The “hormone like” FGFs exhibit different signaling properties in vitro.

<p><i>Panel 1.</i> In 3T3-L1 fibroblasts over-expressing KL we saw phosphorylation of the FGF target ERK upon treatment with FGF23, while FGF19 and 21 had no effect (A). Conversely, in 3T3-L1/KLB cells we saw no effect of FGF23 but potent signaling with FGF19 or FGF21 treatment (B). When we examined glucose uptake in the 3T3-L1/KL cells we found that only FGF23 lead to its stimulation (C). As we saw with pERK in 3T3-L1/KLB cells FGF19 and FGF21 both increased glucose uptake significantly (D). In 3T3-L1 fibroblasts expressing FGFR4 in the absence of KLB only FGF19 was able to increase glucose uptake (E). Furthermore, in Hep3B cells which show a relative enrichment of FGFR4, FGF19 was significantly more potent than FGF21 in inducing expression of the immediate early gene EGR1 (F). When 3T3-L1 cells were differentiated to become mature adipocytes FGF19 was also more potent than FGF21 even in the absence of FGFR4 expression suggesting a possible unknown factor which is not present on fibroblasts may be affecting FGF19's action in these cells, or vice versa (G). In 3T3-L1 adipocytes treated with either FGF19, FGF21 or a combination of both we did not see any additive or synergistic effects of combination treatment over individual therapy suggesting FGF19 and FGF21 share a common mechanism of action (H). To confirm our initial results regarding the specificity of FGF19 for FGFR4 we turned to L6 cells which have been reported to have extremely low expression of FGFRs and KLs. In parental L6 cells treatment with FGF19 had no effect on the level of ERK phosphorylation, however, when cells were transfected with KLB a small but significant increase in pERK was detected. Furthermore, when FGFR4 was added the response to FGF19 stimulation was magnified. Interestingly, we saw again that in cells transfected with FGFR4 alone FGF19 was also able to induce pERK confirming KLB independent signaling with this ligand can occur (I). In contrast to FGF19, cells treated with FGF21 showed pERK induction only in the presence of KLB with the level of this baseline induction similar to that seen with FGF19 treatment (J).