Background Recent studies suggest that betaKlotho (KLB) and endocrine FGF19 and FGF21 redirect FGFR signaling to regulation of metabolic homeostasis and suppression of obesity and diabetes. However, the identity of the predominant metabolic tissue in which a major FGFR-KLB resides that critically mediates the differential actions and metabolism effects of FGF19 and FGF21 remain unclear. Methodology/Principal Findings We determined the receptor and tissue specificity of FGF21 in comparison to FGF19 by using direct, sensitive and quantitative binding kinetics, and downstream signal transduction and expression of early response gene upon administration of FGF19 and FGF21 in mice. We found that FGF21 binds FGFR1 with much higher affinity than FGFR4 in presence of KLB; while FGF19 binds both FGFR1 and FGFR4 in presence of KLB with comparable affinity. The interaction of FGF21 with FGFR4-KLB is very weak even at high concentration and could be negligible at physiological concentration. Both FGF19 and FGF21 but not FGF1 exhibit binding affinity to KLB. The binding of FGF1 is dependent on where FGFRs are present. Both FGF19 and FGF21 are unable to displace the FGF1 binding, and conversely FGF1 cannot displace FGF19 and FGF21 binding. These results indicate that KLB is an indispensable mediator for the binding of FGF19 and FGF21 to FGFRs that is not required for FGF1. Although FGF19 can predominantly activate the responses of the liver and to a less extent the adipose tissue, FGF21 can do so significantly only in the adipose tissue and adipocytes. Among several metabolic and endocrine tissues, the response of adipose tissue to FGF21 is predominant, and can be blunted by the ablation of KLB or FGFR1. Conclusions Our results indicate that unlike FGF19, FGF21 is unable to bind FGFR4-KLB complex with affinity comparable to FGFR1-KLB, and therefore, at physiological concentration less likely to directly and significantly target the liver where FGFR4-KLB predominantly resides. However, both FGF21 and FGF19 have the potential to activate responses of primarily the adipose tissue where FGFR1-KLB resides.
References
[1]
Beenken A, Mohammadi M (2009) The FGF family: biology, pathophysiology and therapy. Nat Rev Drug Discov 8: 235–253.
[2]
Eswarakumar VP, Lax I, Schlessinger J (2005) Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 16: 139–149.
[3]
Kurosu H, Kuro OM (2009) The Klotho gene family as a regulator of endocrine fibroblast growth factors. Mol Cell Endocrinol 299: 72–78.
[4]
Luo Y, Yang C, Lu W, Xie R, Jin C, et al. (2010) Metabolic regulator {beta}klotho interacts with fibroblast growth factor receptor 4 (FGFR4) to induce apoptosis and inhibit tumor cell proliferation. J Biol Chem 285: 30069–30078.
[5]
McKeehan WL, Wang F, Kan M (1998) The heparan sulfate-fibroblast growth factor family: diversity of structure and function. Prog Nucleic Acid Res Mol Biol 59: 135–176.
[6]
Itoh N, Ornitz DM (2004) Evolution of the Fgf and Fgfr gene families. Trends Genet 20: 563–569.
[7]
Grose R, Dickson C (2005) Fibroblast growth factor signaling in tumorigenesis. Cytokine Growth Factor Rev 16: 179–186.
[8]
Huang X, Yang C, Luo Y, Jin C, Wang F, et al. (2007) FGFR4 prevents hyperlipidemia and insulin resistance but underlies high-fat diet induced fatty liver. Diabetes 56: 2501–2510.
[9]
Ogawa Y, Kurosu H, Yamamoto M, Nandi A, Rosenblatt KP, et al. (2007) BetaKlotho is required for metabolic activity of fibroblast growth factor 21. Proc Natl Acad Sci U S A 104: 7432–7437.
[10]
Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, et al. (2006) Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature 444: 770–774.
[11]
Yu C, Wang F, Kan M, Jin C, Jones RB, et al. (2000) Elevated cholesterol metabolism and bile acid synthesis in mice lacking membrane tyrosine kinase receptor FGFR4. J Biol Chem 275: 15482–15489.
[12]
Luo Y, Lu W, Mohamedali KA, Jang JH, Jones RB, et al. (1998) The glycine box: a determinant of specificity for fibroblast growth factor. Biochemistry 37: 16506–16515.
[13]
Luo Y, Ye S, Kan M, McKeehan WL (2006) Structural specificity in a FGF7-affinity purified heparin octasaccharide required for formation of a complex with FGF7 and FGFR2IIIb. J Cell Biochem 97: 1241–1258.
[14]
Luo Y, Ye S, Kan M, McKeehan WL (2006) Control of FGF7- and FGF1-induced mitogenesis and downstream signaling by distinct heparin octasaccharide motifs. J Biol Chem 97: 21052–21061.
[15]
Ye S, Luo Y, Lu W, Jones RB, Linhardt RJ, et al. (2001) Structural basis for interaction of FGF-1, FGF-2, and FGF-7 with different heparan sulfate motifs. Biochemistry 40: 14429–14439.
[16]
Goetz R, Beenken A, Ibrahimi OA, Kalinina J, Olsen SK, 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.
[17]
Kurosu H, Choi M, Ogawa Y, Dickson AS, Goetz R, 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.
[18]
Ito S, Kinoshita S, Shiraishi N, Nakagawa S, Sekine S, et al. (2000) Molecular cloning and expression analyses of mouse betaklotho, which encodes a novel Klotho family protein. Mech Dev 98: 115–119.
[19]
Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, et al. (1997) Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 390: 45–51.
[20]
Matsumura Y, Aizawa H, Shiraki-Iida T, Nagai R, Kuro-o M, et al. (1998) Identification of the human klotho gene and its two transcripts encoding membrane and secreted klotho protein. Biochem Biophys Res Commun 242: 626–630.
[21]
Lin BC, Wang M, Blackmore C, Desnoyers LR (2007) Liver-specific activities of FGF19 require Klotho beta. J Biol Chem 282: 27277–27284.
[22]
Shiraki-Iida T, Aizawa H, Matsumura Y, Sekine S, Iida A, et al. (1998) Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein. FEBS Lett 424: 6–10.
[23]
Suzuki M, Uehara Y, Motomura-Matsuzaka K, Oki J, Koyama Y, 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.
[24]
Inagaki T, Choi M, Moschetta A, Peng L, Cummins CL, et al. (2005) Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis. Cell Metab 2: 217–225.
[25]
Kharitonenkov A, Shanafelt AB (2008) Fibroblast growth factor-21 as a therapeutic agent for metabolic diseases. BioDrugs 22: 37–44.
[26]
Moore DD (2007) Physiology. Sister act. Science 316: 1436–1438.
[27]
Berglund ED, Li CY, Bina HA, Lynes SE, Michael MD, et al. (2009) Fibroblast growth factor 21 controls glycemia via regulation of hepatic glucose flux and insulin sensitivity. Endocrinology 150: 4084–4093.
[28]
Kharitonenkov A, Shiyanova TL, Koester A, Ford AM, Micanovic R, et al. (2005) FGF-21 as a novel metabolic regulator. J Clin Invest 115: 1627–1635.
[29]
Kharitonenkov A, Wroblewski VJ, Koester A, Chen YF, Clutinger CK, et al. (2007) The metabolic state of diabetic monkeys is regulated by fibroblast growth factor-21. Endocrinology 148: 774–781.
[30]
Xu J, Lloyd DJ, Hale C, Stanislaus S, Chen M, et al. (2009) Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice. Diabetes 58: 250–259.
[31]
Badman MK, Pissios P, Kennedy AR, Koukos G, Flier JS, et al. (2007) Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states. Cell Metab 5: 426–437.
[32]
Hondares E, Rosell M, Gonzalez FJ, Giralt M, Iglesias R, et al. (2010) Hepatic FGF21 expression is induced at birth via PPARalpha in response to milk intake and contributes to thermogenic activation of neonatal brown fat. Cell Metab 11: 206–212.
[33]
Inagaki T, Dutchak P, Zhao G, Ding X, Gautron L, et al. (2007) Endocrine regulation of the fasting response by PPARalpha-mediated induction of fibroblast growth factor 21. Cell Metab 5: 415–425.
[34]
Lundasen T, Hunt MC, Nilsson LM, Sanyal S, Angelin B, et al. (2007) PPARalpha is a key regulator of hepatic FGF21. Biochem Biophys Res Commun 360: 437–440.
[35]
Mai K, Andres J, Biedasek K, Weicht J, Bobbert T, et al. (2009) Free fatty acids link metabolism and regulation of the insulin-sensitizing fibroblast growth factor-21. Diabetes 58: 1532–1538.
[36]
Moyers JS, Shiyanova TL, Mehrbod F, Dunbar JD, Noblitt TW, et al. (2007) Molecular determinants of FGF-21 activity-synergy and cross-talk with PPARgamma signaling. J Cell Physiol 210: 1–6.
[37]
Muise ES, Azzolina B, Kuo DW, El-Sherbeini M, Tan Y, et al. (2008) Adipose fibroblast growth factor 21 is up-regulated by peroxisome proliferator-activated receptor gamma and altered metabolic states. Mol Pharmacol 74: 403–412.
[38]
Wang H, Qiang L, Farmer SR (2008) Identification of a domain within peroxisome proliferator-activated receptor gamma regulating expression of a group of genes containing fibroblast growth factor 21 that are selectively repressed by SIRT1 in adipocytes. Mol Cell Biol 28: 188–200.
[39]
Hojman P, Pedersen M, Nielsen AR, Krogh-Madsen R, Yfanti C, et al. (2009) Fibroblast growth factor-21 is induced in human skeletal muscles by hyperinsulinemia. Diabetes 58: 2797–2801.
[40]
Izumiya Y, Bina HA, Ouchi N, Akasaki Y, Kharitonenkov A, et al. (2008) FGF21 is an Akt-regulated myokine. FEBS Lett 582: 3805–3810.
[41]
Hondares E, Iglesias R, Giralt A, Gonzalez FJ, Giralt M, et al. (2011) Thermogenic activation induces FGF21 expression and release in brown adipose tissue. J Biol Chem 286: 12983–12990.
[42]
Nishimura T, Nakatake Y, Konishi M, Itoh N (2000) Identification of a novel FGF, FGF-21, preferentially expressed in the liver. Biochim Biophys Acta 1492: 203–206.
[43]
Arner P, Pettersson A, Mitchell PJ, Dunbar JD, Kharitonenkov A, et al. (2008) FGF21 attenuates lipolysis in human adipocytes - a possible link to improved insulin sensitivity. FEBS Lett 582: 1725–1730.
[44]
Hotta Y, Nakamura H, Konishi M, Murata Y, Takagi H, et al. (2009) Fibroblast growth factor 21 regulates lipolysis in white adipose tissue but is not required for ketogenesis and triglyceride clearance in liver. Endocrinology 150: 4625–4633.
[45]
Li X, Ge H, Weiszmann J, Hecht R, Li YS, et al. (2009) Inhibition of lipolysis may contribute to the acute regulation of plasma FFA and glucose by FGF21 in ob/ob mice. FEBS Lett 583: 3230–3234.
[46]
Badman MK, Koester A, Flier JS, Kharitonenkov A, Maratos-Flier E (2009) Fibroblast growth factor 21-deficient mice demonstrate impaired adaptation to ketosis. Endocrinology 150: 4931–4940.
[47]
Potthoff MJ, Inagaki T, Satapati S, Ding X, He T, et al. (2009) FGF21 induces PGC-1alpha and regulates carbohydrate and fatty acid metabolism during the adaptive starvation response. Proc Natl Acad Sci U S A 106: 10853–10858.
[48]
Galman C, Lundasen T, Kharitonenkov A, Bina HA, Eriksson M, et al. (2008) The circulating metabolic regulator FGF21 is induced by prolonged fasting and PPARalpha activation in man. Cell Metab 8: 169–174.
[49]
Wente W, Efanov AM, Brenner M, Kharitonenkov A, Koster A, 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.
[50]
Mashili FL, Austin RL, Deshmukh AS, Fritz T, Caidahl K, et al. (2011) Direct effects of FGF21 on glucose uptake in human skeletal muscle: implications for type 2 diabetes and obesity. Diabetes Metab Res Rev 27: 286–297.
[51]
Sarruf DA, Thaler JP, Morton GJ, German J, Fischer JD, et al. (2010) Fibroblast growth factor 21 action in the brain increases energy expenditure and insulin sensitivity in obese rats. Diabetes 59: 1817–1824.
[52]
Fon Tacer K, Bookout AL, Ding X, Kurosu H, John GB, et al. (2010) Research resource: Comprehensive expression atlas of the fibroblast growth factor system in adult mouse. Mol Endocrinol 24: 2050–2064.
[53]
Micanovic R, Raches DW, Dunbar JD, Driver DA, Bina HA, et al. (2009) Different roles of N- and C- termini in the functional activity of FGF21. J Cell Physiol 219: 227–234.
[54]
Yie J, Hecht R, Patel J, Stevens J, Wang W, et al. (2009) FGF21 N- and C-termini play different roles in receptor interaction and activation. FEBS Lett 583: 19–24.
[55]
Kan M, Wu X, Wang F, McKeehan WL (1999) Specificity for fibroblast growth factors determined by heparan sulfate in a binary complex with the receptor kinase. J Biol Chem 274: 15947–15952.
[56]
Wu X, Ge H, Gupte J, Weiszmann J, Shimamoto G, et al. (2007) Co-receptor requirements for fibroblast growth factor-19 signaling. J Biol Chem 282: 29069–29072.
[57]
Wu X, Lemon B, Li X, Gupte J, Weiszmann J, et al. (2008) C-terminal tail of FGF19 determines its specificity toward Klotho co-receptors. J Biol Chem 283: 33304–33309.
[58]
Bhatnagar S, Damron HA, Hillgartner FB (2009) Fibroblast growth factor-19, a novel factor that inhibits hepatic fatty acid synthesis. J Biol Chem 284: 10023–10033.
[59]
Tomlinson E, Fu L, John L, Hultgren B, Huang X, et al. (2002) Transgenic mice expressing human fibroblast growth factor-19 display increased metabolic rate and decreased adiposity. Endocrinology 143: 1741–1747.
[60]
Ito S, Fujimori T, Furuya A, Satoh J, Nabeshima Y (2005) Impaired negative feedback suppression of bile acid synthesis in mice lacking betaKlotho. J Clin Invest 115: 2202–2208.
[61]
Razzaque MS, Sitara D, Taguchi T, St-Arnaud R, Lanske B (2006) Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process. Faseb J 20: 720–722.
[62]
Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, et al. (2006) Regulation of fibroblast growth factor-23 signaling by klotho. J Biol Chem 281: 6120–6123.
[63]
Gupte J, Yang L, Wu X, Weiszmann J, Hecht R, et al. The FGFR D3 domain determines receptor selectivity for fibroblast growth factor 21. J Mol Biol 408: 491–502.
[64]
Itoh N, Ornitz DM (2008) Functional evolutionary history of the mouse Fgf gene family. Dev Dyn 237: 18–27.
[65]
Kharitonenkov A (2009) FGFs and metabolism. Curr Opin Pharmacol 9: 805–810.
[66]
Huang X, Yu C, Jin C, Kobayashi M, Bowles CA, et al. (2006) Ectopic activity of fibroblast growth factor receptor 1 in hepatocytes accelerates hepatocarcinogenesis by driving proliferation and vascular endothelial growth factor-induced angiogenesis. Cancer Res 66: 1481–1490.
[67]
Fu L, John LM, Adams SH, Yu XX, Tomlinson E, et al. (2004) Fibroblast growth factor 19 increases metabolic rate and reverses dietary and leptin-deficient diabetes. Endocrinology 145: 2594–2603.
[68]
Fisher FM, Chui PC, Antonellis PJ, Bina HA, Kharitonenkov A, et al. (2010) Obesity is a fibroblast growth factor 21 (FGF21)-resistant state. Diabetes 59: 2781–2789.
[69]
Zhang X, Yeung DC, Karpisek M, Stejskal D, Zhou ZG, et al. (2008) Serum FGF21 levels are increased in obesity and are independently associated with the metabolic syndrome in humans. Diabetes 57: 1246–1253.
[70]
Huang X, Yang C, Jin C, Luo Y, Wang F, et al. (2008) Resident hepatocyte fibroblast growth factor receptor 4 limits hepatocarcinogenesis. Mol Carcinog 48: 553–562.
[71]
He W, Barak Y, Hevener A, Olson P, Liao D, et al. (2003) Adipose-specific peroxisome proliferator-activated receptor gamma knockout causes insulin resistance in fat and liver but not in muscle. Proc Natl Acad Sci U S A 100: 15712–15717.
[72]
Trokovic R, Trokovic N, Hernesniemi S, Pirvola U, Vogt Weisenhorn DM, et al. (2003) FGFR1 is independently required in both developing mid- and hindbrain for sustained response to isthmic signals. EMBO J 22: 1811–1823.
[73]
Kan M, Zhang GH, Zarnegar R, Michalopoulos G, Myoken Y, et al. (1991) Hepatocyte growth factor/hepatopoietin A stimulates the growth of rat kidney proximal tubule epithelial cells (RPTE), rat nonparenchymal liver cells, human melanoma cells, mouse keratinocytes and stimulates anchorage-independent growth of SV-40 transformed RPTE. Biochem Biophys Res Commun 174: 331–337.
[74]
Kan M, Shi EG, McKeehan WL (1991) Identification and assay of fibroblast growth factor receptors. Methods Enzymol 198: 158–171.
