Separating mitogenic and metabolic activities of fibroblast growth factor 19 (FGF19)

Abstract

FGF19 and FGF21 are distinctive members of the FGF family that function as endocrine hormones. Their potent effects on normalizing glucose, lipid, and energy homeostasis in disease models have made them an interesting focus of research for combating the growing epidemics of diabetes and obesity. Despite overlapping functions, FGF19 and FGF21 have many discrete effects, the most important being that FGF19 has both metabolic and proliferative effects, whereas FGF21 has only metabolic effects. Here we identify the structural determinants dictating differential receptor interactions that explain and distinguish these two physiological functions. We also have generated FGF19 variants that have lost the ability to induce hepatocyte proliferation but that still are effective in lowering plasma glucose levels and improving insulin sensitivity in mice. Our results add valuable insight into the structure-function relationship of FGF19/FGF21 and identify the structural basis underpinning the distinct proliferative feature of FGF19 compared with FGF21. In addition, these studies provide a road map for engineering FGF19 as a potential therapeutic candidate for treating diabetes and obesity.

Fig. 1.

Effects of mutations in heparin-binding regions of FGF19 on FGFR4 interaction/activation. (A) Sequence alignment between FGF19 and FGF21 around the β1-β2 loop and β10–β12 segment. Sequences swapped in the chimeric proteins are underlined. (B) Schematic diagram showing chimeric proteins FGF19-1, -2, and -3. (C) Solid-phase binding assay measuring the interaction between FGFR4 and FGF19 or FGF19-1 in the presence and absence of heparin. (D) L6 cells were transfected with expression vectors for FGFR1c plus βKlotho, FGFR4 plus βKlotho, or FGFR4 alone. After overnight serum starvation, cells were stimulated with vehicle (ctl) or 50 nM recombinant FGF19 or FGF19-1, -2, or -3 for 15 min and were snap-frozen in liquid nitrogen. Cell lysates were prepared for Western blot analysis using antibodies against phosphorylated ERK1/2 (p-ERK) or total ERK1/2 (T-ERK). (E) Semiquantitative analysis of BrdU immunostaining of livers from female FVB mice treated for 6 d with PBS, or 2 mg/kg/d recombinant FGF19 or FGF19-1. The scores assigned to BrdU incorporation for these animals were based on a semiquantitative scale described in Materials and Methods.

Fig. 2.

Effects of residues 38–42 of FGF19 on FGFR4 interaction/activation. (A) Sequence alignment between FGF19 and FGF21 near the N-terminal region. Sequences swapped in the chimeric proteins are underlined. (B) Schematic diagram showing chimeric proteins FGF21/19^38–42 and FGF19/21^41–43. (C) L6 cells were transfected with expression vectors for FGFR1c plus βKlotho, FGFR4 plus βKlotho, or FGFR4 alone. After overnight serum starvation, cells were stimulated with vehicle (ctl) or 50 nM recombinant FGF19, FGF21/19^38–42, or FGF19/21^41–43 for 15 min and were snap-frozen in liquid nitrogen. Cell lysates were prepared for Western blot analysis using antibodies against phosphorylated ERK1/2 (p-ERK) or total ERK1/2 (T-ERK). (D) Solid-phase binding assay measuring the interaction between FGFR4 and FGF21/19^38–42 or FGF19/21^41–43 in the presence or absence of heparin.

Fig. 3.

Combination mutations of residues 38–42 and heparin-binding regions of FGF19. (A) Schematic diagram showing chimeric proteins FGF19-4, -5, and -6. (B) L6 cells were transfected with expression vectors for FGFR1c plus βKlotho, FGFR4 plus βKlotho, or FGFR4 alone. After overnight serum starvation, cells were stimulated with vehicle (ctl) or 50 nM recombinant FGF19 or FGF19-4, -5, or -6 for 15 min and were snap-frozen in liquid nitrogen. Cell lysates were prepared for Western blot analysis using antibodies against phosphorylated ERK1/2 (p-ERK) or total ERK1/2 (T-ERK). (C) Semiquantitative analysis of BrdU immunostaining of livers from female FVB mice treated for 6 d with PBS or with 2 mg/kg/d recombinant FGF19 or FGF19-4, -5, or -6. The scores assigned to BrdU incorporation for these animals were based on a semiquantitative scale described in Materials and Methods. (D) Differentiated 3T3-L1 adipocytes were incubated for 72 h with recombinant FGF19 or FGF19-4 and assayed for glucose uptake. (E) Ob/ob mice were injected with recombinant FGF19 (n = 10) or FGF19-4 (n = 10). Plasma glucose levels were measured between 3 and 7 h after injection and plotted as AUC during this time period.

Fig. 4.

Structural comparison of FGF19 and FGF2/FGFR1. (A) The potential interface between the N-terminal region of FGF19 (magenta) and the Ig-like extracellular domain 3 (D3) of the receptor. (B) The potential interface between the β1-β2 (orange) and β10–β12 (blue) segments of FGF19, heparin, and the Ig-like extracellular domain 2 (D2) of the receptor. (C) Ribbon representation of FGF19 structure superimposed on the complex structure of FGF2/FGFR1. FGF19 (PDB code: 2P23) is shown in cyan. The N-terminal residues ⁴⁰DPI⁴² are shown in magenta. In the ternary complex structure (PDB code: 1FQ9), FGF2 is gray, and FGFR1 is green. Heparin is shown by gray sticks.