Fibroblast growth factor 21 night watch: advances and uncertainties in the field.

Fibroblast growth factor (FGF) 21 belongs to a hormone-like subgroup within the FGF superfamily. The members of this subfamily, FGF19, FGF21 and FGF23, are characterized by their reduced binding affinity for heparin that enables them to be transported in the circulation and function in an endocrine manner. It is likely that FGF21 also acts in an autocrine and paracrine fashion, as multiple organs can produce this protein and its plasma concentration seems to be below the level necessary to induce a pharmacological effect. FGF21 signals via FGF receptors, but for efficient receptor engagement it requires a cofactor, membrane-spanning ßKlotho (KLB). The regulation of glucose uptake in adipocytes was the initial biological activity ascribed to FGF21, but this hormone is now recognized to stimulate many other pathways in vitro and display multiple pharmacological effects in metabolically compromised animals and humans. Understanding of the precise physiology of FGF21 and its potential medicinal role has evolved exponentially over the last decade, yet numerous aspects remain to be defined and others are a source of debate. Here we provide a historical overview of the advances in FGF21 biology focusing on the uncertainties in the mechanism of action as well as the differing viewpoints relating to this intriguing protein.

Physiology and Endocrinology Symposium: FGF21: Insights into mechanism of action from preclinical studies.

Fibroblast growth factor 21 (FGF21) is a multifaceted metabolic regulator which has several potential applications in the treatment of metabolic disease. When administered in vivo, FGF21 exhibits a plethora of actions, modulating metabolic homeostasis in a diverse manner. However, the mechanism and site of action underlying these effects were, until recently, entirely uncertain. Using mouse models lacking either FGF receptor isoform 1 (FGFR1) or ßKlotho (KLB), a transmembrane co-factor critical for FGF21 action, our group and others sought to determine the tissue on which FGF21 acts and the receptor complex responsible for mediating its in vivo efficacy. Importantly, when KLB was ablated from all tissues mice were completely refractory to FGF21 action. Therefore, to determine the precise tissue of action we utilized mice with tissue specific deletion of FGFR1 in either adipose tissue or neurons, respectively. Surprisingly, in animals with neuronal FGFR1 loss there was no change in the metabolic activity of FGF21, suggesting a lack of central FGF21 action in the pharmacologic setting. In contrast, we found dramatic attenuation of metabolic efficacy in mice with adipose-specific FGFR1 ablation following either acute or chronic dosing with recombinant FGF21. Furthermore, several recent studies have suggested that the metabolic effects of FGF21 may occur via modulation of adipokines such as adiponectin and leptin. Importantly, the action of FGF21 via adipose tissue results in alterations in both secretion as well as systemic sensitivity to these factors. Therefore, while FGF21 itself does not seem to directly act on the CNS, leptin and other endocrine mediators may serve as intermediary facilitators of FGF21's secondary central effects downstream of an initial and direct engagement of FGF21 receptor complex in adipose tissue. Further studies are required to delineate the precise mechanistic basis underlying the interplay between peripheral and central FGF21 modes of action in both the physiological and pharmacological settings.

Influence of growth hormone on circulating fibroblast growth factor 21 levels in humans.

OBJECTIVE: Findings from animal studies indicate that growth hormone (GH) may stimulate the production of the putative metabolic regulator fibroblast growth factor 21 (FGF21). We investigated whether circulating FGF21 levels are altered in patients with GH deficiency and characterized how levels of this growth factor are influenced by acute and long-term administration of GH, and the potential relationship between FGF21 and nonesterified fatty acids (NEFAs). DESIGN AND SETTING: GH-deficient patients (n = 9) were studied prior to and during 1 year of replacement with GH. Healthy subjects (n = 8) received an intravenous bolus of GH with or without concomitant oral glucose. Healthy subjects and patients with heterozygous familial hypercholesterolaemia (n = 23) were monitored following increasing doses of GH for 3 weeks. The main outcome measures were serum FGF21 and NEFA levels. Studies were performed at two academic centres. RESULTS: GH-deficient patients had FGF21 levels within the normal range, and GH replacement did not influence circulating FGF21 or NEFA concentrations. Acute GH administration to healthy control subjects did not change FGF21 levels, whereas an oral glucose load increased serum FGF21 by 25% and reduced NEFA levels by 48%. Similar effects were seen on administration of glucose together with GH. However, FGF21 levels increased dose dependently up to 3.7-fold in control subjects treated with GH for 3 weeks; simultaneously NEFA levels were increased by 47%. CONCLUSIONS: GH is not critical for the maintenance of basal serum FGF21 levels in humans, but circulating FGF21 levels increase following administration of GH to healthy individuals. There is no correlation between plasma NEFA and circulating FGF21 levels.

Increased responses to the actions of fibroblast growth factor 21 on energy balance and body weight in a seasonal model of adiposity.

The present study aimed to investigate the actions of fibroblast growth factor 21 (FGF21) on energy balance in a natural model of relative fatness, the Siberian hamster. Hamsters were studied under long days (LD) to promote weight gain, or short days to induce weight loss, and treated with rhFGF21 (3 mg/kg/day) via s.c. minipumps for 14 days. On days 7-9, detailed assessments of ingestive behaviour, metabolic gas exchange and locomotor activity were made. FGF21 caused substantial (P < 0.0001) weight loss in the fat LD state but not in the lean SD state: at the end of the study, FGF21-treated hamsters in LD lost 18% of body weight compared to vehicle controls, which is comparable to the natural body weight loss observed in SD. Epididymal fat pads, a correlate of total carcass fat content, were reduced by 19% in FGF21 treated hamsters in LD, whereas no difference was found in SD. Body weight loss in LD was associated with a reduction in food intake (P < 0.001) and a decreased respiratory exchange ratio (P < 0.001), indicating increased fat oxidation. Treatment with FGF21 maintained the normal nocturnal increase in oxygen consumption and carbon dioxide production into the early light phase in hamsters in LD, indicating increased energy expenditure, although locomotor activity was unaffected. These data suggest a greater efficacy of FGF21 in hamsters in LD compared to those in SD, which is consistent with both the peripheral and possibly central actions of FGF21 with respect to promoting a lean phenotype. The observed differences in FGF21 sensitivity may relate to day length-induced changes in adipose tissue mass.

FGF21: The center of a transcriptional nexus in metabolic regulation.

Fibroblast growth factor 21 (FGF21) is emerging as a key regulator of energy homeostasis and presents a novel target for the development of therapies for the treatment of diabetes, cardiovascular disease and obesity. Recent publications have demonstrated that FGF21 resides downstream of a complex network of transcriptional regulators which modulate its expression in response to a wide array of physiological stimuli or pharmacologic agents. The manner in which these mechanisms are integrated to regulate FGF21 transcription, production and subsequent secretion is poorly understood. While FGF21 is detected in many metabolically active tissues and is regulated by several of the known transcription factors involved in metabolic control little is known about how these pathways are integrated. In this review, we discuss the data presented to date on regulation of FGF21 by a wide array of transcription factors and explore how it relates to metabolic regulation in vivo.

A family of proteins that inhibit signalling through tyrosine kinase receptors.

Phosphotyrosine phosphatases are critical negative or positive regulators in the intracellular signalling pathways that result in growth-factor-specific cell responses such as mitosis, differentiation, migration, survival, transformation or death. The SH2-domain-containing phosphotyrosine phosphatase SHP-2 is a positive signal transducer for several receptor tyrosine kinases (RTKs) and cytokine receptors. To investigate its mechanism of action we purified a tyrosine-phosphorylated glycoprotein which in different cell types associates tightly with SHP-2 and appears to serve as its substrate. Peptide sequencing in conjunction with complementary DNA cloning revealed a new gene family of at least fifteen members designated signal-regulatory proteins (SIRPs). They consist of two subtypes distinguished by the presence or absence of a cytoplasmic SHP-2-binding domain. The transmembrane polypeptide SIRP alpha1 is a substrate of activated RTKs and in its tyrosine-phosphorylated form binds SHP-2 through SH2 interactions and acts as its substrate. It also binds SHP-1 and Grb2 in vitro and has negative regulatory effects on cellular responses induced by growth factors, oncogenes or insulin. Our findings indicate that proteins belonging to the SIRP family generally regulate signals defining different physiological and pathological processes.

PTP1D is a positive regulator of the prolactin signal leading to beta-casein promoter activation.

Stimulation of the prolactin receptor (PRLR), a member of the cytokine/growth hormone receptor family, results in activation of the associated Jak2 tyrosine kinase and downstream signaling pathways. We report that PTP1D, a cytoplasmic protein tyrosine phosphatase containing two Src homology 2 (SH2) domains, physically associates with the PRLR-Jak2 complex and is tyrosine-phosphorylated upon stimulation with prolactin. The formation of the trimeric PRLR-Jak2-PTP1D complex is critical for transmission of a lactogenic signal, while PTP1D phosphorylation is necessary, but not sufficient. The dominant negative inhibitory effect of a phosphatase-deficient mutant on expression of a beta-casein promoter-controlled reporter gene is evidence for an essential role of fully functional PTP1D in the regulation of milk protein gene transcription.

Adapter function of protein-tyrosine phosphatase 1D in insulin receptor/insulin receptor substrate-1 interaction.

Insulin signal transduction involves the multisite docking protein insulin receptor substrate-1 (IRS-1) and a number of Src homology-2 (SH2) domain factors, including p85/p110 phosphatidylinositol 3-kinase, p110 GTPase-activating protein, and the phosphotyrosine-specific phosphatase PTP1D. In transfected baby hamster kidney cells, Rat1 fibroblasts, and normal IM9 lymphoblasts, PTP1D directly binds activated insulin receptor. This interaction is mediated by catalytic domain-proximal SH2 determinants of the phosphatase and phosphotyrosine 1146 of the activated insulin receptor. While the receptor and the phosphatase do not serve as substrates for each other, their interaction promotes IRS-1 binding to the receptor, indicating that PTP1D functions as an adapter for insulin receptor and IRS-1. The formation of a multiprotein signaling complex involving the insulin receptor, PTP1D, and IRS-1 enhances cellular glucose uptake, a critical process in the physiological action of insulin.

Protein-tyrosine phosphatase 1D modulates its own state of tyrosine phosphorylation.

The insulin receptor-mediated signal transduction pathway involves insulin receptor substrate 1 and a variety of proteins containing Src homology-2 (SH2) domains, such as phosphatidylinositol 3-kinase, Grb2, and protein-tyrosine phosphatase 1D (PTP1D). Upon insulin stimulation of baby hamster kidney cells overexpressing the IR, the catalytically inactive mutant of PTP1D, C463A, becomes tyrosine-phosphorylated and coprecipitates with Grb2. Tyrosine phosphorylation of this mutant is significantly reduced when wild type PTP1D is coexpressed. Substitution of tyrosine residues 546 and 584 with phenylalanine abrogates tyrosine phosphorylation of the catalytically inactive mutant and abolishes its interaction with Grb2.

Association of SH2 domain protein tyrosine phosphatases with the epidermal growth factor receptor in human tumor cells. Phosphatidic acid activates receptor dephosphorylation by PTP1C.

The SH2 domain protein tyrosine phosphatases (PTPases) PTP1C and PTP1D were found associated with epidermal growth factor (EGF) receptor which was purified from A431 cell membranes by several steps of chromatography. Both PTPases also associated with the EGF receptor upon exposure of immunoprecipitated receptor to lysates of MCF7 mammary carcinoma cells. The associated PTPases had little activity toward the bound receptor when it was autophosphorylated in vitro. Receptor dephosphorylation could, however, be initiated by treatment of the receptor-PTPase complex with phosphatidic acid (PA). When autophosphorylated EGF receptor was exposed to lysates of PTP1C or PTP1D overexpressing 293 cells, the association of PTP1C but not of PTP1D was enhanced in the presence of PA. In intact A431 cells, an association of PTP1C and PTP1D with the EGF receptor was detectable by coimmunoprecipitation experiments. PA treatment reduced the phosphorylation state of ligand activated EGF receptors in A431 cells and in 293 cells overexpressing EGF receptors together with PTP1C but not in 293 cells overexpressing EGF receptors alone or together with PTP1D. We conclude that PTP1C but not PTP1D participates in dephosphorylation of activated EGF receptors. A possible role of PA for physiological modulation of EGF receptor signaling is discussed.

Selective down-regulation of the insulin receptor signal by protein-tyrosine phosphatases alpha and epsilon.

Binding of insulin to its receptor (IR) causes rapid autophosphorylation with concomitant activation of its tyrosine kinase which transmits the signal by phosphorylating cellular substrates. The IR activity is controlled by protein-tyrosine phosphatases, but those directly involved in regulating the insulin receptor and its signaling pathways have not yet been identified. Using baby hamster kidney cells overexpressing the IR and a novel insulin-based selection principle, we established stable cell lines with functionally coupled expression of the IR and protein-tyrosine phosphatases. The two closely related protein-tyrosine phosphatases alpha and epsilon were identified as negative regulators of IR tyrosine kinase.

Early lymphocyte activation events are inhibited by antiproliferative synthetic peptide 2438, a fragment of human interferon alpha-2, and immunosuppressant cyclosporine A.

A synthetic peptide (designated 2438) corresponding to the human interferon alpha-2 amino acid sequence 124-138 inhibits proliferation of T-lymphocytes in vitro. Time-course experiments suggest that peptide 2438 affects early stages of lymphocyte activation. Molecular mechanisms of peptide 2438 action were studied. By western-blotting with monoclonal antibodies against phosphotyrosine peptide 2438 was shown to decrease the phosphotyrosine content of an endogenous protein substrate (M.M. = 36 kDa) in human lymphocytes activated with concanavalin A (ConA). Similar effect on tyrosine-specific phosphorylation in mitogen-stimulated lymphocytes was observed with the native interferon or Cyclosporine A (CsA). Calcium fluxes induced by ConA in human lymphocytes were measured using a fluorescent calcium chelator Fura-2. In contrast to CsA, peptide 2438 did not affect the ConA-induced calcium influx in lymphocytes.

Src kinase associates with a member of a distinct subfamily of protein-tyrosine phosphatases containing an ezrin-like domain.

A 6.2-kb full-length clone encoding a distinct protein-tyrosine phosphatase (PTP; EC 3.1.3.48), PTPD1, was isolated from a human skeletal muscle cDNA library. The cDNA encodes a protein of 1174 amino acids with N-terminal sequence homology to the ezrin-band 4.1-merlin-radixin protein family, which also includes the two PTPs H1 and MEG1. The PTP domain is positioned in the extreme C-terminal part of PTPD1, and there is an intervening sequence of about 580 residues without any apparent homology to known proteins separating the ezrin-like and the PTP domains. Thus, PTPD1 and the closely related, partially characterized, PTPD2 belong to the same family as PTPH1 and PTPMEG1, but because of distinct features constitute a different PTP subfamily. Northern blot analyses indicate that PTPD1 and PTPD2 are expressed in a variety of tissues. In transient coexpression experiments PTPD1 was found to be efficiently phosphorylated by and associated with the src kinase pp60src.

D-glyceraldehyde-3-phosphate dehydrogenase purified from rabbit muscle contains phosphotyrosine.

Homogeneous preparations of D-glyceraldehyde-3-phosphate dehydrogenase purified from rabbit muscle were found to contain 0.2-0.7 moles of covalently bound phosphate per mole of the enzyme. With the use of anti-phosphotyrosine antibodies, evidence was obtained that the enzyme is phosphorylated at tyrosine residues.

Interaction of a synthetic peptide of the interferon alpha 2 C-terminal part with human blood leukocytes. II. Effect on protein tyrosine phosphorylation.

Tyrosine phosphorylation in human blood lymphocytes was studied as a function of stimulation with concanavalin A (ConA) and treatment of the cells with interferon alpha 2 (IFN alpha 2) and/or an IFN-derived C-terminal synthetic peptide 2438 (amino acid residues 124-138). Both IFN alpha 2 and the peptide 2438 decreased the level of protein tyrosine phosphorylation in the ConA-stimulated cells. In unstimulated cells, IFN alpha 2 increased, and the peptide 2438 decreased the level of the tyrosine phosphorylation. A possible correlation of these effects with stimulation of cell proliferation is discussed.

Interaction of a synthetic peptide of the interferon alpha-2 C-terminal part with human blood leukocytes. Binding to peripheral blood mononuclear cells.

A biologically active synthetic peptide, 2438, representing the 124-138 amino acid sequence of the human interferon alpha-2 (IFN alpha-2) molecule, which is known to possess IFN-like antiproliferative activity, specifically binds to human blood leukocytes. Scatchard plots reveal two different Kd values, for the 'low' and 'high' affinity binding. The interaction of the 125I-labelled peptide 2438 with the cells is not impaired by human IFN alpha-2 or cholera toxin.

[Autophosphorylation of EGF receptors in the A431 cell line with an increased intracellular concentration of Ca(2+) ions]. / Avtofosforilirovanie retseptorov EFR v kletkakh linii A431 v usloviiakhpovyshennoi vnutrikletochnoi kontsentratsii ionov Ca(2+).

The increase in the intracellular concentration of Ca2+ in A431 cells induced by the calcium ionophore A23187 leads to phosphorylation of epidermal growth factor (EGF) receptors at serine and/or threonine residues. This process is accompanied by the decrease in the level of EGF receptor autophosphorylation at tyrosine residues. Preincubation of cells in a A23187-containing medium in the presence of phorbol-12-myristoyl-13-acetate leads to a further decrease of the phosphotyrosine content in EGF receptors. At increased intracellular concentrations of Ca2+ preincubation of A431 cells with the protein kinase C inhibitor H-7 has no effect on the degree of EGF receptor autophosphorylation. Down-regulation of cellular protein kinase C does not change the A23187-induced effect either. The data obtained suggest that the decreased autophosphorylation of EGF receptors induced by Ca2+ is not due to the activation of cellular protein kinase C.

[The effect of phorbol esters and Ca2+ ions on the process of autophosphorylation of epidermal growth factor receptor in vivo]. / Vliianie forbolovykh éfirov i ionov Ca2+ na protsess avtofosforilirovaniia retseptora epidermal'nogo faktora rosta in vivo.

Monoclonal antibodies against phosphotyrosine were used to study tyrosine phosphorylation in human epidermal carcinoma A431 cells in vivo. Incubation of A431 cells with the epidermal growth factor (EGF) leads to tyrosine phosphorylation of the EGF receptor; the phosphotyrosine content in cellular EGF receptors increases 50-100-fold in the presence of the growth factor. The maximum level of the receptor autophosphorylation is reached on the 5th min and is then held constant during 90-min incubation with EGF. After preincubation of A431 cells with phorbol-12-myristoyl-13-acetate (PMA) or calcium ionophore A23187 the receptor autophosphorylation decreases significantly. After addition of A23187 and EGTA to the preincubation medium the phosphotyrosine content in cellular EGF receptors stimulated by the growth factor reaches the control level i.e., that observed in the absence of the ionophore. After preincubation of cells in the presence of phorbol ester and H-7 (protein kinase C inhibitor) the level of EGF receptor autophosphorylation does not practically differ from that of control.

[Preparation of monoclonal antibodies to phosphotyrosine and their use for identification of phosphotyrosine-containing proteins]. / Poluchenie monoklonal'nykh antitel k fosfotirozinu i ikh ispol'zovanie dlia identifikatsii fosfotirozinsoderzhashchikh belkov.

Protein kinases phosphorylating proteins at tyrosine residues play an essential role in the cell growth regulation and neoplastic transformation. However, the functions of the majority of tyrosine protein kinases are still obscure, thus creating hindrances in the identification and isolation of phosphotyrosine-containing proteins. The use of the phosphotyrosine structural analog, aminobenzyl phosphonate, as a hapten group enabled the preparation of monoclonal antibodies capable of reacting to phosphotyrosine. The phosphotyrosine specificity of six clones of monoclonal antibodies was tested by a competitive solid phase immunoenzymatic assay. Using fluorescence quenching, the values of constants of binding for antibodies of four clones to phosphotyrosine (2.5-4.0 x 10(6) M-1) were determined. Using two independent methods, it was shown that clone B4 antibodies reveal the highest specificity towards phosphotyrosine. An immunoadsorbent based on clone B4 antibodies was obtained; this immunoadsorbent possessed an ability to selectively interact with an EFR receptor phosphorylated at tyrosine residue. Using eluate acid hydrolysis from the immunoadsorbent, it was demonstrated that clone B4 antibodies interact only with the phosphotyrosine-containing proteins. The experimental results are suggestive of clone B4 monoclonal antibody specificity to phosphotyrosine and of the feasibility of their application for the isolation and identification of tyrosine protein kinases and their substrates.