As a single transmembrane protein, KLB plays an important role in regulating bile acid synthesis, as revealed by genetic ablation in normal condition 
. KLB−/− mice exhibited elevated biosynthesis and secretion of bile acids through upregulation of Cyp7a1 expression, a rate-limiting enzyme in the bile acid anabolic pathway. This phenotype resembles those of the FGFR4 and FGF15 (FGF19 in human) knockout mice 
. Mice deficient in KL exhibited a variety of aging-like phenotypes 
, many of which phenocopy those observed in FGF23−/− mice 
. These studies uncovered the biological connections among the conventional HS-FGFR, the KL family, and the FGF19 subfamily that function in convergence in signal transduction pathways for commitment of metabolic regulation 
rather than grossly the growth control. Like canonic FGFs, the endocrine FGF19, FGF21 and FGF23 remain to signal through FGFRs but only in the presence of the KL family. The underlying mechanism for the KL family integration and the consequence resulted from is still unclear. In this study, we found that although FGF19 and FGF21 are unable to bind HS-FGFR alone, they are able to bind through KLB with high affinity. The binding of FGF19 and FGF21 to KLB and FGFR-KLB is not affected by the presence of canonic member FGF1; similarly, the binding of FGF1 to FGFR and FGFR-KLB is also completely resistant to the presence of FGF19 and FGF21. The presence of KLB confers even higher affinity for the FGF19 subfamily on the HS-FGFR complex than KLB alone. These data suggest a mode for the endocrine FGF-initiated FGFR signaling complex formation that is different from that initiated by canonic FGFs.
We speculate that the binding sites on FGFR for FGF1 and FGF19/21 may be not all the same, or may require additional composite ones during the complex assemblage 
. It is also possible that FGF1 could reject the KLB from the complex, or that KLB has no effect on FGF1-FGFR complex formation even when present. The latter possibility appears to be supported by our data 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 
The differences in formation of the active canonic and non-canonic complexes may underlie their downstream functional divergence 
. 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 FGF15−/− 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 
. 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 
. Reports including ours (manuscript in preparation) are consistent on the predominant co-expression of FGFR4-KLB in the liver, hepatocyte or hepatocyte-derived cells, and of FGFR1-KLB in adipose tissues or mature adipocytes 
. 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 comparable to that of FGF19 binding. This differential molecular 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 
; 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 overexpression of constitutively active FGFR1 gain a suppression of bile acids synthesis that is similar to the constitutively active FGFR4 overexpression 
. 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 
. 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 
, and that FGF21 targets primarily the fatty tissues, indicate an emerging endocrine metabolic pathway from the liver to fatty tissues in 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 KLB−/− mice exhibited bile acids phenotype resembling that of FGFR4−/− and FGF15−/−, these mice have not been reported under normal diet condition a metabolic abnormality phenocopying that of FGF21−/−. 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 presence of HS motifs with high affinity, while FGF19 and FGF21 cannot bind directly to the HS-FGFR but in the presence of the transmembrane KLB. It has been shown that the N- and C-terminuses of FGF19 and FGF21 are required for the interaction with FGFR and KLB, respectively; however, it is obvious that the N-terminus alone is not sufficient for direct interaction with FGFR without the presence of KLB 
. These imply that the high affinity binding of FGF19 and FGF21 to KLB, which has been shown in a binary complex with FGFR that is ready to be activated by the binding event 
, is likely the first and key promoting step for the subsequent productive complex formation through more interactions with FGFR that produce further higher affinity and more stability. The integration of KLB into the HS-FGFR for transducing the FGF19 and FGF21 stimulation across the plasma membrane is therefore a hallmark event for their metabolic effects. This integration was visualized by the presence of several possible cross-linking bands. It would be interesting to know how the divergence and specificity in the intracellular phosphorylation and selection of signaling relay adaptors are resulted from the extracellular integration.