FGF21 Activates Preferentially Adipose FGFR1-KLB Figure 3. Dose-dependent differential binding of FGF21, FGF19 and FGF1 to FGFR1-KLB and FGFR4-KLB. (A) Competition ability of FGF21 and FGF1 with FGF19 to bind FGFR-KLB. Graded concentrations as indicated for FGF21 (open square), FGF19 (filled triangle) and FGF1 (filled circle) were added together with 2 ng/ml 125I-FGF19 to the cells expressing FGFR1-KLB (solid line) or FGFR4-KLB (dot line) after 30 ng/ml Tet induction overnight, the remaining specific bindings of 125I-FGF19 were determined as described in Figure 1A. (B) Competition ability of FGF21 with FGF1 to bind FGFR1-KLB. Graded concentrations as indicated for FGF21 (open square), FGF19 (not shown) and FGF1 (filled circle) were added together with 2 ng/ml 125I-FGF1 to the cells expressing FGFR1-KLB (solid line) or FGFR4-KLB (Inset), the remaining specific bindings of 125I-FGF1 under these conditions were determined as described above. doi:10.1371/journal.pone.0033870.g003 are the two major types of tissues being implicated predominantly in their actions. However, current reports are discrepant on whether there is a liver effect of the FGF21 endocrine action. It is also not clear whether there is an direct adipose effect of FGF19, even though it was known that FGF19 exhibited anti-obesigenic and anti-diabetic activities [58,59]. Recombinant FGF21 at 0.5 mg/Kg were injected into mouse intraperitoneally as compared to FGF19 and vehicle PBS, and after 20 min tissues were isolated for qPCR gene expression analyses. FGF21 and FGF19 stimulated c-Fos mRNA expression in mouse liver at 1.3 and 13.8 times over the PBS control, respectively (Figure 7A). The liver response to FGF19 is more than 10 times that to FGF21. In contrast, FGF21 and FGF19 stimulated c-Fos mRNA expression in WAT at 5.1 and 4.3 times over the control, respectively (Figure 7A). The response of WAT to FGF19 is about 80% that to FGF21. The responses of other tissues including pancreas, skeletal and heart muscles and hypothalamus to FGF21 stimulation are relatively very low or undetectable (Figure 7B). Note that the breast, which contains mostly the adipose tissue, is the second highest responder. In all the cases, the response of the liver to FGF21 is the lowest and close to vesicle control. A thorough comparison of differential responses among all aforementioned tissues to FGF21 and FGF19 would further provide insights into their differential biological effects, which will be undertaken in tissues from wild-type, KLB2/2, FGFR1 conditional ablation and FGF21 transgenic mice after crossbreed- ing into a similar strain background. Consistent with the acute c-Fos expression response, treatment of mice with FGF21 for 20 minutes stimulates peak activation of Erk1/2 [28] in the WAT (Figure 8A), but not in the liver and only very weakly in hypothalamus and possibly skeletal muscle as well (Figure 8A). These effects are substantially abolished in mice with gene deletion for the cofactor KLB, as indicated by the percent changes of pErk1/2 relative to total Erk1/2 (Figure 8A). Furthermore, the conditional ablation of FGFR1 in adipocytes in WAT by aP2 Cre (manuscript in preparation) also completely abrogates the Erk1/2 activation by both FGF21 and FGF19 (Figure 8B), as compared to the FGFR1 floxed tissue and PBS vehicle control. In the liver unaffected by the adipocyte-specific ablation of FGFR1, Erk1/2 activation in response to FGF19 remains unchanged, while the response to FGF21 is relatively insignificant or undetectable (Figure 8B), similar to the baseline control from PBS as the delivery vehicle (not shown). This response profile is also consistent with the differential tissue specific expression of FGFR and KLB (Figure 9). The expression of FGFR1 is high in WAT and hypothalamus. FGFR4 is only expressed in the Liver, not in the WAT, hypothalamus and skeletal muscle. In contrast, KLB is expressed high in both WAT and liver, PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33870