We recently identified activating mutations of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) in bladder carcinoma. In this study we assessed the incidence of FGFR3 mutations in a series of 132 bladder carcinomas: 20 carcinoma in situ (CIS), 50 pTa, 19 pT1, and 43 pT2-4. All 48 mutations identified were identical to the germinal activating mutations that cause thanatophoric dysplasia, a lethal form of dwarfism. The S249C mutation, found in 33 of the 48 mutated tumors, was the most common. The frequency of mutations was higher in pTa tumors (37 of 50, 74%) than in CIS (0 of 20, 0%; P < 0.0001), pT1 (4 of 19, <em>21</em>%; P < 0.0001) and pT2-4 tumors (7 of 43, 16%; P < 0.0001). FGFR3 mutations were detected in 27 of 32 (84%) G1, 16 of 29 (55%) G2, and 5 of 71 (7%) G3 tumors. This association between FGFR3 mutations and low grade was highly significant (P < 0.0001). FGFR3 is the first gene found to be mutated at a high frequency in pTa tumors. The absence of FGFR3 mutations in CIS and the low frequency of FGFR3 mutations in pT1 and pT2-4 tumors are consistent with the model of bladder tumor progression in which the most common precursor of pT1 and pT2-4 tumors is CIS.

Obesity increases the risk for metabolic, cardiovascular, chronic inflammatory, and several malignant diseases and, therefore, may contribute to shortened lifespan. Adipokines are peptides that signal the functional status of adipose tissue to targets in the brain, liver, pancreas, immune system, vasculature, muscle, and other tissues. Secretion of adipokines, including leptin, adiponectin, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), retinol-binding protein 4 (RBP4), dipeptidyl peptidase 4 (DPP-4), bone morphogenetic protein (BMP)-4, BMP-7, vaspin, apelin, and progranulin, is altered in adipose tissue dysfunction and may contribute to a spectrum of obesity-associated diseases. Adipokines are promising candidates both for novel pharmacological treatment strategies and as diagnostic tools, provided that we can develop a better understanding of the function and molecular targets of the more recently discovered adipokines.

A 6.75-kilobase human hepatoma-derived basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) cDNA was cloned and sequenced. An amino-terminal sequence generated from a purified hepatoma bFGF was found to correspond to the nucleotide sequence and to begin 8 amino acids upstream from the putative methionine start codon thought to initiate a 154-amino acid bFGF translation product. This sequence suggests that a form of bFGF of at least 163 amino acids exists. The hepatoma cDNA was transcribed in vitro into RNA; in vitro translation of this RNA generated three forms of bFGF with molecular masses of 18, <em>21</em>, and 22.5 kDa. By use of in vitro mutagenesis, it was found that the 22.5-kDa bFGF and possibly the <em>21</em>-kDa form were initiated with CUG start codons. The 18-kDa bFGF was initiated with an AUG codon. By transfecting into COS cells human hepatoma bFGF cDNA and a construct from which the AUG initiator was eliminated, it was found that the higher molecular mass forms of bFGF were as biologically active as the 18-kDa form.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF-<em>21</em>) is a metabolic regulator that can influence glucose and lipid control in diabetic rodents and primates. We demonstrate that betaKlotho is an integral part of an activated FGF-<em>21</em>-betaKlotho-FGF receptor (FGFR) complex thus a critical subunit of the FGF-<em>21</em> receptor. Cells lacking betaKlotho did not respond to FGF-<em>21</em>; the introduction of betaKlotho to these cells conferred FGF-<em>21</em>-responsiveness and recapitulated the entire scope of FGF-<em>21</em> signaling observed in naturally responsive cells. Interestingly, FGF-<em>21</em>-mediated effects are heparin independent suggesting that betaKlotho plays a role in FGF-<em>21</em> activity similar to the one played by heparin in the signaling of conventional FGFs. Moreover, in addition to conferring specificity for FGF-<em>21</em>, betaKlotho appears to support FGF-19 activity and mediates the receptor selectivity profile of FGF-19. All together, these results indicate that betaKlotho and FGFRs form the cognate FGF-<em>21</em> receptor complex, mediating FGF-<em>21</em> cellular specificity and physiological effects.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em>, a structural relative of FGF23 that regulates phosphate homeostasis, is a regulator of insulin-independent glucose transport in adipocytes and plays a role in the regulation of body weight. It also regulates ketogenesis and adaptive responses to starvation. We report that in a reconstituted receptor activation assay system using BaF3 cells, which do not endogenously express any type of FGF receptor (FGFR) or heparan sulfate proteoglycan, FGF<em>21</em> alone does not activate FGFRs and that betaKlotho is required for FGF<em>21</em> to activate two specific FGFR subtypes: FGFR1c and FGFR3c. Coexpression of betaKlotho and FGFR1c on BaF3 cells enabled FGF<em>21</em>, but not FGF23, to activate receptor signaling. Conversely, coexpression of FGFR1c and Klotho, a protein related to betaKlotho, enabled FGF23 but not FGF<em>21</em> to activate receptor signaling, indicating that expression of betaKlotho/Klotho confers target cell specificity on FGF<em>21</em>/FGF23. In all of these cases, heparin enhanced the activation but was not essential. In 3T3-L1 adipocytes, up-regulation of glucose transporter (GLUT) expression by FGF<em>21</em> was associated with expression of betaKlotho, which was absent in undifferentiated 3T3-L1 <em>fibroblasts</em>. It is thus suggested that betaKlotho expression is a crucial determinant of the FGF<em>21</em> specificity of the target cells upon which it acts in an endocrine fashion.

We have found that the spontaneous migration of bovine aortic endothelial cells from the edge of a denuded area in a confluent monolayer is dependent upon the release of endogenous basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). Cell movement is blocked by purified polyclonal rabbit IgG to bFGF as well as affinity purified anti-bFGF IgG and anti-bFGF F(ab')2 fragments. The inhibitory effect of the immunoglobulins is dependent upon antibody concentration, is reversible, is overcome by the addition of recombinant bFGF, and is removed by affinity chromatography of the antiserum through a column of bFGF-Sepharose. Cell movement is also reversibly inhibited by the addition of protamine sulfate and suramin; two agents reported to block bFGF binding to its receptor. The addition of recombinant bFGF to wounded monolayers accelerates the movement of cells into the denuded area. Transforming <em>growth</em> <em>factor</em> beta which has been shown to antagonize several other effects of bFGF also inhibits cell movement. The anti-bFGF IgG prevents the movement of bovine capillary endothelial cells, BHK-<em>21</em>, NIH 3T3, and human skin <em>fibroblasts</em> into a denuded area. Antibodies to bFGF, as well as suramin and protamine sulfate also suppress the basal levels of plasminogen activator and DNA synthesis in bovine aortic endothelial cells.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>) functions as a metabolic regulator. The FGF<em>21</em> transcript is reported to be abundantly expressed in liver, but little is known about the regulation of FGF<em>21</em> expression in other tissues. In this study, we show that levels of FGF<em>21</em> protein expression were similar in skeletal muscle and liver from fasted mice. FGF<em>21</em> transcript and protein expression were upregulated in gastrocnemius muscle of skeletal muscle-specific Akt1 transgenic mice. Serum concentration of FGF<em>21</em> was also increased by Akt1 transgene activation. In cultured skeletal muscle cells, FGF<em>21</em> expression and secretion was regulated by insulin, Akt transduction and LY294002. These data indicate that skeletal muscle is a source of FGF<em>21</em> and that its expression is regulated by a phosphatidylinosistol 3-kinase (PI3-kinase)/Akt1 signaling pathway-dependent mechanism.

Platelet-derived <em>growth</em> <em>factor</em> (PDGF) and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) markedly potentiate tissue repair in vivo. In the present experiments, both in vitro and in vivo responses to PDGF and TGF-beta were tested to identify mechanisms whereby these <em>growth</em> <em>factors</em> might each enhance the wound-healing response. Recombinant human PDGF B-chain homodimers (PDGF-BB) and TGF-beta 1 had identical dose-response curves in chemotactic assays with monocytes and <em>fibroblasts</em> as the natural proteins from platelets. Single applications of PDGF-BB (2 micrograms, 80 pmol) and TGF-beta 1 (20 micrograms, 600 pmol) were next applied to linear incisions in rats and each enhanced the strength required to disrupt the wounds at 5 d up to <em>21</em>2% of paired control wounds. Histological analysis of treated wounds demonstrated an in vivo chemotactic response of macrophages and <em>fibroblasts</em> to both PDGF-BB and to TGF-beta 1 but the response to TGF-beta 1 was significantly less than that observed with PDGF-BB. Marked increases of procollagen type I were observed by immunohistochemical staining in <em>fibroblasts</em> in treated wounds during the first week. The augmented breaking strength of TGF-beta 1 was not observed 2 and 3 wk after wounding. However, the positive influence of PDGF-BB on wound breaking strength persisted through the 7 wk of testing. Furthermore, PDGF-BB-treated wounds had persistently increased numbers of <em>fibroblasts</em> and granulation tissue through day <em>21</em>, whereas the enhanced cellular influx in TGF-beta 1-treated wounds was not detectable beyond day 7. Wound macrophages and <em>fibroblasts</em> from PDGF-BB-treated wounds contained sharply increased levels of immunohistochemically detectable intracellular TGF-beta. Furthermore, PDGF-BB in vitro induced a marked, time-dependent stimulation of TGF-beta mRNA levels in cultured normal rat kidney <em>fibroblasts</em>. The results suggest that TGF-beta transiently attracts <em>fibroblasts</em> into the wound and may stimulate collagen synthesis directly. In contrast, PDGF is a more potent chemoattractant for wound macrophages and <em>fibroblasts</em> and may stimulate these cells to express endogenous <em>growth</em> <em>factors</em>, including TGF-beta, which, in turn, directly stimulate new collagen synthesis and sustained enhancement of wound healing over a more prolonged period of time.

In mammals, excess energy is stored primarily as triglycerides, which are mobilized when energy demands arise. This review mainly focuses on the role of long chain fatty acids (LCFAs) in regulating energy metabolism as ligands of peroxisome proliferator-activated receptors (PPARs). PPAR-alpha expressed primarily in liver is essential for metabolic adaptation to starvation by inducing genes for beta-oxidation and ketogenesis and by downregulating energy expenditure through <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>. PPAR-delta is highly expressed in skeletal muscle and induces genes for LCFA oxidation during fasting and endurance exercise. PPAR-delta also regulates glucose metabolism and mitochondrial biogenesis by inducing FOXO1 and PGC1-alpha. Genes targeted by PPAR-gamma in adipocytes suggest that PPAR-gamma senses incoming non-esterified LCFAs and induces the pathways to store LCFAs as triglycerides. Adiponectin, another important target of PPAR-gamma may act as a spacer between adipocytes to maintain their metabolic activity and insulin sensitivity. Another topic of this review is effects of skin LCFAs on energy metabolism. Specific LCFAs are required for the synthesis of skin lipids, which are essential for water barrier and thermal insulation functions of the skin. Disturbance of skin lipid metabolism often causes apparent resistance to developing obesity at the expense of normal skin function.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a novel metabolic regulator shown to improve glycemic control. However, the molecular and functional mechanisms underlying FGF<em>21</em>-mediated improvements in glycemic control are not completely understood. We examined FGF<em>21</em> effects on insulin sensitivity and glucose fluxes upon chronic (daily injection for 8 d) and acute (6 h infusion) administration in ob/+ and ob/ob mice. Results show that chronic FGF<em>21</em> ameliorated fasting hyperglycemia in ob/ob mice via increased glucose disposal and improved hepatic insulin sensitivity. Acute FGF<em>21</em> suppressed hepatic glucose production, increased liver glycogen, lowered glucagon, and improved glucose clearance in ob/+ mice. These effects were blunted in ob/ob mice. Neither chronic nor acute FGF<em>21</em> altered skeletal muscle or adipose tissue glucose uptake in either genotype. In conclusion, FGF<em>21</em> has potent glycemic effects caused by hepatic changes in glucose flux and improved insulin sensitivity. Thus, these studies define mechanisms underlying anti-hyperglycemic actions of FGF<em>21</em> and support its therapeutic potential.

Fibroblast growth factors (Fgfs) are polypeptide growth factors with diverse functions. Fgf21, a unique member of the Fgf family, is expected to function as a metabolic regulator in an endocrine manner. Hepatic Fgf21 expression was increased by fasting. The phenotypes of hepatic Fgf21 transgenic or knockdown mice and high-fat, low-carbohydrate ketogenic diet-fed mice suggests that Fgf21 stimulates lipolysis in the white adipose tissue during normal feeding and is required for ketogenesis and triglyceride clearance in the liver during fasting. However, the physiological roles of Fgf21 remain unclear. To elucidate the physiological roles of Fgf21, we generated Fgf21 knockout (KO) mice by targeted disruption. Fgf21 KO mice were viable, fertile, and seemingly normal. Food intake, oxygen consumption, and energy expenditure were also essentially unchanged in Fgf21 KO mice. However, hypertrophy of adipocytes, decreased lipolysis in adipocytes, and decreased blood nonesterified fatty acid levels were observed when Fgf21 KO mice were fed normally. In contrast, increased lipolysis in adipocytes and increased blood nonesterified fatty acid levels were observed in Fgf21 KO mice by fasting for 24 h, indicating that Fgf21 stimulates lipolysis in the white adipose tissue during feeding but inhibits it during fasting. In contrast, unexpectedly, hepatic triglyceride levels were essentially unchanged in Fgf21 KO mice. In addition, ketogenesis in Fgf21 KO mice was not impaired by fasting for 24 h. The present results indicate that Fgf21 regulates lipolysis in adipocytes in response to the metabolic state but is not required for ketogenesis and triglyceride clearance in the liver.

OBJECTIVE <em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>21</em> is highly expressed in the liver and regulates hepatic glucose production and lipid metabolism in rodents. However, its role in the pathogenesis of type 2 diabetes in humans remains to be defined. The aim of this study was to quantitate circulating plasma FGF-<em>21</em> levels and examine their relationship with insulin sensitivity in subjects with varying degrees of obesity and glucose tolerance. RESEARCH DESIGN AND METHODS Forty-one subjects (8 lean with normal glucose tolerance [NGT], 9 obese with NGT, 12 with impaired fasting glucose [IFG]/impaired glucose tolerance [IGT], and 12 type 2 diabetic subjects) received an oral glucose tolerance test (OGTT) and a hyperinsulinemic-euglycemic clamp (80 mU/m(2) per min) combined with 3-[(3)H] glucose infusion. RESULTS Subjects with type 2 diabetes, subjects with IGT, and obese subjects with NGT were insulin resistant compared with lean subjects with NGT. Plasma FGF-<em>21</em> levels progressively increased from 3.9 +/- 0.3 ng/ml in lean subjects with NGT to 4.9 +/- 0.2 in obese subjects with NGT to 5.2 +/- 0.2 in subjects with IGT and to 5.3 +/- 0.2 in type 2 diabetic subjects. FGF-<em>21</em> levels correlated inversely with whole-body (primarily reflects muscle) insulin sensitivity (r = -0.4<em>21</em>, P = 0.007) and directly with the hepatic insulin resistance index (r = 0.344, P = 0.034). FGF-<em>21</em> levels also correlated with measures of glycemia (fasting plasma glucose [r = 0.312, P = 0.05], 2-h plasma glucose [r = 0.414, P = 0.01], and A1C [r = 0.325, P = 0.04]). CONCLUSIONS Plasma FGF-<em>21</em> levels are increased in insulin-resistant states and correlate with hepatic and whole-body (muscle) insulin resistance. FGF-<em>21</em> may play a role in pathogenesis of hepatic and whole-body insulin resistance in type 2 diabetes.

Recombinant <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>21</em> has antihyperglycemic, antihyperlipidemic, and antiobesity effects in diabetic rodent and monkey models. Previous studies were confined to measuring steady-state effects of FGF<em>21</em> following subchronic or chronic administration. The present study focuses on the kinetics of biological actions of FGF<em>21</em> following a single injection and on the associated physiological and cellular mechanisms underlying FGF<em>21</em> actions. We show that FGF<em>21</em> resulted in rapid decline of blood glucose levels and immediate improvement of glucose tolerance and insulin sensitivity in two animal models of insulin resistance (ob/ob and DIO mice). In ob/ob mice, FGF<em>21</em> led to a 40-60% decrease in blood glucose, insulin, and amylin levels within 1 h after injection, and the maximal effects were sustained for more than 6 h despite the 1- to 2-h half-life of FGF<em>21</em>. In DIO mice, FGF<em>21</em> reduced fasting blood glucose and insulin levels and improved glucose tolerance and insulin sensitivity within 3 h of treatment. The acute improvement of glucose metabolism was associated with a 30% reduction of hepatic glucose production and an increase in peripheral glucose turnover. FGF<em>21</em> appeared to have no direct effect on ex vivo pancreatic islet insulin or glucagon secretion. However, it rapidly induced typical FGF signaling in liver and adipose tissues and in several hepatoma-derived cell lines and differentiated adipocytes. FGF<em>21</em> was able to inhibit glucose release from H4IIE hepatoma cells and stimulate glucose uptake in 3T3-L1 adipocytes. We conclude that the acute glucose-lowering and insulin-sensitizing effects of FGF<em>21</em> are potentially associated with its metabolic actions in liver and adipose tissues.

Adipose tissue is a metabolically responsive endocrine organ that secretes a myriad of adipokines. Antidiabetic drugs such as peroxisome proliferator-activated receptor (PPAR) gamma agonists target adipose tissue gene expression and correct hyperglycemia via whole-body insulin sensitization. The mechanism by which altered gene expression in adipose tissue affects liver and muscle insulin sensitivity (and thus glucose homeostasis) is not fully understood. One possible mechanism involves the alteration in adipokine secretion, in particular the up-regulation of secreted factors that increase whole-body insulin sensitivity. Here, we report the use of transcriptional profiling to identify genes encoding for secreted proteins the expression of which is regulated by PPARgamma agonists. Of the 379 genes robustly regulated by two structurally distinct PPARgamma agonists in the epididymal white adipose tissue (EWAT) of db/db mice, 33 encoded for known secreted proteins, one of which was FGF21. Although FGF21 was recently reported to be up-regulated in cultured adipocytes by PPARgamma agonists and in liver by PPARalpha agonists and induction of ketotic states, we demonstrate that the protein is transcriptionally up-regulated in adipose tissue in vivo by PPARgamma agonist treatment and under a variety of physiological conditions, including fasting and high fat diet feeding. In addition, we found that circulating levels of FGF21 protein were increased upon treatment with PPARgamma agonists and under ketogenic states. These results suggest a role for FGF21 in mediating the antidiabetic activities of PPARgamma agonists.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>) is a hormone secreted by the liver during fasting that elicits diverse aspects of the adaptive starvation response. Among its effects, FGF<em>21</em> induces hepatic fatty acid oxidation and ketogenesis, increases insulin sensitivity, blocks somatic <em>growth</em> and causes bone loss. Here we show that transgenic overexpression of FGF<em>21</em> markedly extends lifespan in mice without reducing food intake or affecting markers of NAD+ metabolism or AMP kinase and mTOR signaling. Transcriptomic analysis suggests that FGF<em>21</em> acts primarily by blunting the <em>growth</em> hormone/insulin-like <em>growth</em> <em>factor</em>-1 signaling pathway in liver. These findings raise the possibility that FGF<em>21</em> can be used to extend lifespan in other species.DOI:http://dx.doi.org/10.7554/eLife.00065.001.

BACKGROUND

Tissue fibrosis is considered a dysregulated wound-healing response. Fibronectin containing extra type III domain A (EDA) is implicated in the regulation of wound healing. EDA-containing fibronectin is deposited during wound repair, and its presence precedes that of collagen.

OBJECTIVE

To investigate the role of EDA-containing fibronectin in lung fibrogenesis.

METHODS

Primary lung fibroblasts from patients with idiopathic pulmonary fibrosis or from patients undergoing resection for lung cancer were assessed for EDA-containing fibronectin and alpha-smooth muscle actin (alpha-SMA) expression. Mice lacking the EDA domain of fibronectin and their wild-type littermates were challenged with the bleomycin model of lung fibrosis. Primary lung fibroblasts from these mice were assayed in vitro to determine the contribution of EDA-containing fibronectin to fibroblast phenotypes.

RESULTS

Idiopathic pulmonary fibrosis lung fibroblasts produced markedly more EDA-containing fibronectin and alpha-SMA than control fibroblasts. EDA-null mice failed to develop significant fibrosis 21 days after bleomycin challenge, whereas wild-type controls developed the expected increase in total lung collagen. Histologic analysis of EDA-null lungs after bleomycin showed less collagen and fewer alpha-SMA-expressing myofibroblasts compared with that observed in wild-type mice. Failure to develop lung fibrosis in EDA-null mice correlated with diminished activation of latent transforming growth factor (TGF)-beta and decreased lung fibroblast responsiveness to active TGF-beta in vitro.

CONCLUSIONS

The data show that EDA-containing fibronectin is essential for the fibrotic resolution of lung injury through TGF-beta activation and responsiveness, and suggest that EDA-containing fibronectin plays a critical role in tissue fibrogenesis.

OBJECTIVE

To investigate the therapeutic potential of mesenchymal stromal cells (MSCs) in the chronic model of experimental autoimmune encephalomyelitis (EAE).

METHODS

Mesenchymal stromal cells were obtained from the bone marrow of naïve C57BL and green fluorescent protein-transgenic mice and cultured with Eagle minimum essential medium/alpha medium after removal of adhering cells. Following 2 to 3 passages, MSCs were injected intraventricularly or intravenously into mice in which chronic EAE had been induced with myelin oligodendrocyte glycoprotein 35-55 peptide.

RESULTS

In 8 separate experiments, the intravenously and intraventricularly injected green fluorescent protein-positive MSCs were attracted to the areas of central nervous system inflammation and expressed galactocerebroside, O4, glial fibrillary acidic protein, and beta-tubulin type III. The clinical course of chronic EAE was ameliorated in MSC-treated animals (0% mortality; mean [SE] maximal EAE score, 1.76 [1.01] and 1.8 [0.46] in the intraventricular and intravenous groups, respectively, vs 13% and <em>21</em>% mortality and 2.80 [0.79] and 3.42 [0.54] mean maximal score in the controls). A strong reduction in central nervous system inflammation, accompanied by significant protection of the axons (86%-95% intact axons vs 45% in the controls) was observed in the animals injected with MSCs (especially following intraventricular administration). Mesenchymal stromal cells injected intravenously were detected in the lymph nodes and exhibited systemic immunomodulatory effects, downregulating proliferation of lymphocytes in response to myelin antigens and mitogens. Mesenchymal stromal cells cultured with <em>fibroblast</em> <em>growth</em> <em>factor</em> and brain-derived neurotrophic <em>factor</em> in vitro acquired neuronal-lineage cell morphology and expressed beta-tubulin type III, nestin glial fibrillary acidic protein, and O4.

CONCLUSIONS

Our results indicate that stem cells derived from bone marrow may provide a feasible and practical way for neuroprotection, immunomodulation, and possibly remyelination and neuroregeneration in diseases such as multiple sclerosis.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), a hormone primarily secreted by the liver in response to peroxisome proliferator-activated receptor-α (PPARα) activation, has recently been shown to possess beneficial effects on lipid metabolism and hepatic steatosis in animal models. This study investigated the association of FGF<em>21</em> with nonalcoholic fatty liver disease (NAFLD) in Chinese patients.

METHODS

Serum FGF<em>21</em> levels were determined by enzyme-linked immunosorbent assay (ELISA) in 224 NAFLD and 124 control subjects, and their association with parameters of adiposity, glucose, and lipid profiles and levels of liver injury markers was studied. Besides serum concentrations, the mRNA expression of FGF<em>21</em> in the liver tissue was also quantified by real-time PCR in 17 subjects with different degrees of steatosis, and was correlated with the levels of intrahepatic lipid. The protein levels of FGF<em>21</em> were determined by quantitative ELISA.

RESULTS

Serum FGF<em>21</em> levels in patients with NAFLD (402.38 pg/ml [242.03, 618.25]) were significantly higher than those in control subjects (198.62 pg/ml [134.96, 412.62]) (p<0.01). In human liver tissues, FGF<em>21</em> mRNA expression increased with the degree of steatosis. Both FGF<em>21</em> mRNA expression and serum FGF<em>21</em> concentrations were positively correlated with intrahepatic triglyceride (TG) having r = 0.692 and r = 0.662, respectively, at p<0.01. Furthermore, the increased expression of FGF<em>21</em> was accompanied by elevated protein levels in liver tissues.

CONCLUSIONS

These results support the role of FGF<em>21</em> as a key regulator of hepatic lipid metabolism in humans, and suggest that serum FGF<em>21</em> can be potentially used as a biomarker for NAFLD.

Formal proof for an involvement of autocrine stimulation in the disturbed <em>growth</em> of malignant cells has been difficult to obtain, in part due to lack of precise methods of assessing <em>growth</em> <em>factor</em> production and receptor occurrence. In this study we have analyzed the mRNA levels for two <em>growth</em> <em>factors</em> and the corresponding receptors in a number of established human malignant glioma cell lines. Twenty-one tested lines all contained transcripts for the platelet-derived <em>growth</em> <em>factor</em> (PDGF) A chain while 16-17 of <em>21</em> expressed the c-sis/PDGF B chain gene; these two genes were expressed independently of each other. PDGF receptor transcripts were present in 15-16 of the <em>21</em> lines. Transcripts for the epidermal <em>growth</em> <em>factor</em> receptor were found in all 15 tested lines, in 2 of them at high levels, and the corresponding ligand transforming <em>growth</em> <em>factor</em>-alpha was found in 11 of 15 lines. No amplification or structural rearrangements of the genes, as analyzed by Southern blot hybridization, could explain the varying expression of PDGF A and B chain transcripts or the elevated levels of epidermal <em>growth</em> <em>factor</em> receptor mRNA. A correlation was found between cell morphology and expression of <em>growth</em> <em>factor</em> and receptor mRNA in these lines. The highest amount of PDGF receptor transcripts was found in cells with <em>fibroblast</em>-like morphology, and c-sis/B chain transcripts were found in small cell types and in cells with astrocyte-like morphology, while no clear relationship was found between PDGF receptor and A chain transcript levels or between morphology and A chain transcripts. It is possible that the findings reflect a coordinated expression of these genes in the progenitor cells. In conclusion, the data imply the existence of two possible autocrine loops in human malignant glioma lines, affecting the PDGF and epidermal <em>growth</em> <em>factor</em> receptor pathways.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF-23) is critical to the pathogenesis of a distinct group of renal phosphate wasting disorders: tumor-induced osteomalacia, X-linked hypophosphatemia, and autosomal dominant and autosomal recessive hypophosphatemic rickets. Excess circulating FGF-23 is responsible for their major phenotypic features which include hypophosphatemia due to renal phosphate wasting and inappropriately low serum 1,25(OH)2D concentrations. To characterize the effects of FGF-23 on renal sodium-phosphate (Na/P(i)) cotransport and vitamin D metabolism, we administered FGF-23(R176Q) to normal mice. A single injection (0.33 microg/g body wt) induced significant hypophosphatemia, 20 and 29% decreases (P < 0.001) in brush-border membrane (BBM) Na/Pi cotransport at 5 and 17 h after injection, respectively, and comparable decreases in the abundance of type IIa Na/P(i) cotransporter protein in BBM. Multiple injections (6, 12, and 24 mug/day for 4 days) induced dose-dependent decreases (38, 63, and 75%, respectively) in renal abundance of 1alpha-hydroxylase mRNA (P < 0.05). To determine whether FGF-23(R176Q) exerts a direct action on 1alpha-hydroxylase gene expression, we examined its effects in cultured human (HKC-8) and mouse (MCT) renal proximal tubule cells. FGF-23(R176Q) (1 to 10 ng/ml) induced a dose-dependent decrease in 1alpha-hydroxylase mRNA with a maximum suppression of 37% (P < 0.05). Suppression was detectable after 6 h of exposure and maximal after <em>21</em> h. In MCT cells, FGF-23(R176Q) suppressed 1alpha-hydroxylase mRNA and activated the ERK1/2 signaling pathway. The MAPK inhibitor PD98059 effectively abolished FGF-23-induced suppression of 1alpha-hydroxylase mRNA by blocking signal transduction via ERK1/2. These novel findings provide evidence that FGF-23 directly regulates renal 1alpha-hydroxylase gene expression via activation of the ERK1/2 signaling pathway.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a peptide hormone that is synthesized by several organs and regulates energy homeostasis. Excitement surrounding this relatively recently identified hormone is based on the documented metabolic beneficial effects of FGF<em>21</em>, which include weight loss and improved glycemia. The biology of FGF<em>21</em> is intrinsically complicated owing to its diverse metabolic functions in multiple target organs and its ability to act as an autocrine, paracrine, and endocrine <em>factor</em>. In the liver, FGF<em>21</em> plays an important role in the regulation of fatty acid oxidation both in the fasted state and in mice consuming a high-fat, low-carbohydrate ketogenic diet. FGF<em>21</em> also regulates fatty acid metabolism in mice consuming a diet that promotes hepatic lipotoxicity. In white adipose tissue (WAT), FGF<em>21</em> regulates aspects of glucose metabolism, and in susceptible WAT depots, it can cause browning. This peptide is highly expressed in the pancreas, where it appears to play an anti-inflammatory role in experimental pancreatitis. It also has an anti-inflammatory role in cardiac muscle. Although typically not expressed in skeletal muscle, FGF<em>21</em> is induced in situations of muscle stress, particularly mitochondrial myopathies. FGF<em>21</em> has been proposed as a novel therapeutic for metabolic complications such as diabetes and fatty liver disease. This review aims to interpret and delineate the ever-expanding complexity of FGF<em>21</em> physiology.

Induced human <em>fibroblasts</em> produce several mRNAs encoding interferon (IFN) activity. We previously cloned cDNA for a 1.3-kb RNA designated IFN-beta 2 and distinct from the 0.9-kb IFN-beta 1 mRNA. In vitro transcription--translation mapping of the full-length IFN-beta 2 cDNA sequence, shows that it encodes a 23.7-kd protein of <em>21</em>2 amino acids. This cDNA, fused to the SV40 early gene promoter, was transfected and amplified in Chinese hamster ovary cells and clones were obtained which constitutively produce human interferon activity. Two IFN-beta 2 genomic clones were isolated and their expression in hamster and mouse cells also produces biologically active rIFN-beta 2. Specific immunoassays show that IFN-beta 2 secreted by DNA-transformed rodent cells is a processed <em>21</em>-kd protein, whose activity is cross-neutralized by antibodies to human IFN-beta 1 but not to IFN-alpha or gamma. The immunoassay also demonstrates the induction of IFN-beta 2 secretion by <em>fibroblasts</em> in response to <em>growth</em>-regulatory cytokines, such as interleukin-1 and tumor necrosis <em>factor</em>. The function of this IFN-beta 2 as an autoregulatory inhibitor of cell <em>growth</em> is discussed.

OBJECTIVE

MicroRNAs are a class of small ribonucleotides regulating gene/protein targets by transcript degradation or translational inhibition. Transforming <em>growth</em> <em>factor</em>-β (TGF-β) is involved in cardiac fibrosis partly by stimulation of endothelial-to-mesenchymal transition (EndMT). Here, we investigated whether microRNA (miR)-<em>21</em>, a microRNA enriched in <em>fibroblasts</em> and involved in general fibrosis, has a role in cardiac EndMT.

RESULTS

TGF-β treatment of endothelial cells significantly increased miR-<em>21</em> expression and induced EndMT characterized by suppression of endothelial and increase of fibroblast markers. Overexpression of miR-<em>21</em> alone also stimulated EndMT. Importantly, miR-<em>21</em> blockade by transfection of specific microRNA inhibitors partly prevented TGF-β-induced EndMT. Mechanistically, miR-<em>21</em> silenced phosphatase and tensin homolog in endothelial cells, resulting in activation of the Akt-pathway. Akt inhibition partly restored TGF-β-mediated loss of endothelial markers during EndMT. In vivo, pressure overload of the left ventricle led to increased expression of miR-<em>21</em> in sorted cardiac endothelial cells, which displayed molecular and phenotypic signs of EndMT. This was attenuated by treatment of mice subjected to left ventricular pressure overload with an antagomir against miR-<em>21</em>.

CONCLUSIONS

TGF-β-mediated EndMT is regulated at least in part by miR-<em>21</em> via the phosphatase and tensin homolog/Akt pathway. In vivo, antifibrotic effects of miR-<em>21</em> antagonism are partly mediated by blocking EndMT under stress conditions.

BACKGROUND

Muscle biopsy is the gold standard for diagnosis of mitochondrial disorders because of the lack of sensitive biomarkers in serum. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>) is a <em>growth</em> <em>factor</em> with regulatory roles in lipid metabolism and the starvation response, and concentrations are raised in skeletal muscle and serum in mice with mitochondrial respiratory chain deficiencies. We investigated in a retrospective diagnostic study whether FGF-<em>21</em> could be a biomarker for human mitochondrial disorders.

METHODS

We assessed samples from adults and children with mitochondrial disorders or non-mitochondrial neurological disorders (disease controls) from seven study centres in Europe and the USA, and recruited healthy volunteers (healthy controls), matched for age where possible, from the same centres. We used ELISA to measure FGF-<em>21</em> concentrations in serum or plasma samples (abnormal values were defined as >200 pg/mL). We compared these concentrations with values for lactate, pyruvate, lactate-to-pyruvate ratio, and creatine kinase in serum or plasma and calculated sensitivity, specificity, and positive and negative predictive values for all biomarkers.

RESULTS

We analysed serum or plasma from 67 patients (41 adults and 26 children) with mitochondrial disorders, 34 disease controls (22 adults and 12 children), and 74 healthy controls. Mean FGF-<em>21</em> concentrations in serum were 820 (SD 1151) pg/mL in adult and 1983 (1550) pg/mL in child patients with respiratory chain deficiencies and 76 (58) pg/mL in healthy controls. FGF-<em>21</em> concentrations were high in patients with mitochondrial disorders affecting skeletal muscle but not in disease controls, including those with dystrophies. In patients with abnormal FGF-<em>21</em> concentrations in serum, the odds ratio of having a muscle-manifesting mitochondrial disease was 132·0 (95% CI 38·7-450·3). For the identification of muscle-manifesting mitochondrial disease, the sensitivity was 92·3% (95% CI 81·5-97·9%) and specificity was 91·7% (84·8-96·1%). The positive and negative predictive values for FGF-<em>21</em> were 84·2% (95% CI 72·1-92·5%) and 96·1 (90·4-98·9%). The accuracy of FGF-<em>21</em> to correctly identify muscle-manifesting respiratory chain disorders was better than that for all conventional biomarkers. The area under the receiver-operating-characteristic curve for FGF-<em>21</em> was 0·95; by comparison, the values for other biomarkers were 0·83 lactate (p=0·037, 0·83 for pyruvate (p=0·015), 0·72 for the lactate-to-pyruvate ratio (p=0·0002), and 0·77 for creatine kinase (p=0·013).

CONCLUSIONS

Measurement of FGF-<em>21</em> concentrations in serum identified primary muscle-manifesting respiratory chain deficiencies in adults and children and might be feasible as a first-line diagnostic test for these disorders to reduce the need for muscle biopsy.

BACKGROUND

Sigrid Jusélius Foundation, Jane and Aatos Erkko Foundation, Molecular Medicine Institute of Finland, University of Helsinki, Helsinki University Central Hospital, Academy of Finland, Novo Nordisk, Arvo and Lea Ylppö Foundation.