Fibroblast growth factors (FGF) 19, 21 and 23 are characterized by being endocrinely secreted and require co-receptor α-klotho or β-klotho (BKL) for binding and activation of the FGF receptors (FGFR). FGF15 is the rodent orthologue of human FGF19, but the two proteins share only 52% amino acid identity. Despite the physiological role of FGF21 and FGF19 being quite different, both lower blood glucose (BG) when administered to diabetic mice. The present study was designed to clarify why two human proteins with distinct physiological functions both lower BG in db/db mice and if the mouse orthologue FGF15 has similar effect to FGF19 and FGF21. Recombinant human FGF19, -21 and a mouse FGF15 variant (C110S) were expressed and purified from Escherichia coli. While rhFGF19 (recombinant human fibroblast growth factor 19) and rhFGF21 (recombinant human fibroblast growth factor) bound FGFRs in complex with both human and mouse BKL, rmFGF15CS (recombinant mouse fibroblast growth factor 15 C110S) only bound the FGFRs when combined with mouse BKL. Recombinant hFGF21 and rhFGF19, but not rmFGF15CS, increased glucose uptake in mouse adipocytes, while rhFGF19 and rmFGF15CS potently decreased Cyp7a1 expression in rat hepatocytes. The lack of effect of rmFGF15CS on glucose uptake in adipocytes was associated with rmFGF15CS's inability to signal through the FGFR1c/mouse BKL complex. In db/db mice, only rhFGF19 and rhFGF21 decreased BG while rmFGF15CS and rhFGF19, but not rhFGF21, increased total cholesterol. These data demonstrate receptor- and species-specific differential activity of FGF15 and FGF19 which should be taken into consideration when FGF19 is used as a substitute for FGF15.

Introduction

Fibroblast growth factors (FGFs) such as FGF19, FGF21 and FGF23 differ from the classical FGFs in that they lack the heparin-binding domain which enable them to escape the cellular matrix, enter circulation and act as endocrine hormones [1]. The endocrine FGFs require the co-receptor α-klotho or β-klotho (BKL) [2,3], for binding and activation of the FGF receptors (FGFRs). Consequently, the expression patterns of these co-receptors direct the tissue-specific activity of the endocrine FGFs [24]. It is well described that FGF19, 21 and 23 are all involved in the regulation of various metabolic processes [5].

FGF21 is highly expressed in the liver and in the pancreas [6] but is also found in adipose tissue and skeletal muscle [7]. The liver is the major contributor of circulating FGF21 [8], and both expression and release are increased in response to various metabolic stressors such as starvation [9], lack of protein [10] and high blood glucose (BG) [11]. FGF21 binds to and activates the short c-isoform of the FGFR 1 and 3 in the presence of BKL [12], but has also been reported to bind the FGFR4/BKL complex [2] although no signalling through the FGFR4 has been observed [4]. The major target tissues of FGF21 are the adipose tissues (white and brown adipocytes) [13,14] and specific regions of the central nervous system [15]. Pharmacological doses of recombinant rhFGF21 (recombinant human fibroblast growth factor) normalize body weight, hyperglycaemia and dyslipidaemia in animal models of obesity and type 2 diabetes [1618]. In humans, recombinant analogues of FGF21 have been shown to reduce body weight and dyslipidaemia [19,20].

FGF19 and FGF15 are orthologues [21], but share only 52% amino acid identity. FGF19/15 regulates bile acid metabolism [22] and are expressed in the ileal enterocytes in response to bile acids via activation of the farnesoid X receptor (FXR) [23,24]. FGF19 binds and activates the FGFR4/BKL complex, but FGF19 can also bind to the short c-isoform of FGFR 1, -2 and -3 [3,4]. To our knowledge, there are at present no data showing the receptor preference of FGF15. The major target tissues of FGF19/15 expression are the liver and gall bladder; two tissues which also display high expression of FGFR4 and BKL. In the liver, FGF19/FGF15 suppress the expression of the rate-limiting enzyme in the conversion of cholesterol to bile acids, cholesterol 7 α-hydroxylase (Cyp7a1) [25], thus providing negative feedback to high bile acid levels [26]. FGF19/15 also controls refilling of bile acids into the gall bladder [27]. Administration of rhFGF19 (recombinant human fibroblast growth factor 19) has been shown to increase plasma triglycerides (TGs) and plasma low-density lipoprotein (LDL) cholesterol in mice [28] in agreement with the inhibitory effect of FGF19 on hepatic Cyp7a1. Similarly, an optimized rhFGF19 analogue (NGM282) suppressed the level of bile acid intermediates (C4) and increased plasma LDL cholesterol in humans [29].

Pharmacological doses of both rhFGF19 and rhFGF21 lower BG in diabetic mice, while adeno-associated viral (AAV) delivery of FGF15 does not induce BG lowering in db/db mice [30]. While the metabolic effects of pharmacological doses of FGF19 and FGF21 previously have been directly compared [31], a direct comparison of a recombinant mouse FGF15 analogue (FGF15C110S hereafter rmFGF15CS, recombinant mouse fibroblast growth factor 15 C110S), rhFGF19 and rhFGF21, has not previously been described. The study was designed to clarify why FGF19, but not FGF15, lowers BG in mice. The study reveals important differences in receptor selectivity and metabolic effects of rmFGF15CS versus rhFGF19 and highlights the potential pitfalls using rhFGF19 as a substitute for FGF15.

Materials and methods

Expression and purification of FGF15, -19 and -21

Human rFGF21 and rFGF19 and the murine analogue of FGF15 were produced in Escherichia coli. An N-terminal methionine was added as proteins are expressed in E. coli. Production was carried out at Novo Nordisk A/S, by methods essentially described already (WO2016102562). Briefly, the proteins were expressed in inclusion bodies of E. coli BL21(DE3) cells, purified by standard chromatographical techniques and transferred to phosphate-buffered saline. The identity, purity and stability of the proteins were confirmed by a variety of protein chemical and biophysical techniques. It was impossible to obtain recombinant metFGF15 and therefore a single mutation; C110S was introduced to increase expression and purification of metFGF15C110S. Despite this, the expression yield of rmFGF15CS was very low. A model of the rmFGF15CS is shown in Supplementary Figure S1 and was generated using the Schrödingers advanced homology tool and the crystal structure of FGF19 (pdb:2p23) [32] as a template.

Biotinylated rhFGF21

A cysteine was introduced at position 122C and the analogue was treated with excess N-iodoacetyl-N-biotinylhexylenediamine followed by purification by anion exchange. This analogue was shown to behave like rhFGF21 in 3T3-L1 adipocytes (data not shown). Biotinylated FGF21 was used in the binding assay as described below.

Binding assay

Biotinylated rhFGF21 was coupled to streptavidin donor beads cat. #6760002 (5 mg) from PerkinElmer, and the ectodomain of human FGFR4 or receptor 1c fused to Fc cat. #685-FR-050 and cat. #658-FR-050 from R&D Systems was coupled to Protein A acceptor beads cat. #6760137M from PerkinElmer (method adapted from Smith et al. [34]). A signal, detected as increase in light at 520–620 nm, is generated when either mouse (cat. #2619-KB, R&D Systems) or human BKL (cat. #5889-KB, R&D Systems) protein is added, bringing the donor and acceptor beads in close proximity (Supplementary Figure S2). This signal (measured as counts per second (cps)) can be inhibited by adding increasing doses of rmFGF15CS, rhFGF19 or rhFGF21 for competition with biotinylated rhFGF21 giving an indirect measure of binding affinity. Since the FGFR domains D2 and D3 (FGF-binding domain) are close to 100% conserved between mouse and human receptors (Table 1), the human FGFRs were used in all assays while the human BKL was replaced with mouse BKL to study binding to the mouse receptor complex.

Table 1
Percentage identity compared with human sequence

The sequence alignment as well as percentage identity were calculated using AlignX (a component of Vector NTI Advance® 11.5.1 from Invitrogen).

Percentage identity with human Human Chimpanzee Rhesus monkey Rat Mouse 
FGF21 100 99 96 81 81 
FGF19 FGF15 100 96 98–99 51 52 
FGFR1c (D2/D3) 100 100 100 1001 100 
FGFR4 (D2/D3) 100 99 1001 96 97 
β-klotho (extracellular) 100 99 97 82 79 
Percentage identity with human Human Chimpanzee Rhesus monkey Rat Mouse 
FGF21 100 99 96 81 81 
FGF19 FGF15 100 96 98–99 51 52 
FGFR1c (D2/D3) 100 100 100 1001 100 
FGFR4 (D2/D3) 100 99 1001 96 97 
β-klotho (extracellular) 100 99 97 82 79 
1

One amino acid difference.

Glucose uptake

Murine 3T3-L1 preadipocytes were differentiated into adipocytes as previously described [35]. The day before the adipocytes were used for experiments, and the media were replaced with normal growth media without insulin. To determine the effect of rmFGF15CS, rhFGF19 and rhFGF21 on glucose uptake, the cells were stimulated with increasing concentrations of FGFs for ∼22 h followed by 1-h uptake of 3H-deoxy-glucose (TRK672, Amersham). The cells were washed intensively before Triton X (Sigma, X-100) was added to lyse the cells and the intracellular 3H-2-deoxy-glucose levels were determined by the addition of Microscient 40 (PerkinElmer, #6013641) and counted in a Topcounter.

pERK signalling

HEK293 cells were transfected with BKL from mouse Bkl and cultured as recently described [36]. Cells were stimulated with increasing concentrations of FGFs for 12 min and pErk were determined using the Surefire assay according to the manufacturer's instructions (PerkinElmer, # TGRES10H) and as recently described [36].

Primary hepatocytes and mRNA expression

Primary rat hepatocytes were isolated and cultured as previously described [37]. To determine the effect of rmFGF15CS, rhFGF21 and rhFGF19 on regulation of Cyp7a1 expression, the hepatocytes were incubated with three concentrations of the FGFs for ∼22 h. Cells were harvested and RNA was purified using the RNeasy mini-kit (Qiagen, #74106) according to instructions from the manufacturer. The cDNA was synthesized using iScript (Bio-Rad, #1708891), also according to manufacturer's instructions. The mRNA expression of Cyp7a1 was determined using Taqman probes (Applied Biosystems, Cyp7a1, Rn00564065_m1) and normalized to β-actin (Applied Biosystems, Rn00667869_m1) and analysed using the method.

Receptor (Fgfr1c and Klb) mRNA expression in 3T3-L1 mouse adipocytes, rat liver and HEK293 cells was measured as previously described [38] using the LNA (locked nuclear acid) probe-based system from Roche.

Animals

Eight-week-old db/db C57BL6/JbomTac-KS male mice were purchased from Taconic Europe (Skensved, Denmark) and acclimatized for 2 weeks before the study was initiated. The mice had free access to standard chow (Altromin 1314, Brogaarden, Denmark) and tap water throughout the study. The mice were kept in a climate controlled room at 24 ± 2°C with a 12-h light–dark cycle (lights on at 6 a.m.). Body weight was recorded daily at the beginning of the light period and at the time of the first subcutaneous injection of study drug or vehicle. All experimental procedures were conducted in accordance with internationally accepted principles for the care and use if laboratory animals and were approved by the Danish Ethical Committee for Animal Research and the internal Ethical Review Council for Novo Nordisk A/S.

Pharmacological treatment of recombinant human FGF21, human FGF19 or mouse FGF15

Recombinant hFGF21 compared with rhFGF19

Animals were dosed subcutaneously twice daily with 1 mg/kg rhFGF21 or rhFGF19 for 7 days. BG was measured before dosing on the first day (day 1) and 2 h after dosing on days 1, 6 and 7. Also on day 7 (2-h post-dosing), plasma [ethylenediaminetetraacetic acid (EDTA)] was collected for biochemical analysis. Body weight was measured before the first dose (day 1) and again prior dosing on day 7.

Recombinant hFGF19 compared with rmFGF15CS

Animals were dosed twice daily with 1 mg/kg rhFGF19, 1 mg/kg rmFGF15CS or 3 mg/kg rmFGF15CS for 5 days. BG was measured before dosing at the first day (day 1) and 2 h after dosing on days 1, 4 and 5. Also, on day 5 (2-h post-dosing), plasma (EDTA) was collected for biochemical analysis. Body weight was measured before the first dose (day 1) and again prior dosing on day 5.

Biochemical analyses

Plasma glucose was sampled from the tip of the tail and determined by the glucose oxidation method with ABTS as the substrate. Blood for determination of total bile acids (TBAs), TG and total cholesterol was collected from orbital venous plexus and centrifuged at 4°C at 6000 g in 6 min before plasma was collected and frozen at −80°C for later analysis. Assays for cholesterol (CHOL2; #03039773190, Roche), TG (TRIGL #05171407190, Roche) and TBA assay (# DZ042A-K, Diazyme) were determined on a Hitachi 912 or Cobas 6000 analyzer.

Statistics

One-way or two-way ANOVA with repeated measures using Bonferroni versus vehicle post hoc testing was used to evaluate differences between and within groups (GraphPad Prism 7.0). Significance was accepted at P < 0.05. Data are presented as mean ± SEM.

Results

FGFs and FGFR homology

Mouse and human FGF21 share ∼80% amino acid identity while mouse FGF15 and human FGF19 only share 52% amino acid identity and are orthologues (Table 1). FGFRs are receptor tyrosine kinases that contain an extracellular FGF-binding domain (D2 and D3), a transmembrane domain and an intracellular tyrosine kinase domain [33]. There are four different FGFRs (FGFR1–4) and two splice variants of receptor 1–3. The predominant receptor complex for FGF15 and 19 is FGFR4/BKL [3], whereas FGF21 signalling is mainly mediated by the FGFR1c/BKL complex. As summarized in Table 1, the FGF-binding domains (D2 and D3) of FGFR1c and FGFR4 are highly conserved between species while the extracellular domain of BKL varies and is ∼80% preserved between human and mouse.

FGF19 and FGF21 bind FGFR1c and FGFR4 in the presence of both mouse and human BKL while FGF15 only binds the two FGFR in the presence of mouse BKL

Generation of FGFR1c- and FGFR4-binding assays allowed us to determine and compare the relative affinity of rmFGF15CS, rhFGF19 and rhFGF21 to various FGF/BKL complexes. As shown in Figure 1, rhFGF19 and rhFGF21 bound the human BKL and FGFR1c with approximately the same relative affinity (Figure 1A), while rmFGF15CS did not bind FGFR1 nor FGFR4 in the presence of human BKL (Figure 1A,C). Recombinant hFGF19 bound the human BKL and FGFR4 with more than 100-fold higher relative affinity than rhFGF21 (Figure 1C), while rmFGF15CS and rhFGF19 had almost same affinity for mouse BKL and FGFR4 (Figure 1D). Recombinant mFGF15 bound FGFR1c/mouse BKL with 2-fold higher affinity than FGF21, but with lower affinity than FGF19 (Figure 1B). None of the proteins bound FGFRs in the absence of BKL (data not shown). In summary, rhFGF21 and rhFGF19 bound mouse and human BKL in complex with both FGFR1c and FGFR4, while rmFGF15CS only bound FGFR1c and FGFR4 in the presence of mouse BKL. Importantly, rhFGF19 bound mouse BKL and FGFR1c and FGFR4 with 5- to 40-fold higher affinity than rhFGF21. The IC50 values of the binding data are summarized in Table 2.

Representative binding data on FGF15, FGF19 and FGF21.

Figure 1.
Representative binding data on FGF15, FGF19 and FGF21.

Representative binding data of FGF15, -19 and -21 to (A) human β-klotho receptor and human FGFR1c, (B) mouse β-klotho receptor and human FGFR1c, (C) human β-klotho receptor and human FGFR4 and (D) mouse β-klotho receptor and human FGFR4. Data are mean ± SEM. Average of IC50 and pIC50 from 2–5 independent experiments are given in Table 2.

Figure 1.
Representative binding data on FGF15, FGF19 and FGF21.

Representative binding data of FGF15, -19 and -21 to (A) human β-klotho receptor and human FGFR1c, (B) mouse β-klotho receptor and human FGFR1c, (C) human β-klotho receptor and human FGFR4 and (D) mouse β-klotho receptor and human FGFR4. Data are mean ± SEM. Average of IC50 and pIC50 from 2–5 independent experiments are given in Table 2.

Table 2
Binding affinities for FGF21, FGF19 and FGF15 on either mouse or human β-klotho (BKL) in combination with human FGFR1c or human FGFR4

Data are mean ± SEM (n = 2–5). Binding affinities are IC50 values (nM); values in parentheses are plC50 ± SEM.

 Human BKL Mouse BKL 
Human FGFR4 Human FGFR1c Human FGFR4 Human FGFR1c 
FGF15 No binding No binding 24 (7.6 ± 0.03) 23 (7.6 ± 0.03) 
FGF19 15 (7.8 ± 0.02) 269 (6.6 ± 0.09) 11 (8.0 ± 0.04) 8 (8.1 ± 0.02) 
FGF21 6486 (5.2 ± 0.06) 120 (6.9 ± 0.13) 472 (6.3 ± 0.06) 46 (7.3 ± 0.02) 
 Human BKL Mouse BKL 
Human FGFR4 Human FGFR1c Human FGFR4 Human FGFR1c 
FGF15 No binding No binding 24 (7.6 ± 0.03) 23 (7.6 ± 0.03) 
FGF19 15 (7.8 ± 0.02) 269 (6.6 ± 0.09) 11 (8.0 ± 0.04) 8 (8.1 ± 0.02) 
FGF21 6486 (5.2 ± 0.06) 120 (6.9 ± 0.13) 472 (6.3 ± 0.06) 46 (7.3 ± 0.02) 

FGF19 and FGF21 increase glucose uptake in murine adipocytes while FGF15 and FGF19 suppress Cyp7a1 in primary rat hepatocytes

Mouse 3T3-L1 adipocytes express Klb and Fgfr1c at the mRNA level (Figure 2A), and as expected, rhFGF19 and rhFGF21 were able to induce glucose uptake in this cell type with respective EC50 values of ∼0.08 nM (pEC50 10.19 ± 0.17, n = 4) and ∼0.3 nM (pEC50 9.04 ± 0.12, n = 4) (Figure 2B). Surprisingly, rmFGF15CS was unable to increase glucose uptake in mouse adipocytes, despite it binding to mouse BKL and FGFR1c (Figure 1B). Therefore, rmFGF15CS was tested in HEK293 cells overexpressing mouse Klb using FGF21 as control. HEK293 cells have a high endogenous expression of FGFR1c (Figure 2C) and as seen in Figure 2D, FGF21 induced Erk phosphorylation in these cells while rmFGF15CS did not. Rat hepatocytes express high levels of Klb and Fgfr4 (Figure 2E), and in primary cultured rat hepatocytes overnight incubation with rmFGF15CS and rhFGF19 decreased Cyp7a1 expression with maximal effect observed already at 1 nM (Figure 2F). Recombinant hFGF21 did also decrease Cyp7a1 but with ∼100-fold less potency compared with rhFGF19 in agreement with the FGFR4/mBKL-binding data (Table 2). In summary, only rhFGF19 and rhFGF21 induced glucose uptake in mouse adipocytes, while rmFGF15CS and rhFGF19 were equipotent, and superior to rhFGF21, in their ability to suppress Cyp7a1 mRNA in rat hepatocytes.

Comparison of FGF15, -19 and -21 in vitro.

Figure 2.
Comparison of FGF15, -19 and -21 in vitro.

(A) FGF receptor mRNA expression in 3T3-L1 adipocytes (mouse), Abbreviations: N.D., not detected, (n = 3). (B) Representative data of glucose uptake in 3T3-L1 adipocytes (mouse) after 24 h of stimulation (n = 1). Average EC50 values on four independent experiments; FGF21; 0.3 nM (pEC50: 9.04 ± 0.12), FGF19; 0.08 nM (pEC50: 10.19 ± 0.17), and FGF15; no glucose uptake. (C) FGF receptor mRNA expression in HEK293 cells (n = 3). (D) Representative pERK signalling in HEK293 cells overexpressing mouse BKL. Average EC50 values on three independent experiments; FGF21; 0.6 nM (pEC50: 9.2 ± 0.09), FGF15; 2.6 nM (pEC50: 8.6 ± 0.09). (E) FGF receptor mRNA expression in rat liver (n = 3). (F) Cyp7a1 mRNA expression in primary rat hepatocytes after 24 h of stimulation (n = 3). Data are mean ± SEM. *P < 0.05, **P < 0.01. Student's t-test.

Figure 2.
Comparison of FGF15, -19 and -21 in vitro.

(A) FGF receptor mRNA expression in 3T3-L1 adipocytes (mouse), Abbreviations: N.D., not detected, (n = 3). (B) Representative data of glucose uptake in 3T3-L1 adipocytes (mouse) after 24 h of stimulation (n = 1). Average EC50 values on four independent experiments; FGF21; 0.3 nM (pEC50: 9.04 ± 0.12), FGF19; 0.08 nM (pEC50: 10.19 ± 0.17), and FGF15; no glucose uptake. (C) FGF receptor mRNA expression in HEK293 cells (n = 3). (D) Representative pERK signalling in HEK293 cells overexpressing mouse BKL. Average EC50 values on three independent experiments; FGF21; 0.6 nM (pEC50: 9.2 ± 0.09), FGF15; 2.6 nM (pEC50: 8.6 ± 0.09). (E) FGF receptor mRNA expression in rat liver (n = 3). (F) Cyp7a1 mRNA expression in primary rat hepatocytes after 24 h of stimulation (n = 3). Data are mean ± SEM. *P < 0.05, **P < 0.01. Student's t-test.

FGF19 and FGF21 lower BG while FGF15 and FGF19 regulate bile acid metabolism in db/db mice

The leptin signalling-deficient db/db and ob/ob mice are hyperphagic and develop diabetes. Previous published data show good anti-diabetic effect of systemic administration of rhFGF21 [39] and rhFGF19 [40] in these models, while the effect of FGF15 has only been studied by AAV overexpression and here no effect on BG was observed [30]. In the present study, the three FGFs were compared across two different cohorts of mice. In the first group of diabetic db/db mice, the effect of rhFGF21 was compared with that of rhFGF19. Both FGFs were dosed at 1 mg/kg twice daily for 7 days and both decreased plasma glucose (Figure 3A), while no change in body weight was observed (data not shown). Recombinant hFGF19 increased plasma TG and total cholesterol decreased TBA (Figure 3B–D). Next, the effect of rhFGF19 was compared with rmFGF15CS in the second cohort of db/db mice. Because rmFGF15CS was shown to be ∼2- to 3-fold less potent than rhFGF19 in our binding assay, we chose to test two doses of rmFGF15CS: 1 and 3 mg/kg given subcutaneously twice daily. Owing to low yield of rmFGF15CS expressed in E. coli, it was only possible to dose the mice for 5 days. As seen in Figure 3E, rhFGF19 significantly lowered BG on day 5, while no effect of rmFGF15CS was observed. Neither rmFGF15CS nor rhFGF19 lowered bodyweight after 5 days of treatment (data not shown). Only the highest dose of rmFGF15CS (3 mg/kg) increased plasma TG (Figure 3F), while both doses of rmFGF15CS increased plasma total cholesterol (Figure 3G). Plasma bile acids were also lowered, but this was only significant in the rhFGF19-treated group (Figure 3H). In conclusion, rhFGF19 and rhFGF21 decreased BG in db/db mice, while rhFGF19 and rmFGF15CS increased plasma cholesterol. Recombinant hFGF19 furthermore decreased bile acids while the effect of rmFGF15 on bile acids did not reached statistical significance.

Comparison of FGF19 versus FGF21 and FGF19 versus FGF15 in diabetic db/db mice.

Figure 3.
Comparison of FGF19 versus FGF21 and FGF19 versus FGF15 in diabetic db/db mice.

(A–D) Male diabetic db/db mice were dosed 1 mg/kg of FGF19 or FGF21 subcutaneously for 7 days. Data are mean ± SEM, n = 5 per group. (A) Blood glucose was measured 2 h after dosing at days 1, 6 and 7. ***P < 0.001 two-way ANOVA post hoc Bonferroni versus vehicle. Plasma triglycerides (B), cholesterol (C) and total bile acids (D) were measured 2 h after dosing at day 7. *P < 0.05, ***P < 0.001, two-way ANOVA or one-way ANOVA versus vehicle, when appropriate. (E–H) Male diabetic db/db mice were dosed with FGF19 (1 mg/kg) or two different doses of FGF15 (1 and 3 mg/kg) for 5 days. Data are mean ± SEM, n = 5 per group. (A) Blood glucose was measured 2 h after dosing at day 0 and day 5. Plasma triglycerides (B), cholesterol (C) and total bile acids (D) were measured 2 h after dosing at day 5. *P < 0.05, ***P < 0.001, two-way ANOVA or one-way ANOVA multiple comparisons versus vehicle, when appropriate.

Figure 3.
Comparison of FGF19 versus FGF21 and FGF19 versus FGF15 in diabetic db/db mice.

(A–D) Male diabetic db/db mice were dosed 1 mg/kg of FGF19 or FGF21 subcutaneously for 7 days. Data are mean ± SEM, n = 5 per group. (A) Blood glucose was measured 2 h after dosing at days 1, 6 and 7. ***P < 0.001 two-way ANOVA post hoc Bonferroni versus vehicle. Plasma triglycerides (B), cholesterol (C) and total bile acids (D) were measured 2 h after dosing at day 7. *P < 0.05, ***P < 0.001, two-way ANOVA or one-way ANOVA versus vehicle, when appropriate. (E–H) Male diabetic db/db mice were dosed with FGF19 (1 mg/kg) or two different doses of FGF15 (1 and 3 mg/kg) for 5 days. Data are mean ± SEM, n = 5 per group. (A) Blood glucose was measured 2 h after dosing at day 0 and day 5. Plasma triglycerides (B), cholesterol (C) and total bile acids (D) were measured 2 h after dosing at day 5. *P < 0.05, ***P < 0.001, two-way ANOVA or one-way ANOVA multiple comparisons versus vehicle, when appropriate.

Discussion

In the present study, we compared the in vitro and in vivo metabolic effects of the two orthologues FGF15 and FGF19 with those of FGF21 using recombinant proteins. Our receptor-binding data showed several differences in the relative affinities of these three endocrine FGFs. It was evident that rmFGF15CS cannot bind human BKL while rhFGF21 and rhFGF19 bound mouse BKL with higher affinity than human BKL. The difference in relative binding affinity towards the receptor complexes must rely in the binding interaction with BKL as the FGFR D2/D3 domains are highly conserved between species, as recently confirmed by the elucidation of the crystal structure [41]. Taken together, these data suggest that sequence differences in the FGF ligand-binding region of BKL result in higher affinities of rhFGF21 and rhFGF19 towards mouse BKL compared with the human BKL. In agreement with our data, rhFGF21 has been shown to bind mouse BKL with ∼20-fold higher affinities than human BKL based on surface plasmon resonance data [42]. However, the story is more complex as such, as the binding affinities of rhFGF19 towards the FGFR4/hBKL and FGFR4/mBKL complexes are within the same range, indicating that the FGFR subtype also affects the receptor affinity.

Surprisingly, rmFGF15CS which binds the FGFR1c/mBKL complex did not signal in cellular systems expressing these receptors (adipocytes and HEK293/mBKL cells). It is currently unclear why receptor binding of rmFGF15CS to FGFR1c/mBKL does not result in downstream signalling. A limitation of the present study is that we only interrogated activation of Erk; however, while other signalling pathways may be activated, we also did not observe induction of glucose uptake in murine adipocytes in response to rmFGF15CS. Similar observations have been made for rhFGF21 that has been shown to bind the FGFR4/BKL complex [2], but lacks the ability to initiate signalling through this receptor complex [2,4]. Interestingly, another study [43] using a receptor-binding assay based on 125I-FGF19 showed that FGF21 cannot bind FGFR4/BKL. Importantly, Yang et al. [43] address the binding of FGF21 indirectly through its ability to displace 125I-FGF19, whereas our binding assay detects a direct binding between biotinylated FGF21 and FGFR4/BKL; therefore, the two studies are not directly comparable and not necessarily in conflict. In agreement with previous studies, we found that rhFGF19 is ∼10-fold more potent than rhFGF21 in inducing glucose uptake in mouse 3T3-L1 adipocytes [4]. The BG-lowering effect of rhFGF19 in mice may therefore be through the same mechanism as with rhFGF21 which mediates increased glucose uptake into adipose tissue by up-regulation of Glut1 [44]. However, rhFGF19 has also been described to decrease Pepck (phosphoenolpyruvate carboxykinase) mRNA expression and thereby decrease hepatic glucose production [45], which may also have contributed to the BG-lowering effect in vivo. Oppositely, rhFGF21 has been shown to acutely increase Pepck expression [46]. In vivo rhFGF19 at 1 mg/kg has much more pronounced effect on plasma TG and cholesterol than 3 mg/kg rmFGF15CS. This is, however, not in agreement with our in vitro data where rmFGF15CS and rhFGF19 bound FGFR4/mBKL and suppressed Cyp7a1 mRNA expression in rat hepatocytes with approximately same affinity and potency. Therefore, we cannot exclude that different pharmacokinetic (PK) properties of rhFGF19 and rmFGF15CS may have affected our in vivo data as no PK was determined of the recombinant proteins in the present study. The half-life (t½) of rhFGF21 and rhFGF19 is ∼1–2 h in mice [18,47], while the t½ of rmFGF15CS was not determined due to the absence of a reliable FGF15 ELISA at the time of the study.

In agreement with our FGFR4/mBKL-binding data, rhFGF19 and rmFGF15CS are ∼100-fold more potent than rhFGF21 in inhibiting Cyp7a1 mRNA expression in hepatocytes. These data corroborate previous findings that rhFGF21 inhibits Cyp7a1 expression in human hepatocytes with 1000-fold lower potency than rhFGF19 [48]. Hepatocytes express only very low levels of FGFR1c, and as FGF21 cannot signal through the FGFR4/BKL complex [4], the effect of FGF21 on hepatocytes may be mediated via the FGFR3c/BKL complex. In vivo, we did not observe any effect of rhFGF21 (1 mg/kg) on bile acids; however, Chen et al. [48] have observed effect of rhFGF21 on bile acid metabolism in mice when rhFGF21 was dosed at 3 mg/kg. In the present study, we have not compared recombinant mouse FGF21 with rhFGF21, but rmFGF21 has been found to have similar glucose- and lipid-lowering effects as rhFGF21 in mice [49]. Mouse and human FGF21 share ∼80% amino acid identity and future studies will show if rmFGF21 like rmFGF15CS does not bind human BKL. The extra complexity of FGF19 and FGF21 binding to FGFR2c/BKL and FGFR3c/BKL is out of scope of the present paper and future studies using receptor selective analogues or FGFR isoform-specific knockout mice are needed to address the contribution of these receptors to the biology.

Recombinant mFGF15CS did not increase glucose uptake in 3T3-L1 adipocytes and no glucose-lowering effect was seen in db/db mice. However, the lack of effect of rmFGF15CS on BG was not due to rmFGF15CS being inactive, as it induced a clear suppression of Cyp7a1 mRNA in rat hepatocytes and increased total cholesterol in vivo (db/db mice). However, we cannot exclude that the biological activity and potency of wild-type recombinant mouse FGF15 may differ from the rmFGF15CS analogue we have used. Recently, it has been shown that FGF15 appears to exist as a homodimer in its native state [30], but here we have demonstrated that the FGF15 analogue rmFGF15CS retained activity without the ability to form a dimer due to mutation of cysteine 110 to a serine. Our findings using rmFGF15CS are in agreement with Zhou et al. [30] who showed that AAV-mediated expression of FGF15 and FGF19 repressed bile acid synthesis in vivo but only FGF19 lowered BG in db/db mice.

In conclusion, our data demonstrate that in contrast with rhFGF19, rmFGF15CS cannot signal through the FGFR1c/BKL complex to increase glucose uptake in mouse adipocytes or lower BG in db/db mice. The effect of FGF19 is overlapping with rhFGF21's effect on glucose uptake in adipocytes and the BG-lowering effect in db/db mice. Recombinant hFGF19 and rmFGF15CS both suppressed Cyp7a1 mRNA expression via activation of the FGFR4/BKL complex in rat hepatocytes and increased total cholesterol in mice. The data indicate that differential receptor selectivity of the FGF15/FGF19 orthologues causes differences in metabolic activity. Our data are summarized in Figure 4 and illustrate the degree of overlap of the complex biology of FGF15, FGF19 and FGF21. The present study highlights an important receptor and species differences between FGF15 and FGF19 and shows how data should be interpreted cautiously when studying effects of rhFGF19 in murine models.

Schematic summary of data.

Figure 4.
Schematic summary of data.

Recombinant mFGF15 and rhFGF19 bind and signal through the FGFR4/BKL complex which regulated Cyp7a1 expression in hepatocytes causing a decreased in plasma bile acids and increased in plasma cholesterol in db/db mice. Recombinant hFGF19 and rhFGF21 bind and signal through the FGFR1c/BKL complex, increase glucose uptake in adipocytes and decrease blood glucose in db/db mice. The dotted line indicates that at 100 nM of FGF21, a suppression of CYP/A1 was observed in hepatocytes in vitro, but this did not cause changes in bile acid metabolism in db/db mice.

Figure 4.
Schematic summary of data.

Recombinant mFGF15 and rhFGF19 bind and signal through the FGFR4/BKL complex which regulated Cyp7a1 expression in hepatocytes causing a decreased in plasma bile acids and increased in plasma cholesterol in db/db mice. Recombinant hFGF19 and rhFGF21 bind and signal through the FGFR1c/BKL complex, increase glucose uptake in adipocytes and decrease blood glucose in db/db mice. The dotted line indicates that at 100 nM of FGF21, a suppression of CYP/A1 was observed in hepatocytes in vitro, but this did not cause changes in bile acid metabolism in db/db mice.

Abbreviations

     
  • AAV

    adeno-associated virus

  •  
  • BG

    blood glucose

  •  
  • BKL

    β-klotho

  •  
  • Cyp7a1

    cholesterol 7 alpha-hydroxylase

  •  
  • D2 and D3

    FGF-binding domain

  •  
  • EDTA

    ethylenediaminetetraacetic acid

  •  
  • FGF

    fibroblast growth factors

  •  
  • rhFGF21

    recombinant human fibroblast growth factor

  •  
  • rmFGF15CS

    recombinant mouse fibroblast growth factor 15 C110S

  •  
  • rhFGF19

    recombinant human fibroblast growth factor 19

  •  
  • FGFR

    fibroblast growth factors receptor

  •  
  • LDL

    low-density lipoprotein

  •  
  • Pepck

    phosphoenolpyruvate carboxykinase

  •  
  • TBA

    total bile acids

  •  
  • TG

    triglycerides.

Acknowledgments

We thank Kirsten Vinkel Haugegaard, Sine L. Rosenfalck Døhn, Ying Lv, Helle Nygaard, Line Kristensen and Elene Hother Carlsen from the department of Type 2 Diabetes and GLP1 in vitro and in vivo pharmacology for their skilled technical assistance. B.A. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Author Contribution

A.M.K.H., S.G.V., K.L., X.Z., G.T., D.H., X.Z., H.T., K.S., T.T., K.R. and B.A. created the data (proteins production, binding assays, in vitro and in vivo studies). B.A. conceived the idea and wrote the manuscript. A.H. and S.G. contributed to and reviewed/edited the manuscript.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript. All authors are employed by Novo Nordisk A/S.

References

References
1
Fukumoto
,
S.
(
2008
)
Actions and mode of actions of FGF19 subfamily members
.
Endocr. J.
55
,
23
31
2
Ogawa
,
Y.
,
Kurosu
,
H.
,
Yamamoto
,
M.
,
Nandi
,
A.
,
Rosenblatt
,
K.P.
,
Goetz
,
R.
et al. 
(
2007
)
Betaklotho is required for metabolic activity of fibroblast growth factor 21
.
Proc. Natl Acad. Sci. U.S.A.
104
,
7432
7437
3
Lin
,
B.C.
,
Wang
,
M.
,
Blackmore
,
C.
and
Desnoyers
,
L.R.
(
2007
)
Liver-specific activities of FGF19 require Klotho beta
.
J. Biol. Chem.
282
,
27277
27284
4
Kurosu
,
H.
,
Choi
,
M.
,
Ogawa
,
Y.
,
Dickson
,
A.S.
,
Goetz
,
R.
,
Eliseenkova
,
A.V.
et al. 
(
2007
)
Tissue-specific expression of betaKlotho and fibroblast growth factor (FGF) receptor isoforms determines metabolic activity of FGF19 and FGF21
.
J. Biol. Chem.
282
,
26687
26695
5
Beenken
,
A.
and
Mohammadi
,
M.
(
2012
)
The structural biology of the FGF19 subfamily
.
Adv. Exp. Med. Biol.
728
,
1
24
6
Fon Tacer
,
K.
,
Bookout
,
A.L.
,
Ding
,
X.
,
Kurosu
,
H.
,
John
,
G.B.
,
Wang
,
L.
et al. 
. (
2010
)
Research resource: comprehensive expression atlas of the fibroblast growth factor system in adult mouse
.
Mol. Endocrinol.
24
,
2050
2064
7
Vienberg
,
S.G.
,
Brons
,
C.
,
Nilsson
,
E.
,
Astrup
,
A.
,
Vaag
,
A.
and
Andersen
,
B.
(
2012
)
Impact of short-term high-fat feeding and insulin-stimulated FGF21 levels in subjects with low birth weight and controls
.
Eur. J. Endocrinol.
167
,
49
57
8
Markan
,
K.R.
,
Naber
,
M.C.
,
Ameka
,
M.K.
,
Anderegg
,
M.D.
,
Mangelsdorf
,
D.J.
,
Kliewer
,
S.A.
et al. 
(
2014
)
Circulating FGF21 is liver derived and enhances glucose uptake during refeeding and overfeeding
.
Diabetes
63
,
4057
4063
9
Inagaki
,
T.
,
Dutchak
,
P.
,
Zhao
,
G.
,
Ding
,
X.
,
Gautron
,
L.
,
Parameswara
,
V.
et al. 
(
2007
)
Endocrine regulation of the fasting response by PPARalpha-mediated induction of fibroblast growth factor 21
.
Cell Metab.
5
,
415
425
10
Laeger
,
T.
,
Henagan
,
T.M.
,
Albarado
,
D.C.
,
Redman
,
L.M.
,
Bray
,
G.A.
,
Noland
,
R.C.
et al. 
(
2014
)
FGF21 is an endocrine signal of protein restriction
.
J. Clin. Invest.
124
,
3913
3922
11
von Holstein-Rathlou
,
S.
,
BonDurant
,
L.D.
,
Peltekian
,
L.
,
Naber
,
M.C.
,
Yin
,
T.C.
,
Claflin
,
K.E.
et al. 
(
2016
)
FGF21 mediates endocrine control of simple sugar intake and sweet taste preference by the liver
.
Cell Metab.
23
,
335
343
12
Suzuki
,
M.
,
Uehara
,
Y.
,
Motomura-Matsuzaka
,
K.
,
Oki
,
J.
,
Koyama
,
Y.
,
Kimura
,
M.
et al. 
(
2008
)
Betaklotho is required for fibroblast growth factor (FGF) 21 signaling through FGF receptor (FGFR) 1c and FGFR3c
.
Mol. Endocrinol.
22
,
1006
1014
13
BonDurant
,
L.D.
,
Ameka
,
M.
,
Naber
,
M.C.
,
Markan
,
K.R.
,
Idiga
,
S.O.
,
Acevedo
,
M.R.
et al. 
(
2017
)
FGF21 regulates metabolism through adipose-dependent and -independent mechanisms
.
Cell Metab.
25
,
935
944.e4
14
Lan
,
T.
,
Morgan
,
D.A.
,
Rahmouni
,
K.
,
Sonoda
,
J.
,
Fu
,
X.
,
Burgess
,
S.C.
et al. 
(
2017
)
FGF19, FGF21, and an FGFR1/β-Klotho-activating antibody act on the nervous system to regulate body weight and glycemia
.
Cell Metab.
26
,
709
718.e3
15
Owen
,
B.M.
,
Ding
,
X.
,
Morgan
,
D.A.
,
Coate
,
K.C.
,
Bookout
,
A.L.
,
Rahmouni
,
K.
et al. 
(
2014
)
FGF21 acts centrally to induce sympathetic nerve activity, energy expenditure, and weight loss
.
Cell Metab.
20
,
670
677
16
Coskun
,
T.
,
Bina
,
H.A.
,
Schneider
,
M.A.
,
Dunbar
,
J.D.
,
Hu
,
C.C.
,
Chen
,
Y.
et al. 
. (
2008
)
Fibroblast growth factor 21 corrects obesity in mice
.
Endocrinology
149
,
6018
6027
17
Kharitonenkov
,
A.
,
Shiyanova
,
T.L.
,
Koester
,
A.
,
Ford
,
A.M.
,
Micanovic
,
R.
,
Galbreath
,
E.J.
et al. 
(
2005
)
FGF-21 as a novel metabolic regulator
.
J. Clin. Invest.
115
,
1627
1635
18
Kharitonenkov
,
A.
,
Wroblewski
,
V.J.
,
Koester
,
A.
,
Chen
,
Y.F.
,
Clutinger
,
C.K.
,
Tigno
,
X.T.
et al. 
(
2007
)
The metabolic state of diabetic monkeys is regulated by fibroblast growth factor-21
.
Endocrinology
148
,
774
781
19
Talukdar
,
S.
,
Zhou
,
Y.
,
Li
,
D.
,
Rossulek
,
M.
,
Dong
,
J.
,
Somayaji
,
V.
et al. 
(
2016
)
A long-acting FGF21 molecule, PF-05231023, decreases body weight and improves lipid profile in non-human primates and type 2 diabetic subjects
.
Cell Metab.
23
,
427
440
20
Gaich
,
G.
,
Chien
,
J.Y.
,
Fu
,
H.
,
Glass
,
L.C.
,
Deeg
,
M.A.
,
Holland
,
W.L.
et al. 
(
2013
)
The effects of LY2405319, an FGF21 analog, in obese human subjects with type 2 diabetes
.
Cell Metab.
18
,
333
340
21
Wright
,
T.J.
,
Ladher
,
R.
,
McWhirter
,
J.
,
Murre
,
C.
,
Schoenwolf
,
G.C.
and
Mansour
,
S.L.
(
2004
)
Mouse FGF15 is the ortholog of human and chick FGF19, but is not uniquely required for otic induction
.
Dev. Biol.
269
,
264
275
22
Inagaki
,
T.
,
Choi
,
M.
,
Moschetta
,
A.
,
Peng
,
L.
,
Cummins
,
C.L.
,
McDonald
,
J.G.
et al. 
(
2005
)
Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis
.
Cell Metab.
2
,
217
225
23
Jung
,
D.
,
Inagaki
,
T.
,
Gerard
,
R.D.
,
Dawson
,
P.A.
,
Kliewer
,
S.A.
,
Mangelsdorf
,
D.J.
et al. 
(
2007
)
FXR agonists and FGF15 reduce fecal bile acid excretion in a mouse model of bile acid malabsorption
.
J. Lipid Res.
48
,
2693
2700
24
Walters
,
J.R.
(
2014
)
Bile acid diarrhoea and FGF19: new views on diagnosis, pathogenesis and therapy
.
Nat. Rev. Gastroenterol. Hepatol.
11
,
426
434
25
Jones
,
S.
(
2008
)
Mini-review: endocrine actions of fibroblast growth factor 19
.
Mol. Pharm.
5
,
42
48
26
Cyphert
,
H.A.
,
Ge
,
X.
,
Kohan
,
A.B.
,
Salati
,
L.M.
,
Zhang
,
Y.
and
Hillgartner
,
F.B.
(
2012
)
Activation of the farnesoid X receptor induces hepatic expression and secretion of fibroblast growth factor 21
.
J. Biol. Chem.
287
,
25123
25138
27
Kir
,
S.
,
Kliewer
,
S.A.
and
Mangelsdorf
,
D.J.
(
2011
)
Roles of FGF19 in liver metabolism
.
Cold Spring Harb. Symp. Quant. Biol.
76
,
139
144
28
Wu
,
X.
,
Ge
,
H.
,
Baribault
,
H.
,
Gupte
,
J.
,
Weiszmann
,
J.
,
Lemon
,
B.
et al. 
(
2013
)
Dual actions of fibroblast growth factor 19 on lipid metabolism
.
J. Lipid Res.
54
,
325
332
29
Harrison
,
S.A.
,
Rinella
,
M.E.
,
Abdelmalek
,
M.F.
,
Trotter
,
J.F.
,
Paredes
,
A.H.
,
Arnold
,
H.L.
et al. 
(
2018
)
NGM282 for treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial
.
Lancet
391
,
1174
1185
30
Zhou
,
M.
,
Luo
,
J.
,
Chen
,
M.
,
Yang
,
H.
,
Learned
,
R.M.
,
DePaoli
,
A.M.
et al. 
(
2017
)
Mouse species-specific control of hepatocarcinogenesis and metabolism by FGF19/FGF15
.
J. Hepatol.
66
,
1182
1192
31
Adams
,
A.C.
,
Coskun
,
T.
,
Rovira
,
A.R.
,
Schneider
,
M.A.
,
Raches
,
D.W.
,
Micanovic
,
R.
et al. 
(
2012
)
Fundamentals of FGF19 & FGF21 action in vitro and in vivo
.
PLoS ONE
7
,
e38438
32
Goetz
,
R.
,
Beenken
,
A.
,
Ibrahimi
,
O.A.
,
Kalinina
,
J.
,
Olsen
,
S.K.
,
Eliseenkova
,
A.V.
et al. 
(
2007
)
Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members
.
Mol. Cell Biol.
27
,
3417
3428
33
Ornitz
,
D.M.
and
Itoh
,
N.
(
2015
)
The fibroblast growth factor signaling pathway
.
Wiley Interdiscip. Rev. Dev. Biol.
4
,
215
266
34
Smith
,
R.
,
Duguay
,
A.
,
Weiszmann
,
J.
,
Stanislaus
,
S.
,
Belouski
,
E.
,
Cai
,
L.
et al. 
(
2013
)
A novel approach to improve the function of FGF21
.
BioDrugs
27
,
159
166
35
Zebisch
,
K.
,
Voigt
,
V.
,
Wabitsch
,
M.
and
Brandsch
,
M.
(
2012
)
Protocol for effective differentiation of 3T3-L1 cells to adipocytes
.
Anal. Biochem.
425
,
88
90
36
Agrawal
,
A.
,
Parlee
,
S.
,
Perez-Tilve
,
D.
,
Li
,
P.
,
Pan
,
J.
,
Mroz
,
P.A.
et al. 
(
2018
)
Molecular elements in FGF19 and FGF21 defining KLB/FGFR activity and specificity
.
Mol. Metab.
13
,
45
55
37
Andersen
,
B.
,
Rassov
,
A.
,
Westergaard
,
N.
and
Lundgren
,
K.
(
1999
)
Inhibition of glycogenolysis in primary rat hepatocytes by 1, 4-dideoxy-1,4-imino-d-arabinitol
.
Biochem. J.
342
,
545
550
38
Nygaard
,
E.B.
,
Vienberg
,
S.G.
,
Orskov
,
C.
,
Hansen
,
H.S.
and
Andersen
,
B.
(
2012
)
Metformin stimulates FGF21 expression in primary hepatocytes
.
Exp. Diabetes Res.
2012
,
465282
39
Wente
,
W.
,
Efanov
,
A.M.
,
Brenner
,
M.
,
Kharitonenkov
,
A.
,
Koster
,
A.
,
Sandusky
,
G.E.
et al. 
(
2006
)
Fibroblast growth factor-21 improves pancreatic beta-cell function and survival by activation of extracellular signal-regulated kinase 1/2 and Akt signaling pathways
.
Diabetes
55
,
2470
2478
40
Strack
,
A.M.
and
Myers
,
R.W.
(
2004
)
Modulation of metabolic syndrome by fibroblast growth factor 19 (FGF19)?
Endocrinology
145
,
2591
2593
41
Lee
,
S.
,
Choi
,
J.
,
Mohanty
,
J.
,
Sousa
,
L.P.
,
Tome
,
F.
,
Pardon
,
E.
et al. 
(
2018
)
Structures of β-klotho reveal a ‘zip code’-like mechanism for endocrine FGF signalling
.
Nature
553
,
501
505
42
Stanislaus
,
S.
,
Hecht
,
R.
,
Yie
,
J.
,
Hager
,
T.
,
Hall
,
M.
,
Spahr
,
C.
et al. 
(
2017
)
A novel Fc-FGF21 with improved resistance to proteolysis, increased affinity towards β-Klotho and enhanced efficacy in mice and cynomolgus monkeys
.
Endocrinology
158
,
1314
1327
43
Yang
,
C.
,
Jin
,
C.
,
Li
,
X.
,
Wang
,
F.
,
McKeehan
,
W.L.
and
Luo
,
Y.
(
2012
)
Differential specificity of endocrine FGF19 and FGF21 to FGFR1 and FGFR4 in complex with KLB
.
PLoS ONE
7
,
e33870
44
Ge
,
X.
,
Chen
,
C.
,
Hui
,
X.
,
Wang
,
Y.
,
Lam
,
K.S.
and
Xu
,
A.
(
2011
)
Fibroblast growth factor 21 induces glucose transporter-1 expression through activation of the serum response factor/Ets-like protein-1 in adipocytes
.
J. Biol. Chem.
286
,
34533
34541
45
Potthoff
,
M.J.
,
Boney-Montoya
,
J.
,
Choi
,
M.
,
He
,
T.
,
Sunny
,
N.E.
,
Satapati
,
S.
et al. 
(
2011
)
FGF15/19 regulates hepatic glucose metabolism by inhibiting the CREB-PGC-1alpha pathway
.
Cell Metab.
13
,
729
738
46
Potthoff
,
M.J.
,
Inagaki
,
T.
,
Satapati
,
S.
,
Ding
,
X.
,
He
,
T.
,
Goetz
,
R.
et al. 
(
2009
)
FGF21 induces PGC-1alpha and regulates carbohydrate and fatty acid metabolism during the adaptive starvation response
.
Proc. Natl Acad. Sci. USA
106
,
10853
10858
47
Alvarez-Sola
,
G.
,
Uriarte
,
I.
,
Latasa
,
M.U.
,
Fernandez-Barrena
,
M.G.
,
Urtasun
,
R.
,
Elizalde
,
M.
et al. 
(
2017
)
Fibroblast growth factor 15/19 (FGF15/19) protects from diet-induced hepatic steatosis: development of an FGF19-based chimeric molecule to promote fatty liver regeneration
.
Gut
66
,
1818
1828
48
Chen
,
M.M.
,
Hale
,
C.
,
Stanislaus
,
S.
,
Xu
,
J.
and
Veniant
,
M.M.
(
2018
)
FGF21 acts as a negative regulator of bile acid synthesis
.
J. Endocrinol.
237
,
139
152
49
Veniant
,
M.M.
,
Hale
,
C.
,
Helmering
,
J.
,
Chen
,
M.M.
,
Stanislaus
,
S.
,
Busby
,
J.
et al. 
(
2012
)
FGF21 promotes metabolic homeostasis via white adipose and leptin in mice
.
PLoS ONE
7
,
e40164

Supplementary data