Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for reducing excess hepatic glucose production in patients with type 2 diabetes. Despite extensive efforts, there is currently no human therapeutic that directly inhibits the glucagon/glucagon receptor pathway. We undertook a novel approach by generating high-affinity human monoclonal antibodies (mAbs) to the human glucagon receptor (GCGR) that display potent antagonistic activity in vitro and in vivo. A single injection of a lead antibody, mAb B, at 3 mg/kg, normalized blood glucose levels in ob/ob mice for 8 days. In addition, a single injection of mAb B dose-dependently lowered fasting blood glucose levels without inducing hypoglycemia and improved glucose tolerance in normal C57BL/6 mice. In normal cynomolgus monkeys, a single injection improved glucose tolerance while increasing glucagon and active glucagon-like peptide-1 levels. Thus, the anti-GCGR mAb could represent an effective new therapeutic for the treatment of type 2 diabetes.
Several human diseases are characterized by defects in the synthesis and secretion of the apolipoprotein (apo) B-containing lipoproteins. Familial hypobetalipoproteinemia is caused by mutations in the apo-B gene and is characterized by abnormally low plasma concentrations of apo-B and low-density lipoprotein (LDL) cholesterol. Another apo-B deficiency syndrome, abetalipoproteinemia, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP). MTP is a microsomal protein that is thought to transfer lipids to the apo-B protein as it is translated, allowing it to attain the proper conformation for lipoprotein assembly. A third apo-B deficiency syndrome, Anderson's disease (or chylomicron retention disease), is characterized by the inability to secrete apo-B-containing chylomicrons from the intestine but an apparently normal capacity to secrete lipoproteins from the liver. To more fully understand these human apo-B deficiency syndromes, our laboratory has generated and characterized gene-targeted mouse models. This review summarizes what has been learned from these animal models.
Leptin has been shown to have a wide repertoire of peripheral effects, some of which are mediated through the central nervous system and others that are induced through a direct action on target tissues. There is now evidence showing that leptin exerts some of its metabolic effects acting directly on peripheral tissues. The role of leptin has expanded from a narrow position in obesity to effects on biological processes, such as diabetes, appetite, thermogenesis, the immune system and reproduction. Here in a first part, we review preclinical evidence for direct effects on specific tissues (neurons, liver and muscle) and metabolic pathways. In a second part we review clinical evidence for leptin effects. In particular we review the effects of recombinant human leptin in lean, obese, diabetic subjects and in patients with congenital leptin deficiency or lipoatrophic diabetes. Additionally, while clinic leptin has not shown dramatic effects in obese / diabetic subjects with measurable serum leptin, in states of leptin deficiency treatment with leptin has been shown to have profound effects on body weight and appetite and insulin resistance.
Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic β-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.
The genes for apolipoprotein B and microsomal triglyceride transfer protein are expressed in mouse and human heart tissue. Why the heart would express these "lipoprotein assembly" genes has been unclear. Here we demonstrate that the beating mouse heart actually secretes spherical lipoproteins. Moreover, increased cardiac production of lipoproteins (e.g., in mice that express a human apolipoprotein B transgene) was associated with increased triglyceride secretion from the heart and decreased stores of triglycerides within the heart. Increased cardiac production of lipoproteins also reduced the pathological accumulation of triglycerides that occurs in the hearts of mice lacking long-chain acyl coenzyme A dehydrogenase. In contrast, blocking heart lipoprotein secretion (e.g., in heart-specific microsomal triglyceride transfer protein knockout mice) increased cardiac triglyceride stores. Thus, heart lipoprotein secretion helps regulate cardiac triglyceride stores and may protect the heart from the detrimental effects of surplus lipids.
Circulating levels of fibroblast growth factor 21 (FGF21), a metabolic regulator of glucose, lipid, and energy homeostasis, are elevated in obese diabetic subjects, raising questions about potential FGF21 resistance. Here we report tissue expression changes in FGF21 and its receptor components, and we describe the target-organ and whole-body responses to FGF21 in ob/ob and diet-induced obese (DIO) mice. Plasma FGF21 concentrations were elevated 8- and 16-fold in DIO and ob/ob mice, respectively, paralleling a dramatic increase in hepatic FGF21 mRNA expression. Concurrently, expression levels of βKlotho, FGF receptor (FGFR)-1c, and FGFR2c were markedly down-regulated in the white adipose tissues (WAT) of ob/ob and DIO mice. However, dose-response curves of recombinant human FGF21 (rhFGF21) stimulation of ERK phosphorylation in the liver and WAT were not right shifted in disease models, although the magnitude of induction in ERK phosphorylation was partially attenuated in DIO mice. Whole-body metabolic responses were preserved in ob/ob and DIO mice, with disease models being more sensitive and responsive than lean mice to the glucose-lowering and weight-loss effects of rhFGF21. Endogenous FGF21 levels, although elevated in diseased mice, were below the half-maximal effective concentrations of rhFGF21, suggesting a state of relative deficiency. Hepatic and WAT FGF21 mRNA expression levels declined after rhFGF21 treatment in the absence of the increased expression levels of βKlotho and FGFR. We conclude that overt FGF21 resistance was not evident in the disease models, and increased hepatic FGF21 expression as a result of local metabolic changes is likely a major cause of elevated circulating FGF21 levels.
11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is the enzyme that converts cortisone to cortisol. A growing body of evidence suggests that selective inhibition of 11β-HSD1 could potentially treat metabolic syndrome as well as type 2 diabetes. Through modification of our initial lead 1, we have discovered trifluoromethyl thiazolone 17. This compound had a Ki of 22 nM, possessed low in vivo clearance, and showed a 91% inhibition of adipose 11β-HSD1 enzymatic activity in a mouse ex vivo pharmacodynamic model.
FGF19 is a unique member of the fibroblast growth factor (FGF) family of secreted proteins that regulates bile acid homeostasis and metabolic state in an endocrine fashion. Here we investigate the cell surface receptors required for signaling by FGF19. We show that βKlotho, a single-pass transmembrane protein highly expressed in liver and fat, induced ERK1/2 phosphorylation in response to FGF19 treatment and significantly increased the interactions between FGF19 and FGFR4. Interestingly, our results show that αKlotho, another Klotho family protein related to βKlotho, also induced ERK1/2 phosphorylation in response to FGF19 treatment and increased FGF19-FGFR4 interactions in vitro, similar to the effects of βKlotho. In addition, heparin further enhanced the effects of both αKlotho and βKlotho in FGF19 signaling and interaction experiments. These results suggest that a functional FGF19 receptor may consist of FGF receptor (FGFR) and heparan sulfate complexed with either αKlotho or βKlotho. FGF19 is a unique member of the fibroblast growth factor (FGF) family of secreted proteins that regulates bile acid homeostasis and metabolic state in an endocrine fashion. Here we investigate the cell surface receptors required for signaling by FGF19. We show that βKlotho, a single-pass transmembrane protein highly expressed in liver and fat, induced ERK1/2 phosphorylation in response to FGF19 treatment and significantly increased the interactions between FGF19 and FGFR4. Interestingly, our results show that αKlotho, another Klotho family protein related to βKlotho, also induced ERK1/2 phosphorylation in response to FGF19 treatment and increased FGF19-FGFR4 interactions in vitro, similar to the effects of βKlotho. In addition, heparin further enhanced the effects of both αKlotho and βKlotho in FGF19 signaling and interaction experiments. These results suggest that a functional FGF19 receptor may consist of FGF receptor (FGFR) and heparan sulfate complexed with either αKlotho or βKlotho. The fibroblast growth factors (FGFs) 2The abbreviations used are:FGFfibroblast growth factorsFGFRFGF receptorERKextracellular signal-regulated kinaseGFPgreen fluorescent protein. constitute a structurally related family of 22 proteins (1Zhang X. Ibrahimi O.A. Olsen S.K. Umemori H. Mohammadi M. Ornitz D.M. J. Biol. Chem. 2006; 281: 15694-15700Abstract Full Text Full Text PDF PubMed Scopus (883) Google Scholar). This family of secreted proteins has been implicated in a variety of functions including angiogenesis, mitogenesis, vertebrate and invertebrate development, cellular differentiation, wound healing/repair, and metabolic regulations (2Eswarakumar V.P. Lax I. Schlessinger J. Cytokine Growth Factor Rev. 2005; 16: 139-149Crossref PubMed Scopus (1465) Google Scholar, 3Powers C.J. McLeskey S.W. Wellstein A. Endocr.-Relat. Cancer. 2000; 7: 165-197Crossref PubMed Scopus (1111) Google Scholar). FGFs can be grouped into seven subfamilies based on their sequence similarities and functional properties (4Ornitz D.M. Itoh N. Genome Biol. 2001; 2 (REVIEWS3005.1-3005.12)Crossref PubMed Google Scholar). Four tyrosine kinase receptors have been identified for FGFs (FGFR1–4), each containing an extracellular ligand binding domain, a single transmembrane domain, and an intracellular tyrosine kinase domain (5Mohammadi M. Olsen S.K. Ibrahimi O.A. Cytokine Growth Factor Rev. 2005; 16: 107-137Crossref PubMed Scopus (550) Google Scholar). Alternative RNA splicing of one of two unique exons in FGFR1–3 results in the two different, b and c, receptor isoforms (2Eswarakumar V.P. Lax I. Schlessinger J. Cytokine Growth Factor Rev. 2005; 16: 139-149Crossref PubMed Scopus (1465) Google Scholar). Because most FGFs only function in an autocrine or paracrine fashion, the tissue distribution of these FGFs determines the tissue specific functions for most of the FGF family members (2Eswarakumar V.P. Lax I. Schlessinger J. Cytokine Growth Factor Rev. 2005; 16: 139-149Crossref PubMed Scopus (1465) Google Scholar, 4Ornitz D.M. Itoh N. Genome Biol. 2001; 2 (REVIEWS3005.1-3005.12)Crossref PubMed Google Scholar).The FGF19 subfamily contains three members, FGF19, FGF21, and FGF23. In contrast to other FGFs, which require heparin or heparan sulfate for high affinity receptor binding and activation, FGF19 subfamily members do not bind heparin with high affinity (6Goetz R. Beenken A. Ibrahimi O.A. Kalinina J. Olsen S.K. Eliseenkova A.V. Xu C. Neubert T.A. Zhang F. Linhardt R.J. Yu X. White K.E. Inagaki T. Kliewer S.A. Yamamoto M. Kurosu H. Ogawa Y. Kuro-o M. Lanske B. Razzaque M.S. Mohammadi M. Mol. Cell. Biol. 2007; 27: 3417-3428Crossref PubMed Scopus (414) Google Scholar). In addition, FGF19 subfamily members contain intramolecular disulfide bonds, which may function to increase their stability in plasma and allow them to function as hormones (7Harmer N.J. Pellegrini L. Chirgadze D. Fernandez-Recio J. Blundell T.L. Biochemistry. 2004; 43: 629-640Crossref PubMed Scopus (103) Google Scholar). Indeed, although FGF19 is not expressed in liver or gallbladder, it can regulate hepatic bile acid metabolism and control gallbladder filling (8Holt J.A. Luo G. Billin A.N. Bisi J. McNeill Y.Y. Kozarsky K.F. Donahee M. Wang D.Y. Mansfield T.A. Kliewer S.A. Goodwin B. Jones S.A. Genes Dev. 2003; 17: 1581-1591Crossref PubMed Scopus (523) Google Scholar, 9Inagaki T. Choi M. Moschetta A. Peng L. Cummins C.L. McDonald J.G. Luo G. Jones S.A. Goodwin B. Richardson J.A. Gerard R.D. Repa J.J. Mangelsdorf D.J. Kliewer S.A. Cell Metab. 2005; 2: 217-225Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar, 10Houten S.M. Cell Metab. 2006; 4: 423-424Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 11Choi M. Moschetta A. Bookout A.L. Peng L. Umetani M. Holmstrom S.R. Suino-Powell K. Xu H.E. Richardson J.A. Gerard R.D. Mangelsdorf D.J. Kliewer S.A. Nat. Med. 2006; 12: 1253-1255Crossref PubMed Scopus (231) Google Scholar). Furthermore, transgenic animals overexpressing FGF19 from skeletal muscle and animals injected with recombinant protein display improved insulin sensitivity, reduced adiposity, and increased metabolic rate (12Fu L. John L.M. Adams S.H. Yu X.X. Tomlinson E. Renz M. Williams P.M. Soriano R. Corpuz R. Moffat B. Vandlen R. Simmons L. Foster J. Stephan J.P. Tsai S.P. Stewart T.A. Endocrinology. 2004; 145: 2594-2603Crossref PubMed Scopus (424) Google Scholar, 13Tomlinson E. Fu L. John L. Hultgren B. Huang X. Renz M. Stephan J.P. Tsai S.P. Powell-Braxton L. French D. Stewart T.A. Endocrinology. 2002; 143: 1741-1747Crossref PubMed Scopus (287) Google Scholar). In addition, FGF21 has been shown to regulate glucose and lipid metabolism in an endocrine fashion (14Kharitonenkov A. Shiyanova T.L. Koester A. Ford A.M. Micanovic R. Galbreath E.J. Sandusky G.E. Hammond L.J. Moyers J.S. Owens R.A. Gromada J. Brozinick J.T. Hawkins E.D. Wroblewski V.J. Li D.S. Mehrbod F. Jaskunas S.R. Shanafelt A.B. J. Clin. Investig. 2005; 115: 1627-1635Crossref PubMed Scopus (1584) Google Scholar), and FGF23 may function as a phosphaturic hormone (15Fukagawa M. Nii-Kono T. Kazama J.J. Curr. Opin. Nephrol. Hypertens. 2005; 14: 325-329Crossref PubMed Scopus (45) Google Scholar). These unique features of the FGF19 subfamily suggest that they may interact with their receptors differently from the canonical FGFs and may require different receptor complexes to function.The unique receptor requirements were first noted from studies with FGF23 (16Urakawa I. Yamazaki Y. Shimada T. Iijima K. Hasegawa H. Okawa K. Fujita T. Fukumoto S. Yamashita T. Nature. 2006; 444: 770-774Crossref PubMed Scopus (1421) Google Scholar, 17Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar). The αKlotho gene encodes a 130-kDa single-pass transmembrane protein with a short cytoplasmic domain. Since FGF23-deficient mice (Fgf23–/– mice) and αKlotho-deficient mice (αKloth–/– mice) share remarkable similarity in phenotypes, including shortened life span, growth retardation, infertility, muscle atrophy, hypoglycemia, and vascular calcification in the kidneys (18Shimada T. Kakitani M. Yamazaki Y. Hasegawa H. Takeuchi Y. Fujita T. Fukumoto S. Tomizuka K. Yamashita T. J. Clin. Investig. 2004; 113: 561-568Crossref PubMed Scopus (1246) Google Scholar, 19Yoshida T. Fujimori T. Nabeshima Y. Endocrinology. 2002; 143: 683-689Crossref PubMed Scopus (169) Google Scholar), it was hypothesized that FGF23 and αKlotho function through a common signal transduction pathway. Direct evidence for αKlotho as a co-receptor for FGF23 came from recent biochemical and cellular studies (16Urakawa I. Yamazaki Y. Shimada T. Iijima K. Hasegawa H. Okawa K. Fujita T. Fukumoto S. Yamashita T. Nature. 2006; 444: 770-774Crossref PubMed Scopus (1421) Google Scholar, 17Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar). It was shown that although FGF23 alone has poor affinity for FGFRs and did not promote efficient activation of FGFRs in cells, αKlotho was able to function as an essential cofactor for the activation of FGFR signaling by FGF23. This suggests that FGF23 binds an αKlotho-FGFR complex with higher affinity than to either receptor alone. Coexpression of αKlotho in HEK293 and Chinese hamster ovary cells significantly enhanced the ability of FGF23 to induce phosphorylation of FGF receptor substrate and ERK1/2 in these cells (16Urakawa I. Yamazaki Y. Shimada T. Iijima K. Hasegawa H. Okawa K. Fujita T. Fukumoto S. Yamashita T. Nature. 2006; 444: 770-774Crossref PubMed Scopus (1421) Google Scholar, 17Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar). Similar observations have recently been reported for FGF21. Instead of requiring αKlotho as a cofactor, FGF21 utilizes another Klotho family protein, βKlotho, as a coreceptor for signaling (20Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (465) Google Scholar). Therefore, the presence of βKlotho confers high affinity binding of FGF21 to FGFRs and allows cells coexpressing βKlotho and FGFRs to respond to FGF21 and activate signal transduction pathways.Although earlier observations suggest that heparin has low affinity interaction with FGF19 and may act as a weak cofactor for FGF19 (21Xie M.H. Holcomb I. Deuel B. Dowd P. Huang A. Vagts A. Foster J. Liang J. Brush J. Gu Q. Hillan K. Goddard A. Gurney A.L. Cytokine. 1999; 11: 729-735Crossref PubMed Scopus (229) Google Scholar), whether additional factors were required or could modulate FGF19-FGFR interactions and the effect of heparin on receptor complex formation have not been defined. Here, we show that both αKlotho and βKlotho may function as coreceptors for FGF19. The presence of αKlotho or βKlotho together with relatively high concentrations of heparin confers the strongest binding of FGF19 to FGFR and results in the highest level of ERK1/2 phosphorylation following receptor activation.EXPERIMENTAL PROCEDURESPlasmids and Proteins—Full-length human βKlotho, αKlotho, and extracellular domains of human βKlotho and αKlotho were cloned into the pTT14 expression vector. pFA2-Elk1 and pFR-Luc plasmids used in the ERK luciferase reporter assay were from Stratagene. Renilla luciferase, pRL-TK (Promega), was used as an internal control reporter. Recombinant FGF19 and FGFR-Fc fusion proteins were purchased from R&D.Cell Culture and Transfections—HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were transfected with the expression vector plasmids using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol.Conditioned Medium Preparation—HEK293 cells were transfected with expression vectors encoding the extracellular domains of βKlotho and αKlotho, and the medium was collected 3 days after transfection. Expression of soluble βKlotho and αKlotho was confirmed by Western blot using anti-βKlotho and anti-αKlotho antibodies (R&D). Medium from cells transfected with vector encoding green florescent protein (GFP) was also collected as a negative control.Pull-down Assay—To analyze the interaction between FGF19 and FGF receptors, recombinant FGF19 (0.5 μg) was mixed with FGFR1c-Fc, FGFR2c-Fc, FGFR3c-Fc, or FGFR4-Fc fusion proteins (0.5 μg) in conditioned medium and applied to 50 μl of protein G-Sepharose at 4 °C for 2 h. The beads were washed three times with phosphate-buffered saline and then suspended in SDS sample buffer and subjected to Western blot analysis with anti-FGF19 antibody (R&D).ERK Activation Reporter Assay—HEK293 cells were seeded in 96-well plates (105 cells/well). For each well, the transfection was done by mixing 10 ng of pFA2-Elk1, 150 ng of pFR-Luc, and 10 ng of pRL-TK reporter plasmids and 150 ng of βKlotho expression vector in 25 μl of OptiMEM (Invitrogen) with 0.5 μl of Lipofectamine 2000 in 25 μl of OptiMEM. On the following day, the cells were cultured in Dulbecco's modified Eagle's medium without serum but with 0.2% bovine serum albumin overnight. The cells were incubated with various concentrations of FGF19 for 5 h, and the luciferase activity was measured using DualGlo (Promega), according to the manufacturer's instructions.Western Blot Analysis of the FGF Signaling Pathway—HEK293 cells were transfected in 6-well plates and starved in serum-free medium overnight the day after transfection. For heparitinase treatment, 0.2 units/ml heparitinase (Sigma) was incubated with the cells for 3 h before FGF19 was added. After treatment with various concentrations of recombinant human FGF19 for 15 min, cells were snap-frozen in liquid nitrogen, and cell lysates were prepared in SDS sample buffer and subjected to Western blot analysis using anti-phospho-p44/42 mitogen-activated protein (MAP) kinase (anti-phospho-ERK1/2) antibody (Cell Signaling) and anti-ERK antibody (Cell Signaling).RESULTS AND DISCUSSIONSβKlotho and Heparin Enhance FGF19 Activation of FGFR Signaling—Since βKlotho is highly expressed in the liver and mice deficient in βKlotho exhibit a dramatic elevation of bile acid synthesis, we tested the possibility that βKlotho might function as a co-receptor for FGF19. We used a luciferase reporter assay system based on ERK1/2 phosphorylation (22Kawasaki H. Springett G.M. Toki S. Canales J.J. Harlan P. Blumenstiel J.P. Chen E.J. Bany I.A. Mochizuki N. Ashbacher A. Matsuda M. Housman D.E. Graybiel A.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13278-13283Crossref PubMed Scopus (309) Google Scholar) to test the activation of the FGF signaling pathway by FGF19 in HEK293 cells, which normally do not respond to FGF19 treatment. In HEK293 cells transfected with only control GFP vector, FGF19 did not activate any luciferase reporter expression (Fig. 1). To test whether βKlotho is required for FGF19 activation of FGF signaling, we transfected HEK293 cells with full-length human βKlotho and stimulated the cells with various concentrations of FGF19. As shown in Fig. 1, βKlotho-expressing cells responded to FGF19 in a dose-dependent manner with an EC50 value of 54 nm. Although FGF19 has been reported to have significantly lower heparin binding affinity (6Goetz R. Beenken A. Ibrahimi O.A. Kalinina J. Olsen S.K. Eliseenkova A.V. Xu C. Neubert T.A. Zhang F. Linhardt R.J. Yu X. White K.E. Inagaki T. Kliewer S.A. Yamamoto M. Kurosu H. Ogawa Y. Kuro-o M. Lanske B. Razzaque M.S. Mohammadi M. Mol. Cell. Biol. 2007; 27: 3417-3428Crossref PubMed Scopus (414) Google Scholar), adding heparin at a relatively high concentration of 20 μg/ml to the cells further enhanced the FGF19 response (Fig. 1). Heparin alone at this concentration had no effect on the reporter expression (Fig. 1). These results suggest that FGF19 may signal through the complex formed by βKlotho, heparin, and FGF receptors.βKlotho and Heparin Enhance the Binding of FGF19 to FGFR4—The mammalian FGFRs are encoded by four distinct genes (FGFR1 through FGFR4). A major alternative mRNA splicing event within one of the three immunoglobulin-like extracellular domains of FGFR1–3 generates the b and c isoforms. It has been shown that both βKlotho and αKlotho preferentially bind to the c isoforms of FGFR1–3 and FGFR4 (17Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar, 20Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (465) Google Scholar). FGF21 interacted with FGFR1c, FGFR2c, and FGFR4 complexed with βKlotho (20Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (465) Google Scholar), and FGF23 interacted with FGFR1c, FGFR3c, and FGFR4 complexed with αKlotho (17Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar). To investigate the effects of βKlotho and heparin on the binding of FGF19 to the various FGFRs, we used pull-down experiments to test whether FGF19 can be co-precipitated by Fc-FGFR fusion proteins complexed to protein G beads. In the presence of 20 μg/ml heparin, we demonstrated the binding of FGF19 to FGFR4 but did not detect significant binding to FGFR1c, FGFR2c, and FGFR3c (Fig. 2A, left panel). When conditioned medium containing the extracellular domain of βKlotho was added to the immunoprecipitation reaction, the FGF19-FGFR4 interaction was increased, but still no significant interactions could be observed between FGF19 and the other FGFRs (Fig. 2A, right panel). These results are consistent with the previous findings that FGF19 specifically interacts with FGFR4 (21Xie M.H. Holcomb I. Deuel B. Dowd P. Huang A. Vagts A. Foster J. Liang J. Brush J. Gu Q. Hillan K. Goddard A. Gurney A.L. Cytokine. 1999; 11: 729-735Crossref PubMed Scopus (229) Google Scholar); however, they do not necessarily rule out interactions between FGF19 and other receptors under different conditions. To further dissect the individual roles played by βKlotho and heparin on the interactions between FGF19 and FGFR4, we tested the additional combination of heparin and βKlotho. In the absence of βKlotho and heparin, no significant interactions were observed between FGF19 and FGFR4 (Fig. 2B, left panel). The addition of either soluble βKlotho conditioned media or heparin alone increased the interaction between FGF19 and FGFR4 (Fig. 2B, middle two panels). The strongest signal was detected when both βKlotho and heparin were present in the immunoprecipitation solution (Fig. 2B, right panel). These results suggest that although either βKlotho or heparin alone could potentiate interactions between FGF19 and FGFR4, together they show an even greater effect and perhaps may work synergistically with FGFR4 to provide the high affinity receptor complex for FGF19.FIGURE 2FGF19 interaction with FGFRs requires both βKlotho and heparin. A, FGF19 binds to FGFR4. Interactions between FGF19 and Fc-FGF fusion receptors were analyzed by a pull-down assay. In the presence of 20 μg/ml heparin and soluble βKlotho, FGF19 binds to FGFR4. The left panel and the right panel are on the same gel with the same exposure. 1c, FGFR1c; 2c, FGFR2c; 3c, FGFR3c; 4, FGFR4. B, FGF19 requires either heparin or βKlotho to bind to FGFR4. βKlotho is needed for the binding between FGF19 and FGFR4 in the absence of heparin. Heparin alone can also enhance the interaction between FGF19 and FGFR4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)αKlotho May Also Potentiate FGF19 Signaling—The Klotho component requirements for FGF21 and FGF23 co-receptor complexes appear very specific; FGF21 only works through βKlotho, and FGF23 only works through αKlotho (16Urakawa I. Yamazaki Y. Shimada T. Iijima K. Hasegawa H. Okawa K. Fujita T. Fukumoto S. Yamashita T. Nature. 2006; 444: 770-774Crossref PubMed Scopus (1421) Google Scholar, 17Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar, 20Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (465) Google Scholar). We wanted to test the specificity of FGF19 toward the Klotho proteins. An experiment similar to that performed for βKlotho (Fig. 2B) was also established for αKlotho. In the absence of αKlotho and heparin, similar to the results seen in Fig. 2B, no significant interaction was observed between FGF19 and FGFR4 (Fig. 3, left panel). The addition of either soluble αKlotho conditioned medium or heparin alone increased the interactions between FGF19 and FGFR4 (Fig. 3, middle two panels). Again, similar to what was observed for βKlotho, the strongest signal was detected when both αKlotho and heparin were present in the immunoprecipitation solution (Fig. 3, right panel). These results suggest that, in contrast to FGF21 and FGF23, which are specific for one form of Klotho protein, FGF19 is able to interact with FGFR complexes that are formed with either αKlotho or βKlotho.FIGURE 3αKlotho enhances the binding of FGF19 to FGFR4. FGFR4 can interact with FGF19 in the pull-down assay in the presence of either αKlotho or heparin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further investigate the role αKlotho plays in FGF19 signaling, we used Western blotting to measure ERK1/2 phosphorylation in HEK293 cells transfected with αKlotho or βKlotho treated with recombinant FGF19. As shown in Fig. 4, unlike FGF21 and FGF23, which only respond to one of the Klotho proteins (16Urakawa I. Yamazaki Y. Shimada T. Iijima K. Hasegawa H. Okawa K. Fujita T. Fukumoto S. Yamashita T. Nature. 2006; 444: 770-774Crossref PubMed Scopus (1421) Google Scholar, 17Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar, 20Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (465) Google Scholar), FGF19 is able to activate ERK1/2 phosphorylation in cells transfected with either βKlotho or αKlotho (Fig. 4). Heparin also seemed to stimulate ERK1/2 phosphorylation in the presence of αKlotho as it did in the presence of βKlotho (Figs. 2B and 4). FGF19 was not able to activate ERK1/2 phosphorylation in HEK293 cells without Klotho proteins (Fig. 1 and data not shown). Therefore, it appears that at least in vitro, FGFRs/heparin complexed with either βKlotho or αKlotho may serve as receptor complexes for FGF19.FIGURE 4FGF19 actives ERK1/2 phosphorylation in HEK293 cells transfected withαKlotho orβKlotho. HEK293 cells were transfected with expression vectors for αKlotho or βKlotho. After being starved with serum-free medium overnight, the cells were stimulated with vehicle or 50 nm recombinant FGF19 for 15 min and snap-frozen in liquid nitrogen. Heparin (20 μg/ml) was added just before the FGF19 treatment. Cell lysates were processed for Western blot with antibodies against phosphorylated ERK1/2 (pERK1/2) or total ERK1/2 (ERK1/2).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Both heparin and heparan sulfate are known to stimulate FGF signal transduction by increasing the affinity between the FGFs and FGFRs (23Pellegrini L. Curr. Opin. Struct. Biol. 2001; 11: 629-634Crossref PubMed Scopus (236) Google Scholar); however, only heparan sulfate is present on cell surfaces. In our pull-down assay, we also found that heparin enhanced the binding of FGF19 and FGFR4 independent of αKlotho or βKlotho. However, in the ERK1/2 phosphorylation assay using HEK293 cells, FGF19 could not activate FGF signaling with heparin alone (Fig. 1). One potential explanation is that although either Klotho or heparin can stabilize the FGF19-FGFR complex, Klotho is fundamentally required for the activation of FGFR by FGF19, perhaps by inducing a specific conformation change in the receptor, which activates downstream signaling pathways. The other possibility is that the presence of endogenous heparan sulfate on the cell surface masked the effects of exogenously added heparin at the concentrations tested in the absence of Klotho proteins. To assess the possible contribution of endogenous heparan sulfate to the FGF19 signaling and provide evidence that the observed effects of heparin are physiologically relevant, we tested the effects of heparitinase treatment on FGF19-induced ERK1/2 phosphorylation in βKlotho-transfected HEK293 cells. As shown in Fig. 5, heparitinase treatment significantly suppressed the FGF19 signaling in the absence of added heparin as observed by the reduced ERK1/2 phosphorylation levels. This result suggests that endogenous heparan sulfate could contribute to FGF19 signaling and that FGFRs/heparan sulfate complexed with Klotho may serve as receptor complexes for FGF19.FIGURE 5Effects of heparitinase treatment on FGF19 activation in HEK293 cells. HEK293 cells were transfected with expression vectors for βKlotho. After being serum-starved overnight, cells were treated with 0.2 units/ml heparitinase for 3 h before being stimulated with 100 nm recombinant FGF19 for 15 min and snap-frozen in liquid nitrogen. Cell lysates were processed for Western blot with antibodies against phosphorylated ERK1/2 (pERK1/2) or total ERK1/2 (ERK1/2).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mice deficient in βKlotho share similar phenotypes with mice lacking either FGF15 (murine ortholog of human FGF19) or FGFR4 (9Inagaki T. Choi M. Moschetta A. Peng L. Cummins C.L. McDonald J.G. Luo G. Jones S.A. Goodwin B. Richardson J.A. Gerard R.D. Repa J.J. Mangelsdorf D.J. Kliewer S.A. Cell Metab. 2005; 2: 217-225Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar, 24Ito S. Fujimori T. Furuya A. Satoh J. Nabeshima Y. Nabeshima Y. J. Clin. Investig. 2005; 115: 2202-2208Crossref PubMed Scopus (194) Google Scholar). In the present study, we show that βKlotho enables FGF19 to bind to FGFR4 and activate ERK phosphorylation. Our results strongly suggest that βKlotho acts as a cofactor that converts a canonical FGFR into a specific receptor for FGF19. The expression pattern of βKlotho may therefore restrict the action of FGF19 and thus confer the tissue-specific bioactivity on FGF19. This would be consistent with studies on the other two family members, FGF21 and FGF23 (16Urakawa I. Yamazaki Y. Shimada T. Iijima K. Hasegawa H. Okawa K. Fujita T. Fukumoto S. Yamashita T. Nature. 2006; 444: 770-774Crossref PubMed Scopus (1421) Google Scholar, 17Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar, 20Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (465) Google Scholar). We also discovered that under our in vitro assay conditions, FGF19 has specificity toward αKlotho as well as βKlotho, although no apparent phenotypic overlapping between FGF19 and αKlotho has been reported. Such dual specificity was not found for FGF21 and FGF23 (16Urakawa I. Yamazaki Y. Shimada T. Iijima K. Hasegawa H. Okawa K. Fujita T. Fukumoto S. Yamashita T. Nature. 2006; 444: 770-774Crossref PubMed Scopus (1421) Google Scholar, 17Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar, 20Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (465) Google Scholar). The physiological relevance of FGF19 and αKlotho interactions will be a subject for future studies. The fibroblast growth factors (FGFs) 2The abbreviations used are:FGFfibroblast growth factorsFGFRFGF receptorERKextracellular signal-regulated kinaseGFPgreen fluor
Fibroblast growth factor (FGF) 21 is a natural hormone that modulates glucose, lipid, and energy metabolism. Previously, we engineered an Fc fusion FGF21 variant with two mutations, Fc-FGF21(RG), to extend the half-life and reduce aggregation and in vivo degradation of FGF21. We now describe a new variant developed to reduce the extreme C-terminal degradation and improve the binding affinity to β-Klotho. We demonstrate, by introducing one additional mutation located at the C terminus of FGF21 (A180E), that the new molecule, Fc-FGF21(RGE), has gained many improved attributes. Compared with Fc-FGF21(RG), Fc-FGF21(RGE) has similar in vitro potency, preserves β-Klotho dependency, and maintains FGF receptor selectivity and cross-species reactivity. In vivo, Fc-FGF21(RGE) showed reduced susceptibility to extreme C-terminal degradation and increased plasma levels of the bioactive intact molecule. The circulating half-life of intact Fc-FGF21(RGE) increased twofold compared with that of Fc-FGF21(RG) in mice and cynomolgus monkeys. Additionally, Fc-FGF21(RGE) exhibited threefold to fivefold enhanced binding affinity to coreceptor β-Klotho across mouse, cynomolgus monkey, and human species. In obese and diabetic mouse and cynomolgus monkey models, Fc-FGF21(RGE) demonstrated greater efficacies to Fc-FGF21(RG), resulting in larger and more sustained improvements in multiple metabolic parameters. No increased immunogenicity was observed with Fc-FGF21(RGE). The superior biophysical, pharmacokinetic, and pharmacodynamic properties, as well as the positive metabolic effects across species, suggest that further clinical development of Fc-FGF21(RGE) as a metabolic therapy for diabetic and/or obese patients may be warranted.
C57BLKS/J (BLKS) mice are susceptible to islet exhaustion in insulin-resistant states as compared with C57BL6/J (B6) mice, as observed by the presence of the leptin receptor (Lepr) allele, Leprdb/db. Furthermore, DBA2/J (DBA) mice are also susceptible to β-cell failure and share 25% of their genome with BLKS; thus the DBA genome may contribute to β-cell dysfunction in BLKS mice. Here we show that BLKS mice exhibit elevated insulin secretion, as evidenced by improved glucose tolerance and increased islet insulin secretion compared with B6 mice, and describe interstrain transcriptional differences in glucose response. Transcriptional differences between BLKS and B6 mice were identified by expression profiling of isolated islets from both strains. Genomic mapping of gene expression differences demonstrated a significant association of expression differences with DBA loci in BLKS mice (P = 4×10-27). Two genes, Nicotinamide nucleotide transhydrogenase (Nnt) and Pleiomorphic adenoma gene like 1 (Plagl1), were 4 and 7.2-fold higher respectively in BLKS islets, and may be major contributors to increased insulin secretion by BLKS islets. Contrary to reports for B6 mice, BLKS mice do not harbor a mutant Nnt gene. We detected 16 synonymous polymorphisms and a two-amino acid deletion in the Plagl1 gene in BLKS mice. Several inflammatory glucose-responsive genes are expressed at a higher level in BLKS, suggesting an inflammatory component to BLKS islet dysfunction. This study describes physiological differences between BLKS and B6 mice, and provides evidence for a causative role of the DBA genome in β-cell dysfunction in BLKS mice.
