Jamila Gupte

Treating Diabetes and Obesity with an FGF21-Mimetic Antibody Activating the βKlotho/FGFR1c Receptor Complex

A monoclonal antibody mimic of FGF21 exerts beneficial metabolic effects in obese monkeys. A Metabolic Mimic Losing weight typically requires exercise and a healthy diet. Managing diabetes similarly relies on diet and exercise but also includes insulin therapy. Now, both diabetes and obesity could be treated together by targeting the fibroblast growth factor 21 (FGF21) pathway. Foltz and colleagues show that an antibody mimic of FGF21 works to regulate glucose and insulin homeostasis, leading to weight loss and glucose tolerance in monkeys. The authors first engineered the FGF21-mimetic monoclonal antibody, which they termed “mimAb1.” This antibody was able to activate human and monkey FGF receptor 1c (FGFR1c)/βKlotho signaling similar to its native counterpart, FGF21. In vivo in obese cynomolgus monkeys, mimAb1 treatment led to a decrease in body weight and body mass index (BMI)—a decrease that was maintained for 9 weeks after the second round of treatment. These beneficial effects on metabolism were seen only initially with FGF21, before animals regained weight. Animals treated with mimAb1 also showed a decrease in fasting and fed plasma insulin levels, suggesting an improvement in insulin sensitivity, as well as a reduction in plasma triglyceride and glucose levels. Native FGF21 is difficult to develop as a therapeutic for diabetes and obesity; efforts to date have fallen short. mimAb1 recreates all of the beneficial metabolic effects of FGF21 as measured but is easier to manufacture, has prolonged pharmacokinetics, and has been engineered with high specificity. This mimAb1 will need additional safety and toxicity testing for translation, but early efficacy data in nonhuman primates suggest that this antibody is on its way to helping treat patients with diet-induced obesity and diabetes. Fibroblast growth factor 21 (FGF21) is a distinctive member of the FGF family with potent beneficial effects on lipid, body weight, and glucose metabolism and has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to native FGF21, we have developed a monoclonal antibody, mimAb1, that binds to βKlotho with high affinity and specifically activates signaling from the βKlotho/FGFR1c (FGF receptor 1c) receptor complex. In obese cynomolgus monkeys, injection of mimAb1 led to FGF21-like metabolic effects, including decreases in body weight, plasma insulin, triglycerides, and glucose during tolerance testing. Mice with adipose-selective FGFR1 knockout were refractory to FGF21-induced improvements in glucose metabolism and body weight. These results in obese monkeys (with mimAb1) and in FGFR1 knockout mice (with FGF21) demonstrated the essential role of FGFR1c in FGF21 function and suggest fat as a critical target tissue for the cytokine and antibody. Because mimAb1 depends on βKlotho to activate FGFR1c, it is not expected to induce side effects caused by activating FGFR1c alone. The unexpected finding of an antibody that can activate FGF21-like signaling through cell surface receptors provided preclinical validation for an innovative therapeutic approach to diabetes and obesity.

Co-receptor Requirements for Fibroblast Growth Factor-19 Signaling

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-induced hepatocyte proliferation is mediated through FGFR4 activation.

FGF19 and FGF21, unique members of the fibroblast growth factor (FGF) family, are hormones that regulate glucose, lipid, and energy homeostasis. Increased hepatocyte proliferation and liver tumor formation have also been observed in FGF19 transgenic mice. Here, we report that, in contrast to FGF19, FGF21 does not induce hepatocyte proliferation in vivo. To identify the mechanism for FGF19-induced hepatocyte proliferation, we explored similarities and differences in receptor specificity between FGF19 and FGF21. We find that although both are able to activate FGF receptors (FGFRs) 1c, 2c, and 3c, only FGF19 activates FGFR4, the predominant receptor in the liver. Using a C-terminal truncation mutant of FGF19 and a series of FGF19/FGF21 chimeric molecules, we determined that amino acids residues 38–42 of FGF19 are sufficient to confer both FGFR4 activation and increased hepatocyte proliferation in vivo to FGF21. These data suggest that activation of FGFR4 is the mechanism whereby FGF19 can increase hepatocyte proliferation and induce hepatocellular carcinoma formation.

C-terminal Tail of FGF19 Determines Its Specificity toward Klotho Co-receptors

FGF19 subfamily proteins (FGF19, FGF21, and FGF23) are unique members of fibroblast growth factors (FGFs) that regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis in an endocrine fashion. Their activities require the presence of α or βKlotho, two related single-pass transmembrane proteins, as co-receptors in relevant target tissues. We previously showed that FGF19 can bind to both α and βKlotho, whereas FGF21 and FGF23 can bind only to either βKlotho or αKlotho, respectively in vitro. To determine the mechanism regulating the binding and specificity among FGF19 subfamily members to Klotho family proteins, chimeric proteins between FGF19 subfamily members or chimeric proteins between Klotho family members were constructed to probe the interaction between those two families. Our results showed that a chimera of FGF19 with the FGF21 C-terminal tail interacts only with βKlotho and a chimera with the FGF23 C-terminal tail interacts only with αKlotho. FGF signaling assays also reflected the change of specificity we observed for the chimeras. These results identified the C-terminal tail of FGF19 as a region necessary for its recognition of Klotho family proteins. In addition, chimeras between α and βKlotho were also generated to probe the regions in Klotho proteins that are important for signaling by this FGF subfamily. Both FGF23 and FGF21 require intact α or βKlotho for signaling, respectively, whereas FGF19 can signal through a Klotho chimera consisting of the N terminus of αKlotho and the C terminus of βKlotho. Our results provide the first glimpse of the regions that regulate the binding specificity between this unique family of FGFs and their co-receptors.

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

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

Selective activation of FGFR4 by an FGF19 variant does not improve glucose metabolism in ob/ob mice

FGF19 is a hormone that regulates bile acid and glucose homeostasis. Progress has been made in identifying cofactors for receptor activation. However, several functions of FGF19 have not yet been fully defined, including the actions of FGF19 on target tissues, its FGF receptor specificity, and the contributions of other cofactors, such as heparin. Here, we explore the requirements for FGF19-FGFR/co-receptor interactions and signaling in detail. We show that βKlotho was essential for FGF19 interaction with FGFRs 1c, 2c, and 3c, but FGF19 was able to interact directly with FGFR4 in the absence of βKlotho in a heparin-dependent manner. Further, FGF19 activated FGFR4 signaling in the presence or absence of βKlotho, but activation of FGFRs 1c, 2c, or 3c was completely βKlotho dependent. We then generated an FGF19 molecule, FGF19dCTD, which has a deletion of the C-terminal region responsible for βKlotho interaction. We determined that βKlotho-dependent FGFR1c, 2c, and 3c interactions and activation were abolished, and βKlotho-independent FGFR4 activation was preserved; therefore, FGF19dCTD is an FGFR4-specific activator. This unique FGF19 molecule specifically activated FGFR4-dependent signaling in liver and suppressed CYP7A1 expression in vivo, but was unable to activate signaling in adipose where FGFR4 expression is very low. Interestingly, unlike FGF19, treatment of ob/ob mice with FGF19dCTD failed to improve glucose levels and insulin sensitivity. These results suggest that FGF19-regulated liver bile acid metabolism could be independent of its glucose-lowering effect, and direct FGFR activation in adipose tissue may play an important role in the regulation of glucose homeostasis.

The G-Protein-Coupled Receptor GPR103 Regulates Bone Formation

ABSTRACT GPR103 is a G-protein-coupled receptor with reported expression in brain, heart, kidney, adrenal gland, retina, and testis. It encodes a 455-amino-acid protein homologous to neuropeptide FF2, neuropeptide Y2, and galanin GalR1 receptors. Its natural ligand was recently identified as 26RFa, a novel human RF-amide-related peptide with orexigenic activity. To identify the function of GPR103, we generated GPR103-deficient mice. Homozygous mutant mice were viable and fertile. Their body weight was undistinguishable from that of their wild-type littermates. Histological analysis revealed that GPR103−/− mice exhibited a thinned osteochondral growth plate, a thickening of trabecular branches, and a reduction in osteoclast number, suggestive of an early arrest of osteochondral bone formation. Microcomputed tomography confirmed the reduction in trabecular bone and connective tissue densities in GPR103 knockout animals. Whole-body radiography followed by morphometric analysis revealed a kyphosis in mutant animals. Reverse transcription-PCR analysis showed that GPR103 was expressed in human skull, mouse spine, and several osteoblast cell lines. Dexamethasone, a known inhibitor of osteoblast growth and inducer of osteoblast differentiation, inhibited GPR103 expression in human osteoblast primary cultures. Altogether, these results suggest that osteopenia in GPR103−/− mice may be mediated directly by the loss of GPR103 expression in bone.

Elucidation of signaling and functional activities of an orphan GPCR, GPR81

GPR81 is an orphan G protein-coupled receptor (GPCR) that has a high degree of homology to the nicotinic acid receptor GPR109A. GPR81 expression is highly enriched and specific in adipocytes. However, the function and signaling properties of GPR81 are unknown because of the lack of natural or synthetic ligands. Using chimeric G proteins that convert Gi-coupled receptors to Gq-mediated inositol phosphate (IP) accumulation, we show that GPR81 can constitutively increase IP accumulation in HEK293 cells and suggest that GPR81 couples to the Gi signaling pathway. We also constructed a chimeric receptor that expresses the extracellular domains of cysteinyl leukotriene 2 receptor (CysLT2R) and the intracellular domains of GPR81. We show that the CysLT2R ligand, leukotriene D4 (LTD4), is able to activate this chimeric receptor through activation of the Gi pathway. In addition, LTD4 is able to inhibit lipolysis in adipocytes expressing this chimeric receptor. These results suggest that GPR81 couples to the Gi signaling pathway and that activation of the receptor may regulate adipocyte function and metabolism. Hence, targeting GPR81 may lead to the development of a novel and effective therapy for dyslipidemia and a better side effect profile than nicotinic acid.

Signaling property study of adhesion G-protein-coupled receptors

Adhesion G‐protein‐coupled receptors (GPCR) are special members of GPCRs with long N‐termini containing multiple domains. We overexpressed our collection of receptors together with G‐proteins in mammalian cell lines and measured the concentrations of intracellular signaling molecules, such as inositol phosphate and cAMP. Our results show that a subset of tested adhesion GPCRs has constitutive activities and is capable of coupling to a variety of G‐proteins. In addition, we have identified a small molecule compound that specifically activates one of the subfamily members, GPR97, and the activation was confirmed by an independent GTPγS assay. These findings suggest classical GPCR screening assays could be applied to de‐orphanize these receptors, and provide pharmacological tools to improve understanding of the physiological functions of these receptors.

Characterization of a FGF19 variant with altered receptor specificity revealed a central role for FGFR1c in the regulation of glucose metabolism.

Diabetes and associated metabolic conditions have reached pandemic proportions worldwide, and there is a clear unmet medical need for new therapies that are both effective and safe. FGF19 and FGF21 are distinctive members of the FGF family that function as endocrine hormones. Both have potent effects on normalizing glucose, lipid, and energy homeostasis, and therefore, represent attractive potential next generation therapies for combating the growing epidemics of type 2 diabetes and obesity. The mechanism responsible for these impressive metabolic effects remains unknown. While both FGF19 and FGF21 can activate FGFRs 1c, 2c, and 3c in the presence of co-receptor βKlotho in vitro, which receptor is responsible for the metabolic activities observed in vivo remains unknown. Here we have generated a variant of FGF19, FGF19-7, that has altered receptor specificity with a strong bias toward FGFR1c. We show that FGF19-7 is equally efficacious as wild type FGF19 in regulating glucose, lipid, and energy metabolism in both diet-induced obesity and leptin-deficient mouse models. These results are the first direct demonstration of the central role of the βKlotho/FGFR1c receptor complex in glucose and lipid regulation, and also strongly suggest that activation of this receptor complex alone might be sufficient to achieve all the metabolic functions of endocrine FGF molecules.