FGF21 Activates Preferentially Adipose FGFR1-KLB Figure 6. Dose-dependent activity potential of FGF19 and FGF21. Cells expressing endogenous FGFR4-KLB and FGFR1-KLB as indicated were stimulated by different concentrations of FGF19 and FGF21, respectively, for 10 minutes in 37uC culture. Cell lystes were then used to determine the pERK1/2 levels under these conditions by western-blotting. The pErk1/2 level for each cell type under each stimulation condition is expressed as normalized arbitral units of ratio of pErk1/2 to total Erk1/2. The half maximal effective concentration (EC50) is then calculated in the logarithmic plot. doi:10.1371/journal.pone.0033870.g006 interaction underlies the different tissue response profiles for FGF19 and FGF21. FGF21 only effectively activates the responses of the adipose tissue and adipocytes but not the liver where these binary complexes are differentially present [17,28,44,52]; on the other hand, FGF19 activates the responses of both the liver and adipose tissue and the derived cells. These data are consistent with our previous observation that mice with liver specific overexpres- sion of constitutively active FGFR1 gain a suppression of bile acids synthesis that is similar to the constitutively active FGFR4 overexpression [66]. Our results thus support a direct endocrine metabolic role of FGF21 in fatty tissues but less likely in the liver, while FGF19 may have also a role in fatty tissue, beyond the liver as a primary target [59,67]. The reported effects of FGF21 on the metabolic parameters in the liver are more likely from a secondary indirect response of the liver to the direct metabolic effects of FGF21 on other tissues, in particular the fatty tissues, in a physiological concentration. This is consistent with the central roles of the liver in monitoring, regulating and responding to the ever-changing metabolic states of the whole body and many other individual tissues. The results that FGF21 is primarily expressed in the liver in response to fasting and pathological conditions such as fatty accumulation and the obesity and diabetes [68,69], and that FGF21 targets primarily the fatty tissues, indicate an emerging endocrine metabolic pathway from the liver to fatty tissues in Figure 7. Differential tissue-specific responses to FGF21 and FGF19 as measured by the expression level of early responsive gene c-Fos. Thirty age- and weight-matched mice (26365) were fasted for 24 hrs with water freely available, and then injected intraperitoneally with FGF21, FGF19 (0.5 mg/Kg body weight) and PBS vesicle control as indicated. After 20 min, liver and adipose tissue (A) and several other endocrine and metabolic tissues (B) were isolated and processed for RNA purification. Quantitative PCR was used to assess the expression of c-Fos in response to different treatments (n = 5). doi:10.1371/journal.pone.0033870.g007 regulating the lipid, glucose and energy metabolic homeostasis. However, this doesn’t exclude a possibility that under certain condition, the weakly expressed FGFR2 and/or FGFR3 beyond the dominant FGFR4 may help the liver to respond to FGF21 in an autocrine/paracrine fashion although likely at a much reduced level. Other tissues, such as the breast, hypothalamus, pancreas and muscle may also respond to FGF19/21 either directly or indirectly under certain physiological and altered pathological states. Whether FGF21 has a possible KLB-independent role in vivo or whether FGF21 stimulates the responses of other tissues where KLB expression is low or undetectable, as some studies have suggested for FGF19, is now an open question. Interestingly, although KLB2/2 mice exhibited bile acids phenotype resembling that of FGFR42/2 and FGF152/2, these mice have not been reported under normal diet condition a metabolic abnormality phenocopying that of FGF212/2. This does not exclude such a possibility under other pathological or diet stress conditions. The combination of tissue expression specificity and molecular receptor-cofactor interaction specificity determine the eventual physiological outcome. KLB conceivably has functions that overlap with as well as diverge from FGF21. These functional difference and similarity will be balanced to present an overt phenotype under different physiological and pathological states. Tissue specific ablation approaches should be very useful to dissect these difference and similarity issues. We observed that the binding modes of the canonic FGF1 and endocrine FGF19 and FGF21 to FGFRs likely possess similar as well as different elements. The binding of FGF1 to FGFR-KLB appears to be not affected by the presence of FGF19 or FGF21. Furthermore, FGF1 binds FGFR and FGFR-KLB directly in the PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e33870