patterns in adult tissues of themselves, FGFRs and cofactor KL/ KLB, and by their specific interactions with different combinations of FGFRs and KL/KLB that then form productive signaling complexes. The postprandial FGF19 (FGF15 in mice) is produced in ileum under the regulation of nuclear farnesoid X receptor (FXR), and thought to act in liver hepatocytes where FGFR4-KLB resides to negatively regulate bile acids synthesis [8,21,24]. Vitamin D and the vitamin D receptor regulate the expression of FGF23 in bone that in turn negatively regulates minerals metabolism in the kidney that expresses FGFR1 and KL [10]. As the FGF19, FGF21 has been reported to regulate glucose and lipid metabolism and energy balance [25,26]. Treatment with FGF21 corrects metabolic disorders such as hyperglycemia, hyperlipidemia and insulin resistance in rodent and primate models of diabetes and obesity [27,28,29,30]. However, the predominant endocrine axis in which FGF21 is produced in one tissue and then targets the other tissues underlying these beneficial effects is not clearly understood. It is reported that FGF21 is induced preferentially in the liver under the control of PPARa in response to fasting, ketogenic diet and in type 2 diabetes and obesity [31,32,33,34,35]. It is also expressed in some extra-hepatic metabolic and endocrine tissues. In white adipose tissues and cultured adipocytes, FGF21 expression is upregulated by PPARc agonist or under conditions including fasting and high fat diet feeding [36,37,38]. Skeletal muscle or cultured muscle cells were reported to express and secrete FGF21 under insulin stimulation and dependent on AKT1 pathway [39,40]. Thermogenic activation can induce brown adipose tissue to express FGF21 under ATF2 control [41]. Adult thymus may also expresses FGF21 [42]. On the other hand, several tissues have been implied as the potential targets of FGF21 action. Most reports suggested that in adipocytes and white fat, FGF21 stimulates glucose uptake via upregulation of GLUT1 expression [28,36], but were discrepant on whether it stimulates or inhibits lipolysis [28,43,44,45]. Several reports in FGF21 transgenic and knock- down mice demonstrated that FGF21 may play a role in the physiological response of the liver to fasting and ketogenic diets through stimulating ketogenesis and triglycerides clearance in the liver [33,46,47], but other studies such as with FGF21 ablation did not confirm the observations in the liver and suggested that FGF21 primarily stimulates lipolysis during fed state but inhibits upon fasting in white fat [44]. The effects of FGF21 in the liver are assumed through an autocrine or paracrine action that controls ketogenesis [31,33,48], glycemia via regulating glucose flux and insulin sensitivity [27] and gluconeogenesis through induction of PGC-1alpha expression during prolonged fast [47]. FGF21 may also improve pancreatic b-cells function and survival [49], stimulate skeletal muscle glucose uptake [50], stimulate thermo- genesis of brown fat by increasing the expression of thermogenic genes and enhancing the total and uncoupled respiration and glucose oxidation [32], and stimulate the hypothalamus or central nervous system for energy expenditure and reduction of obesity in rat DIO model [51]. FGFR1 and FGFR4 are two major receptor isotypes implicated by the majority of reports in the action of endocrine FGF21 and FGF19. FGFR1 is predominantly expressed in adipose tissue and adipocytes, and FGFR4 is predominantly in the liver and hepatocytes, all of which co-express the KLB [17,21,23,52]. As measured by crude affinity pull-down of ectodomains of FGFRs or by ERK1/2 activation in vitro, both FGF19 and FGF21 could bind and activate FGFR1 in the presence of KLB [9,17,23,53,54]. Unlike FGF19, although FGF21 could also bind to FGFR4-KLB weakly, it was unable to activate FGFR4-KLB and to stimulate the liver response [17,23,44]. Even though FGF21 is expressed FGF21 Activates Preferentially Adipose FGFR1-KLB predominantly in the liver, it could activate the response of fat tissue or adipocytes but not the liver [17,44]. These results differ from several reports that suggested the stimulatory activity of FGF21 for the liver response [27,31,33,47,48]. To better understand the receptor and tissue specificities of FGF21, which are the core constituents of its endocrine metabolic pathway, in comparison to FGF19 and the canonic FGF1, we employed in this study several approaches including the sensitive radio-labeling and quantitative cell surface receptor competition binding and activation, receptor downstream signaling relay and the expression of the early response gene. We found that in presence of KLB, FGF19 binds and activates both FGFR1 and FGFR4 to a comparable extent, while FGF21 binds and activates the FGFR1 that resides in adipose tissues but comparably not the liver FGFR4. Our results support the idea that among endocrine and metabolic tissues, hepatic FGF21 directly and preferentially acts on fatty tissues but less likely or less significantly on the liver. Results FGF21 interacts only with FGFR1 with high affinity in presence of KLB while FGF19 with both FGFR1 and FGFR4 To gain insights into the receptor and tissue specificity of FGF21 action as compared to FGF19 and canonic FGF1, we first performed in vitro sensitive and quantitative receptor binding kinetic analyses. It has been known the direct contact interaction between transmembrane FGFR1/4 and KLB by mutual affinity pull-down and identification by nano-LC MS/MS [4,28]. We found that FGF19 bound equally well to FGFR1-KLB and FGFR4-KLB, but not to FGFR1 alone although there is 10% binding to FGFR4 alone consistent with previous report (Figure 1A) [4]. Covalent affinity cross-linking revealed three cross-linking bands resulted from 125I-FGF19 bound to FGFR1 and KLB co-expressing cells (Figure 1B, bidirectional arrows), similar to what we observed in the interaction of 125I-FGF19 with FGFR4 and KLB, but not with FGFR1 or FGFR4 alone [4]. The successful covalent affinity crosslinking also indicated that our recombinant FGF19 was sufficiently active for interaction with FGFR-KLB (see also Figure 2 described below), similar to FGF1 with or without KLB. Since iodination with the same procedure appeared to inactivate FGF21, we used the labeled FGF19 as the binding tracer and unlabeled FGF21 as a competitor to probe the receptor binding activity of FGF21. 2 ng/ml of iodinated FGF19 could make equally detectable binding to that of 2 ng/ml iodinated FGF1. 1 mg/ml FGF21 competes with 125I-FGF19 for binding to FGFR1-KLB or KLB alone with over 90% efficiency, indicating both recombinant FGF19 and FGF21 had comparable activity towards FGFR1-KLB and KLB. However, at this high concen- tration there is only 10–20% efficiency for FGF21 to bind FGFR4- KLB. No competition binding can be detected to FGFR4 alone to which 10% of 125I-FGF19 bound as compared to FGFR4-KLB (Figure 1C, bidirectional arrows) [4]. In contrast, although 1 mg/ml unlabeled FGF19 competes with 2 ng/ml 125I-FGF19 completely as predicted, FGF1 had no displacement for 125I- FGF19 bound to the FGFR1-KLB, FGFR4-KLB, or KLB alone at 100 ng/ml (Figure 1C) or 1 mg/ml (not shown). The weakly bound FGF19 to FGFR4 alone can be displaced by 100 ng/ml FGF1 equally well as FGF19 itself but not FGF21. These results are consistent with the previous report that, 125I-labeled FGF1 binds equally well to the surface of cells where FGFR4 are present independent of KLB; in contrast, FGF19 binds to cells that express PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e33870