BetaKlotho is required for metabolic activity of fibroblast growth factor 21

Abstract

Fibroblast growth factor 21 (FGF21) is a liver-derived endocrine factor that stimulates glucose uptake in adipocytes. Here, we show that FGF21 activity depends on betaKlotho, a single-pass transmembrane protein whose expression is induced during differentiation from preadipocytes to adipocytes. BetaKlotho physically interacts with FGF receptors 1c and 4, thereby increasing the ability of these FGF receptors to bind FGF21 and activate the MAP kinase cascade. Knockdown of betaKlotho expression by siRNA in adipocytes diminishes glucose uptake induced by FGF21. Importantly, administration of FGF21 into mice induces MAP kinase phosphorylation in white adipose tissue and not in tissues without betaKlotho expression. Thus, betaKlotho functions as a cofactor essential for FGF21 activity.

Fig. 1.

βKlotho is required for FGF21 signaling. The 293 cells were transfected either with mock vector (Mock) or expression vectors for βKlotho and Klotho, respectively, and then stimulated with vehicle (Control) or 300 ng/ml recombinant human FGF21 or 300 ng/ml FGF23 for 10 min. Cell lysates were processed for immunoblotting with antibodies against phosphorylated FRS2α (pFRS2α), phosphorylated ERK1/2 (pERK1/2), total ERK1/2 (ERK1/2), βKlotho, or Klotho.

Fig. 2.

βKlotho binds to multiple FGFRs. Lysates of 293 cells transfected with expression vectors for βKlotho and V5 epitope-tagged FGFR isoforms were incubated with anti-V5 antibody, and immunoprecipitated proteins were analyzed for the presence of βKlotho (Top) or FGFR (Middle). Antibodies used for immunoprecipitation (i.p.) and immunoblotting (i.b.) are indicated. Schemes for FGFR isoforms used in this work are shown above the results. The difference between b and c isoforms in FGFR1–3 resides in the C-terminal half of the third Ig-like domain (D3) are indicated in the scheme by hatched and black boxes, respectively. Another alternative splicing event occurs within the first Ig-like domain (D1) and acidic box, which generates long (L), middle (M), and short isoforms (S) in FGFR1 and FGFR2. A portion of each cell lysate sample was processed for immunoblotting with anti-βKlotho antibody to confirm even expression of βKlotho among the cell samples (Bottom).

Fig. 3.

FGF21 preferentially binds to the βKlotho–FGFR complex. The 293 cells transfected with FGFR alone or cotransfected with FGFR and βKlotho were lysed, and FGFR or the βKlotho–FGFR complex was pulled down from cell lysate on agarose beads carrying anti-V5 antibody. The beads were then incubated with conditioned medium containing murine FGF21, and bead-bound proteins were analyzed by immunoblotting for the presence of βKlotho, FGF21, and FGFR. A portion of each cell lysate sample was processed for immunoblot analysis with anti-βKlotho antibody to confirm even expression of βKlotho among the cell samples (Bottom).

Fig. 4.

Adipocytes expressing βKlotho respond to FGF21. (A) FGF21 activates FRS2α and ERK1/2 in differentiated 3T3-L1 adipocytes. 3T3-L1 preadipocytes and differentiated adipocytes were stimulated with conditioned medium containing murine FGF21 or murine FGF23. The same volume of conditioned medium from mock-transfected 293 cells was used as a negative control (Control). Recombinant FGF1 (100 ng/ml) was used as a positive control. (B) βKlotho is expressed upon differentiation of preadipocytes to adipocytes. Differentiation of 3T3-L1 preadipocytes was induced, and the cells were stimulated for 10 min with the FGF21-containing conditioned medium on the indicated days after differentiation. Cell lysate was prepared for immunoblot analysis with the antibodies indicated. (C) Dose response and time course of FGF21 signaling in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were stimulated with increasing doses of recombinant human FGF21 for 10 min (Left) or stimulated with 1 μg/ml recombinant human FGF21 for various time periods (Right). Cell lysates were subjected to immunoblot analysis by using the antibodies indicated. (D) Endogenous βKlotho and FGFR1c form a complex in adipocytes. Lysate of 3T3-L1 adipocytes was subjected to immunoprecipitation (i.p.) with anti-FGFR1 antibody or normal IgG followed by immunoblot (i.b.) analysis with anti-βKlotho antibody or anti-FGFR1 antibody. Cell lysate prepared from differentiated 3T3-L1 adipocytes (Upper, representing 5% of the input) or from 293 cells transfected with FGFR1c(L) and FGFR1c(S) (Lower, serving as size markers for these receptors) was directly subjected to immunoblot analysis to verify the presence of βKlotho and FGFR1c, respectively (Cell lysate).

Fig. 5.

FGF21 requires βKlotho to activate FGF signaling, increase glucose uptake, and increase GLUT1 expression in adipocytes. (A) Differentiated 3T3-L1 adipocytes were transfected with four different siRNAs for βKlotho (βKlotho1–4) or nontargeting control siRNAs (Control 1, 2); 2 days after transfection, the cells were stimulated for 10 min with recombinant human FGF21, FGF1, or vehicle. Cell lysates were immunoblotted (i.b.) with antibodies as indicated. (B) Differentiated 3T3-L1 adipocytes transfected with βKlotho or control siRNAs as in A were incubated for 18 h either with recombinant human FGF21 or with vehicle and then assayed for glucose uptake. The results are presented as means ± SD error bars (n = 3). ∗, P < 0.001 by Student's t test. (C) Expression of GLUT1 and GLUT4 was determined by immunoblot analysis by using the cell lysates from B.

Fig. 6.

FGF21 activates FGF signaling in WAT in mice. (A) Expression of βKlotho and Klotho in hindlimb muscle (Muscle), perigonadal fat pad tissue (WAT), and kidney from two wild-type mice was examined by immunoblot analysis. Actin blot is shown as a loading control. (B) Muscle, fat, and renal tissues were excised from mice treated either with recombinant human FGF21 (n = 2) or with vehicle (n = 2). Tissue lysates were prepared for immunoblot analysis by using the antibodies indicated. (C) As in B, except that recombinant human FGF23 was injected into mice.