. Further studies should shed light on the mechanisms by which canonic FGFs and endocrine FGFs coordinate local cell proliferation and metabolic function during developmental stages and in pathophysiological circumstances. This may be through regulating the ratio of FGF receptor to the cofactor KLB, therefore, switching the end-effects between the cell proliferation promoted by FGFR free of KLB and the cell metabolism controlled by the integrative FGFR-KLB complex. It is likely that the integration of KLB and endocrine FGFs in specific tissues alters the major downstream signal effectors or pathways, therefore, results in differential end-effects. Although ileum FGF15/19 plays a primary role in negatively regulating hepatic bile acids synthesis, there is so far no reported adipose tissue phenotype in the FGF152/2 mice. However, FGF19 (FGF15 in mice) administration or overexpression was reported to have a profound impact on adiposity and diabetic parameters, through yet unclear mechanisms in term of tissue and molecular targets [21,58,59]. These effects are markedly similar to those of FGF21 administration or overexpression through regulating lipid, glucose and energy metabolism. Both FGF19 and FGF21 reach to target tissues through the endocrine mechanism, the circulation. In particular, FGF19 has been proposed to reach to the liver through portal vein from intestine; therefore, it likely that through circulation, the adipose tissues will be a secondary target of FGF19, even though a significant portion of FGF19 from intestinal producing site may be trapped in the liver through the dominant enterohepatic circulation. It is conceivable that FGF19 may coordinate metabolism in the adipose tissues and liver in response to the prandial stimulation. This is also supported by our current data; however, more in vivo studies are needed to address this important physiological possibility. FGF21 reportedly has no effect on expression of cyp7a1, a key enzyme in hepatic bile acids synthesis (17), which is a hallmark of FGFR4-KLB function in the liver; however, it plays notable roles in lipid and glucose metabolism and thus is proposed as a novel pharmacotherapy for obesity and diabetes. As of FGF19, the mechanisms underlying the beneficial effects of FGF21 are also unclear. Reports are discrepant on whether FGF21 has roles directly in the liver for regulation of ketogenesis, triglycerides clearance and glucose disposal, and on whether FGF21 stimulates or inhibits lipolysis in white fat [17,27,31,33,44,47]. Reports including ours (manuscript in preparation) are consistent on the predominant co-expression of FGFR4-KLB in the liver, hepato- cyte or hepatocyte-derived cells, and of FGFR1-KLB in adipose tissues or mature adipocytes [17,21]. One of the keys to clarify these important issues is to see how different tissues respond at the early stage of FGF21 stimulation. In this study, we showed that although FGF19 interacts with both binary FGFR1-KLB and FGFR4-KLB with high affinity, FGF21 is able to bind only the FGFR1-KLB but not FGFR4-KLB with a high affinity compa- rable to that of FGF19 binding. This differential molecular PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33870