In Vitro and in Vivo Analyses Reveal Profound Effects of Fibroblast Growth Factor 16 as a Metabolic Regulator.

The discovery of brown adipose tissue (BAT) as a key regulator of energy expenditure has sparked interest in identifying novel soluble factors capable of activating inducible BAT (iBAT) to combat obesity. Using a high content cell-based screen, we identified fibroblast growth factor 16 (FGF16) as a potent inducer of several physical and transcriptional characteristics analogous to those of both "classical" BAT and iBAT. Overexpression of Fgf16 in vivo recapitulated several of our in vitro findings, specifically the significant induction of the Ucp1 gene and UCP1 protein expression in inguinal white adipose tissue (iWAT), a common site for emergent active iBAT. Despite significant UCP1 up-regulation in iWAT and dramatic weight loss, the metabolic improvements observed due to Fgf16 overexpression in vivo were not the result of increased energy expenditure, as measured by indirect calorimetric assessment. Instead, a pattern of reduced food and water intake, combined with feces replete with lipid and bile acid, indicated a phenotype more akin to that of starvation and intestinal malabsorption. Gene expression analysis of the liver and ileum indicated alterations in several steps of bile acid metabolism, including hepatic synthesis and reabsorption. Histological analysis of intestinal tissue revealed profound abnormalities in support of this conclusion. The in vivo data, together with FGF receptor binding analysis, indicate that the in vivo outcome observed is the likely result of both direct and indirect mechanisms and probably involves multiple receptors. These results highlight the complexity of FGF signaling in the regulation of various metabolic processes.

Fibroblast growth factor receptor 4 (FGFR4) deficiency improves insulin resistance and glucose metabolism under diet-induced obesity conditions.

The role of fibroblast growth factor receptor 4 (FGFR4) in regulating bile acid synthesis has been well defined; however, its reported role on glucose and energy metabolism remains unresolved. Here, we show that FGFR4 deficiency in mice leads to improvement in glucose metabolism, insulin sensitivity, and reduction in body weight under high fat conditions. Mechanism of action studies in FGFR4-deficient mice suggest that the effects are mediated in part by increased plasma levels of adiponectin and the endocrine FGF factors FGF21 and FGF15, the latter of which increase in response to an elevated bile acid pool. Direct actions of increased bile acids on bile acid receptors, and other potential indirect mechanisms, may also contribute to the observed metabolic changes. The results described herein suggest that FGFR4 antagonists alone, or in combination with other agents, could serve as a novel treatment for diabetes.

Liver-specific mitochondrial amidoxime-reducing component 1 (Mtarc1) knockdown protects the liver from diet-induced MASH in multiple mouse models.

BACKGROUND: Human genetic studies have identified several mitochondrial amidoxime-reducing component 1 (MTARC1) variants as protective against metabolic dysfunction-associated steatotic liver disease. The MTARC1 variants are associated with decreased plasma lipids and liver enzymes and reduced liver-related mortality. However, the role of mARC1 in fatty liver disease is still unclear. METHODS: Given that mARC1 is mainly expressed in hepatocytes, we developed an N-acetylgalactosamine-conjugated mouse Mtarc1 siRNA, applying it in multiple in vivo models to investigate the role of mARC1 using multiomic techniques. RESULTS: In ob/ob mice, knockdown of Mtarc1 in mouse hepatocytes resulted in decreased serum liver enzymes, LDL-cholesterol, and liver triglycerides. Reduction of mARC1 also reduced liver weight, improved lipid profiles, and attenuated liver pathological changes in 2 diet-induced metabolic dysfunction-associated steatohepatitis mouse models. A comprehensive analysis of mARC1-deficient liver from a metabolic dysfunction-associated steatohepatitis mouse model by metabolomics, proteomics, and lipidomics showed that Mtarc1 knockdown partially restored metabolites and lipids altered by diet. CONCLUSIONS: Taken together, reducing mARC1 expression in hepatocytes protects against metabolic dysfunction-associated steatohepatitis in multiple murine models, suggesting a potential therapeutic approach for this chronic liver disease.

Dual actions of fibroblast growth factor 19 on lipid metabolism.

Elevated triglyceride (TG) and cholesterol levels are risk factors for cardiovascular disease and are often associated with diabetes and metabolic syndrome. Recent reports suggest that fibroblast growth factor (FGF)19 and FGF21 can dramatically improve metabolic dysfunction, including hyperglycemia, hypertriglyceridemia, and hypercholesterolemia. Due to their similar receptor specificities and co-receptor requirements, FGF19 and FGF21 share many common properties and have been thought to be interchangeable in metabolic regulation. Here we directly compared how pharmacological administration of recombinant FGF19 or FGF21 proteins affect metabolism in B6.V-Lep(ob)/J leptin-deficient mice. FGF19 and FGF21 equally improved glucose parameters; however, we observed increased serum TG and cholesterol levels after treatment with FGF19 but not with FGF21. Increases in serum TGs were also observed after a 4-day treatment with FGF19 in C57BL6/J mice on a high-fat diet. This is in contrast to many literature reports that showed significant improvements in hyperlipidemia after chronic treatment with FGF19 or FGF21 in high-fat diet models. We propose that FGF19 has lipid-raising and lipid-lowering actions mediated through different FGF receptors and target tissues, and the results described here provide a potential mechanism that may explain the inconsistency in the reported effects of FGF19 on lipid metabolism.

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 betaKlotho was essential for FGF19 interaction with FGFRs 1c, 2c, and 3c, but FGF19 was able to interact directly with FGFR4 in the absence of betaKlotho in a heparin-dependent manner. Further, FGF19 activated FGFR4 signaling in the presence or absence of betaKlotho, but activation of FGFRs 1c, 2c, or 3c was completely betaKlotho dependent. We then generated an FGF19 molecule, FGF19dCTD, which has a deletion of the C-terminal region responsible for betaKlotho interaction. We determined that betaKlotho-dependent FGFR1c, 2c, and 3c interactions and activation were abolished, and betaKlotho-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.

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.

The G-protein-coupled receptor GPR103 regulates bone formation.

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.

Chronic Over-expression of Fibroblast Growth Factor 21 Increases Bile Acid Biosynthesis by Opposing FGF15/19 Action.

Pharmacological doses of fibroblast growth factor (FGF) 21 effectively normalize glucose, lipid and energy homeostasis in multiple animal models with many benefits translating to obese humans with type 2 diabetes. However, a role for FGF21 in the regulation of bile acid metabolism has not been reported. Herein, we demonstrate AAV-mediated FGF21 overexpression in mice increases liver expression of the key bile acid producing enzyme, Cyp7a1, resulting in an increased bile acid pool. Furthermore, in cholecystectomized mice, FGF21-mediated bile acid pool increase led to increased transit of bile acids into colon. We elucidate that the mechanism of FGF21 induced bile acid changes is mainly through antagonizing FGF15/19 function on liver ßKlotho/FGFR4 receptor complex; thus inhibiting FGF15/19-mediated suppression of Cyp7a1 expression. In conclusion, these data reveal a previously unidentified role for FGF21 on bile acid metabolism and may be relevant to understand the effects of FGF21 analogs in clinical studies.

Soluble CLEC2 Extracellular Domain Improves Glucose and Lipid Homeostasis by Regulating Liver Kupffer Cell Polarization.

The polarization of tissue resident macrophages toward the alternatively activated, anti-inflammatory M2 phenotype is believed to positively impact obesity and insulin resistance. Here we show that the soluble form of the extracellular domain (ECD) of C-type lectin-like receptor 2, CLEC2, regulates Kupffer cell polarization in the liver and improves glucose and lipid parameters in diabetic animal models. Over-expression of Fc-CLEC2(ECD) in mice via in vivo gene delivery, or injection of recombinant Fc-CLEC2(ECD) protein, results in a reduction of blood glucose and liver triglyceride levels and improves glucose tolerance. Furthermore, Fc-CLEC2(ECD) treatment improves cytokine profiles and increases both the M2 macrophage population and the genes involved in the oxidation of lipid metabolism in the liver. These data reveal a previously unidentified role for CLEC2 as a regulator of macrophage polarity, and establish CLEC2 as a promising therapeutic target for treatment of diabetes and liver disease.

Trefoil Factor 3 (TFF3) Is Regulated by Food Intake, Improves Glucose Tolerance and Induces Mucinous Metaplasia.

Trefoil factor 3 (TFF3), also called intestinal trefoil factor or Itf, is a 59 amino acid peptide found as a homodimer predominantly along the gastrointestinal tract and in serum. TFF3 expression is elevated during gastrointestinal adenoma progression and has been shown to promote mucosal wound healing. Here we show that in contrast to other trefoil factor family members, TFF1 and TFF2, TFF3 is highly expressed in mouse duodenum, jejunum and ileum and that its expression is regulated by food intake. Overexpression of TFF3 using a recombinant adeno-associated virus (AAV) vector, or daily administration of recombinant TFF3 protein in vivo improved glucose tolerance in a diet-induced obesity mouse model. Body weight, fasting insulin, triglyceride, cholesterol and leptin levels were not affected by TFF3 treatment. Induction of mucinous metaplasia was observed in mice with AAV-mediated TFF3 overexpression, however, no such adverse histological effect was seen after the administration of recombinant TFF3 protein. Altogether these results suggest that the therapeutic potential of targeting TFF3 to treat T2D may be limited.

Advancing therapeutic discovery through phenotypic screening of the extracellular proteome using hydrodynamic intravascular injection.

OBJECTIVE: Although the human genome encodes ∼ 20,000 protein-coding genes, only a very small fraction of these have been explored as potential targets for therapeutic development. The challenge of identifying and validating new protein targets has contributed to the significant reduction in the productivity of the pharmaceutical industry in the recent decade, highlighting the continued need to find new therapeutic targets. RESEARCH DESIGN AND METHODS: The traditional methods to discover new targets are expensive, low throughput and time consuming, usually taking years to validate or invalidate a target. To address these limitations, as a proof of concept, we explored the hydrodynamic tail vein (HTV) injection as a gene delivery method for direct in vivo phenotypic screening of novel secreted factor targets for Type II diabetes therapeutics. RESULTS: High levels and sustained expression of target proteins were observed in diabetic mouse models tested, allowing us to identify multiple novel hormones that may regulate glucose metabolism. CONCLUSIONS: These results suggest that HTV is a low-cost, high-throughput method for direct in vivo phenotypic drug screening in metabolic disorders and could be applicable to many other disease areas as well. This method if combined with other approaches such as human genetic studies could provide a significant value to future drug discovery.

A novel approach to improve the function of FGF21.

BACKGROUND AND OBJECTIVE: Fibroblast growth factor 21 (FGF21) has potent effects on normalizing glucose, lipid, and energy homeostasis, and represents an attractive novel therapy for type 2 diabetes mellitus and obesity. Approaches to improve the pharmacokinetic properties of FGF21, such as conjugation with polyethylene glycol, have been explored for therapeutic development. However, not only is there room for further pharmacokinetic improvements, additional re-engineering approaches to improve the potency and stability of FGF21 have not been reported. Here, we describe a novel approach to modify and improve the function of FGF21 by altering its C-terminal ßKlotho interaction domain. METHODS: We first identified Avimer proteins that are capable of binding ßKlotho. Then we explored replacing the C-terminal ßKlotho interaction domain of FGF21 with a ßKlotho-binding Avimer protein. RESULTS: Such a ßKlotho-binding Avimer protein was able to fully complement the C-terminal domain function of FGF21. The resulting FGF21-Avimer fusion is functionally indistinguishable from wild type FGF21, and more tolerant of C-terminal modification. CONCLUSION: These results demonstrate a viable strategy to modulate the affinity, potency, and engineering of FGF21, paving the way for further improvements of FGF21 as a therapeutic.

FGF21 can be mimicked in vitro and in vivo by a novel anti-FGFR1c/ß-Klotho bispecific protein.

The endocrine hormone FGF21 has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to the native cytokine, we generated bispecific Avimer polypeptides that bind with high affinity and specificity to one of the receptor and coreceptor pairs used by FGF21, FGFR1c and ß-Klotho. These Avimers exhibit FGF21-like activity in in vitro assays with potency greater than FGF21. In a study conducted in obese male cynomolgus monkeys, animals treated with an FGFR1c/ß-Klotho bispecific Avimer showed improved metabolic parameters and reduced body weight comparable to the effects seen with FGF21. These results not only demonstrate the essential roles of FGFR1c and ß-Klotho in mediating the metabolic effects of FGF21, they also describe a first bispecific activator of this unique receptor complex and provide validation for a novel therapeutic approach to target this potentially important pathway for treating diabetes and obesity.

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.

A unique FGF23 with the ability to activate FGFR signaling through both αKlotho and ßKlotho.

Three fibroblast growth factor (FGF) molecules, FGF19, FGF21, and FGF23, form a unique subfamily that functions as endocrine hormones. FGF19 and FGF21 can regulate glucose, lipid, and energy metabolism, while FGF23 regulates phosphate homeostasis. The FGF receptors and co-receptors for these three FGF molecules have been identified, and domains important for receptor interaction and specificity determination are beginning to be elucidated. However, a number of questions remain unanswered, such as the identification of fibroblast growth factor receptor responsible for glucose regulation. Here, we have generated a variant of FGF23: FGF23-21c, where the C-terminal domain of FGF23 was replaced with the corresponding regions from FGF21. FGF23-21c showed a number of interesting and unexpected properties in vitro. In contrast to wild-type FGF23, FGF23-21c gained the ability to activate FGFR1c and FGFR2c in the presence of ßKlotho and was able to stimulate glucose uptake into adipocytes in vitro and lower glucose levels in ob/ob diabetic mice model to similar extent as FGF21 in vivo. These results suggest that ßKlotho/FGFR1c or FGFR2c receptor complexes are sufficient for glucose regulation. Interestingly, without the FGF23 C-terminal domain, FGF23-21c was still able to activate fibroblast growth factor receptors in the presence of αKlotho. This suggests not only that sequences outside of the C-terminal region may also contribute to the interaction with co-receptors but also that FGF23-21c may be able to regulate both glucose and phosphate metabolisms. This raises an interesting concept of designing an FGF molecule that may be able to address multiple diseases simultaneously. Further understanding of FGF/receptor interactions may allow the development of exciting opportunities for novel therapeutic discovery.

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 ßKlotho 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 ßKlotho/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.

Treating diabetes and obesity with an FGF21-mimetic antibody activating the ßKlotho/FGFR1c receptor complex.

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 ßKlotho with high affinity and specifically activates signaling from the ßKlotho/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 ßKlotho 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.

The FGFR D3 domain determines receptor selectivity for fibroblast growth factor 21.

FGF21 is a member of a unique subfamily of fibroblast growth factors that function as endocrine hormones and regulate a variety of metabolic activities. Unlike paracrine FGFs, FGF21 does not bind heparin and requires ßKlotho as a co-receptor to activate FGFR signaling. In the presence of ßKlotho, FGF21 is able to activate FGFRs 1c, 2c and 3c but not FGFR4. Chimeric FGFR1c/FGFR4 receptors were constructed to identify domains that confer this specificity and to understand regions important for FGF21-induced receptor activation. With these chimeras, we showed that domain 3 of the FGFR1c extracellular domain plays a critical role in specificity determination and receptor activation by FGF21. Furthermore, we were able to narrow down the sequences important for this function to a six amino acid region within domain 3 of FGFR1c. It is interesting to note that this region falls into the ßC'-ßE loop, which has been shown to be important for receptor specificity determination in paracrine FGFs, suggesting a common principle in both endocrine and paracrine FGF receptor interaction and activation.

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 D(4) (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.