Transplant-related lung fibrosis is characterized by excessive fibro-collagenous deposition. Induction of arginase, an enzyme that metabolizes L-arginine to urea and L-ornithine, is vital for collagen synthesis. Pirfenidone is an investigational anti-fibrotic agent shown to be effective in blocking pulmonary fibrosis. The purpose of this study was to determine if pirfenidone was protective against the development of fibro-collagenous injury in rat lung orthotopic transplants through altering L-arginine-arginase metabolic pathways. Lung transplants were performed using Lewis donors and Sprague-Dawley recipients (allografts) or the same strain (isografts). Recipients were given pirfenidone (0.5% chow) 1-<em>21</em>-day post-transplantation. A significantly increased peak airway pressure (PawP) with excessive collagen deposition was found in untreated lung allografts. Pirfenidone treatment decreased PawP and collagen content in lung allografts. The beneficial effects were associated with downregulation of arginase protein expression and activity. In addition, pirfenidone decreased endogenous transforming <em>growth</em> <em>factor</em> (TGF)-beta level in lung allografts, and TGF-beta stimulated arginase activity in a dose-dependent manner in both lung tissue and <em>fibroblasts</em>. These results suggest that pirfenidone inhibits local arginase activity possibly through suppression of endogenous TGF-beta, hence, limiting the development of fibrosis in lung allografts.

BACKGROUND

We previously described increased levels of <em>growth</em> and differentiation <em>factor</em> 15 (GDF-15) in skeletal muscle and serum of patients with mitochondrial diseases. Here we evaluated GDF-15 as a biomarker for mitochondrial diseases affecting children and compared it to <em>fibroblast</em>-<em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>). To investigate the mechanism of GDF-15 induction in these pathologies we measured its expression and secretion in response to mitochondrial dysfunction.

METHODS

We analysed 59 serum samples from 48 children with mitochondrial disease, 19 samples from children with other neuromuscular diseases and 33 samples from aged-matched healthy children. GDF-15 and FGF-<em>21</em> circulating levels were determined by ELISA.

RESULTS

Our results showed that in children with mitochondrial diseases GDF-15 levels were on average increased by 11-fold (mean 4046pg/ml, 1492 SEM) relative to healthy (350, <em>21</em>) and myopathic (350, 32) controls. The area under the curve for the receiver-operating-characteristic curve for GDF-15 was 0.82 indicating that it has a good discriminatory power. The overall sensitivity and specificity of GDF-15 for a cut-off value of 550pg/mL was 67.8% (54.4%-79.4%) and 92.3% (81.5%-97.9%), respectively. We found that elevated levels of GDF-15 and or FGF-<em>21</em> correctly identified a larger proportion of patients than elevated levels of GDF-15 or FGF-<em>21</em> alone. GDF-15, as well as FGF-<em>21</em>, mRNA expression and protein secretion, were significantly induced after treatment of myotubes with oligomycin and that levels of expression of both <em>factor</em>s significantly correlated.

CONCLUSIONS

Our data indicate that GDF-15 is a valuable serum quantitative biomarker for the diagnosis of mitochondrial diseases in children and that measurement of both GDF-15 and FGF-<em>21</em> improves the disease detection ability of either <em>factor</em> separately. Finally, we demonstrate for the first time that GDF-15 is produced by skeletal muscle cells in response to mitochondrial dysfunction and that its levels correlate in vitro with FGF-<em>21</em> levels.

Co-expression of several members of the matrix metalloproteinase (MMP) family is characteristic of human malignant tumours. To investigate the role of stromelysin-2 (MMP-10) in <em>growth</em> and invasion of skin tumours, we studied cutaneous carcinomas with high metastatic capacity (squamous cell carcinomas, SCCs), only locally destructive tumours (basal cell carcinomas, BCCs) and pre-malignant lesions (Bowen's disease and actinic keratosis) using in situ hybridization. Expression of MMP-10 was compared with that of stromelysin-1 (MMP-3) and of MT1-MMP, the expression of which has been shown to correlate with tumour invasiveness. MMP-10 was expressed in 13/<em>21</em> SSCs and 11/19 BCCs only in epithelial laminin-5 positive cancer cells, while premalignant lesions were entirely negative. MT1-MMP mRNA was detected in 19/<em>21</em> SCCs both in epithelial cancer cells and stromal <em>fibroblasts</em> and in 14/18 BCCs only in <em>fibroblasts</em>. The level of MMP-10 was upregulated in a cutaneous SCC cell line (UT-SCC-7) by transforming <em>growth</em> <em>factor</em>-alpha and keratinocyte <em>growth</em> <em>factor</em>, and by interferon-gamma in combination with transforming <em>growth</em> <em>factor</em>-beta1 and tumour necrosis <em>factor</em>-alpha both in UT-SCC-7 and HaCaT cells. Our results show that MMP-10 expression does not correlate with the invasive behaviour of tumours as assessed by their histology and MT1-MMP expression, but may be induced by the wound healing and inflammatory matrix remodelling events associated with skin tumours.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a metabolic regulator of glucose and lipid metabolism. The physiological role of FGF<em>21</em> is not yet fully elucidated, however, administration of FGF<em>21</em> lowers blood glucose in diabetic animals. Moreover, increased levels of FGF<em>21</em> are found in obese and diabetic rodents and humans compared with lean/non-diabetic controls.

METHODS

Adult male rhesus macaque monkeys were chronically maintained on a high-fat diet (HFD) or a standard diet (control, CTR). Plasma levels of FGF<em>21</em>, triglycerides and cholesterol were measured and body weight was record. Glucose-stimulated insulin secretion (GSIS) and glucose clearance was determined during an intravenous glucose tolerance test. Furthermore, expression of FGF<em>21</em> and its receptors were determined in liver, pancreas, three white adipose tissues (WATs) and two skeletal muscles.

RESULTS

A cohort of the high-fat fed monkeys responded to the HFD with increasing body weight, plasma lipids, total cholesterol, GSIS and decreased glucose tolerance. These monkeys were termed HFD sensitive. Another cohort of monkeys did not become obese and maintained normal insulin sensitivity. These animals were defined as HFD resistant. Plasma FGF<em>21</em> levels were significantly increased in all HFD fed monkeys compared with the CTR group. The HFD-sensitive monkeys showed a significant increase in FGF<em>21</em> mRNA expression in all examined tissues compared with CTR, whereas FGF<em>21</em> expression in the HFD-resistant group was only increased in the liver, pancreas and the retroperitoneal WAT. In the WAT, the co-receptor β-klotho was downregulated in the HFD-sensitive monkeys compared with the HFD-resistant group.

CONCLUSIONS

This study demonstrates that HFD changes FGF<em>21</em> and FGF<em>21</em> receptor expression in a tissue-specific manner in rhesus monkeys; differential regulation is moreover observed between HFD-sensitive and -resistant monkeys. Monkeys that maintain normal levels of the FGF<em>21</em> co-receptor β-klotho in the WAT on HFD were protected toward development of dyslipidemia and hyperglycemia.

BACKGROUND

Fenofibrate is a peroxisome proliferator-activated receptor (PPAR)-α agonist that showed beneficial effects on total cardiovascular risk in patients with type 2 diabetes in the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study.

OBJECTIVE

This study aimed to investigate the long-term effect of fenofibrate therapy on three novel biomarkers of cardiovascular risk, namely adipocyte-fatty acid-binding protein (A-FABP), <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), and retinol-binding protein 4 (RBP4), which are all downstream targets of PPAR-α or PPAR-γ, in patients with type 2 diabetes.

METHODS

A total of <em>21</em>6 patients (108 in the fenofibrate group and 108 in the placebo group) were randomly selected from the FIELD study cohort. A-FABP, FGF<em>21</em>, and RBP4 levels were measured in serum samples at both baseline and the fifth year of the study.

RESULTS

Relative to the placebo group, the changes of serum FGF<em>21</em> and RBP4 levels were 85% (P < 0.001) and 10% (P = 0.032) higher in the fenofibrate group, respectively, over 5 yr. Fenofibrate treatment had no detectable effect on serum A-FABP level (P>> 0.05). The effect of fenofibrate treatment on serum FGF<em>21</em>, but not RBP4, remained significant after adjusting for fenofibrate-induced changes in glycosylated hemoglobin, total cholesterol, triglycerides, apolipoprotein A-II, fibrinogen, plasma creatinine, and homocysteine (P = 0.002).

CONCLUSIONS

Long-term fenofibrate treatment could increase serum FGF<em>21</em> levels over 5 yr in patients with type 2 diabetes. Additional studies are needed to investigate the potential role of FGF<em>21</em> in the fenofibrate-mediated reduction of cardiovascular risk.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is an endocrine hormone derived from the liver that exerts pleiotropic effects on the body to maintain overall metabolic homeostasis. During the past decade, there has been an enormous effort made to understand the physiological roles of FGF<em>21</em> in regulating metabolism and to identify the mechanism for its potent pharmacological effects to reverse diabetes and obesity. Through both human and rodent studies, it is now evident that FGF<em>21</em> levels are dynamically regulated by nutrient sensing, and consequently FGF<em>21</em> functions as a critical regulator of nutrient homeostasis. In addition, recent studies using new genetic and molecular tools have provided critical insight into the actions of this endocrine <em>factor</em>. This review examines the numerous functions of FGF<em>21</em> and highlights the therapeutic potential of FGF<em>21</em>-targeted pathways for treating metabolic disease.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>21</em> is a novel regulator of insulin-independent glucose transport in 3T3-L1 adipocytes and has glucose and triglyceride lowering effects in rodent models of diabetes. In this study, we found that FGF-<em>21</em> can significantly attenuate ischemia-reperfusion (I/R) induced damage in H9c2 cells (rat heart). However, it is unclear which signal transduction pathway is involved in the cardioprotective effect of FGF-<em>21</em>. Thus, this study was designed to investigate the potential mechanism induced by FGF-<em>21</em>. The results showed that FGF-<em>21</em> treatment prevented the oxidative stress and apoptosis associated with I/R damage by reducing the levels of superoxide anions, inhibiting glycogen synthase kinase (GSK) 3β by activating Akt phosphorylation, and recovering the levels of ATP synthase pyruvate kinase isozymes M1 and protein kinase C, thereby improving energy supply. In summary, we conclude that FGF-<em>21</em> protects H9c2 cells against I/R injury mainly through the Akt-GSK-3β-caspase-3 dependent pathway, preventing oxidative stress, and recovery of the energy supply.

OBJECTIVE

Circulating <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is an important auto- and endocrine player with beneficial metabolic effects on obesity and diabetes. In humans, thermogenic brown adipose tissue (BAT) was recently suggested as a source of FGF<em>21</em> secretion during cold exposure. Here, we aim to clarify the role of UCP1 and ambient temperature in the regulation of FGF<em>21</em> in mice.

METHODS

Wildtype (WT) and UCP1-knockout (UCP1 KO) mice, the latter being devoid of BAT-derived non-shivering thermogenesis, were exposed to different housing temperatures. Plasma metabolites and FGF<em>21</em> levels were determined, gene expression was analyzed by qPCR, and tissue histology was performed with adipose tissue.

RESULTS

At thermoneutrality, FGF<em>21</em> gene expression and serum levels were not different between WT and UCP1 KO mice. Cold exposure led to highly increased FGF<em>21</em> serum levels in UCP1 KO mice, which were reflected in increased FGF<em>21</em> gene expression in adipose tissues but not in liver and skeletal muscle. Ex vivo secretion assays revealed FGF<em>21</em> release only from BAT, progressively increasing with decreasing ambient temperatures. In association with increased FGF<em>21</em> serum levels in the UCP1 KO mouse, typical FGF<em>21</em>-related serum metabolites and inguinal white adipose tissue morphology and thermogenic gene expression were altered.

CONCLUSIONS

Here we show that the genetic ablation of UCP1 increases FGF<em>21</em> gene expression in adipose tissue. The removal of adaptive nonshivering thermogenesis renders BAT a significant source of endogenous FGF<em>21</em> under thermal stress. Thus, the thermogenic competence of BAT is not a requirement for FGF<em>21</em> secretion. Notably, high endogenous FGF<em>21</em> levels in UCP1-deficient models and subjects may confound pharmacological FGF<em>21</em> treatments.

OBJECTIVE

The aim of this study was to examine the effects of FGF-<em>21</em> over- and underexpression on glucose and lipid metabolism in hepatocytes and adipocytes.

METHODS

FGF-<em>21</em> over-expressive vectors (pcDNA-FGF-<em>21</em>) and FGF-<em>21</em> shRNA-expressing vectors (pGenesil-FGF-<em>21</em>) were transfected into Hepa1-6 hepatocyte and 3T3-L1 adipocyte. The levels of FGF-<em>21</em> in the incubation medium were measured by ELISA. FGF-<em>21</em> protein levels by Western blot analysis, and glucose uptake rates (GUR) by measuring 2-deoxy-d-glucose uptake. The mRNA expression of transcription factors were determined by real-time quantitative PCR.

RESULTS

Transfection of pcDNA-FGF-<em>21</em> significantly increased FGF-<em>21</em> expression in both Hepa1-6 hepatocytes and 3T3-L1 adipocytes (4.8- and 4.2-fold, respectively p<0.05), while transfection of pGenesil-FGF-<em>21</em> significantly decreased FGF-<em>21</em> expressions by 86% and 78%, respectively (p<0.05). In 3T3-L1 adipocytes, the up-regulation of FGF-<em>21</em>markedly increased GUR, decreased intracellular triglyceride (TG) content, up-regulated β-klotho, FGFR1, GLUT-1, IRS-1, ATGL, HSL, and ap2 mRNA expressions (p<0.05). Opposite changes occurred in FGF-<em>21</em> knockdown adipocytes, except for IRS-1. In hepatocyte, FGF-<em>21</em> up-regulation reduced HMGR and PEPCK mRNA expression and increased β-klotho, FGFR4 and LDLr expression (p<0.05), whereas down-regulation had the opposite effects.

CONCLUSIONS

These data suggest that both FGF-<em>21</em> knockdown and over-expression led to changes in lipid levels, GUR and transcription factors involved in glucose and lipid metabolism, but that the mechanisms of FGF-<em>21</em> actions in adipocytes and hepatocytes might be different.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is an emerging metabolic regulator associated with glucose and lipid metabolism. However, previous studies of FGF<em>21</em> have been largely confounded by obesity, and data are limited for advanced outcomes such as coronary artery disease (CAD) and ectopic fat accumulation. We investigated the associations between serum FGF<em>21</em> concentrations and glucose/lipid metabolism, CAD, and pericardial fat deposition in subjects strictly matched for obesity parameters.

METHODS

We enrolled 189 patients who had undergone cardiac multidetector coronary computed tomography. We measured cardiometabolic parameters and serum FGF<em>21</em> levels within body mass index (BMI)-matched groups. Correlations and linear regressions were analysed among serum FGF<em>21</em> levels, pericardial fat volumes and cardiometabolic parameters. Serum FGF<em>21</em> concentrations were compared in patients with and without diabetes, metabolic syndrome (MS) or CAD.

RESULTS

Serum FGF<em>21</em> concentrations were significantly higher in BMI-matched patients with MS (107·2 ± 83·6 vs 82·1 ± 67·4 ng/l without MS, P < 0·05), but not among those with diabetes (84·3 ± 56·4 vs 96·3 ± 98·9 ng/l without diabetes, P = 0·300) or CAD (89·6 ± 65·8 vs 84·2 ± 83·1 ng/l without CAD, P = 0·633). Serum FGF<em>21</em> concentrations correlated positively with triglycerides, low-density lipoprotein-cholesterol, insulin, HOMA-IR and pericardial fat volume. They showed an independent association with pericardial fat volume (β = 0·111 ± 0·053, P < 0·05).

CONCLUSIONS

Serum FGF<em>21</em> concentrations were significantly associated with lipid profiles, insulin resistance, pericardial fat volume and MS, independently of obesity, but not with overt CAD or diabetes.

Both 17beta-estradiol (E2) and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2) stimulate angiogenesis and endothelial cell migration and proliferation. The first goal of this study was to explore the potential link between this hormone and this <em>growth</em> <em>factor</em>. E2-stimulated angiogenesis in SC Matrigel plugs in Fgf2+/+ mice, but not in Fgf2-/- mice. Cell cultures from subcutaneous Matrigel plugs demonstrated that E2 increased both migration and proliferation in endothelial cells from Fgf2+/+ mice, but not from in Fgf2-/- mice. Several isoforms of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2) are expressed: the low molecular weight 18-kDa protein (FGF2lmw) is secreted and activates tyrosine kinase receptors (FGFRs), whereas the high molecular weight (<em>21</em> and 22 kDa) isoforms (FGF2hmw) remains intranuclear, but their role is mainly unknown. The second goal of this study was to explore the respective roles of FGF2 isoforms in the effects of E2. We thus generated mice deficient only in the FGF2lmw (Fgf2lmw-/-). E2 stimulated in vivo angiogenesis and in vitro migration in endothelial cells from Fgf2lmw-/- as it did in Fgf2+/+ mice. E2 increased FGF2hmw protein abundance in endothelial cell cultures from Fgf2+/+ and Fgf2lmw-/- mice. As shown using siRNA transfection, these effects were FGFR independent but involved FGF2-Interacting <em>Factor</em>, an intracellular FGF2hmw partner. This is the first report for a physiological role for the intracellular FGF2hmw found to mediate the effect of E2 on endothelial cell migration via an intracrine action.

BACKGROUND

Angiogenesis is essential for the growth of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Angiogenesis is regulated by angiogenic factors such as vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) 1, 2, and 3 known to be located on vascular endothelial cells (VECs). We hypothesize that VEGFRs are also expressed on HNSCC tumor cells in vitro and in vivo and likely control tumor function in vivo.

METHODS

Immunohistochemical analysis for VEGFR-1 (n = 13), VEGFR-2 (n = 21), and VEGFR-3 (n = 16) was performed on human HNSCC tumor samples. Specimens were analyzed for receptor expression and staining intensity. A cultured oral SCC cell line (SCC-25) and a pharyngeal SCC cell line (FADU) were also studied for receptor expression.

RESULTS

The HNSCC tumor cells expressed VEGFR-1, VEGFR-2, and VEGFR-3 in all specimens evaluated. Staining for all 3 receptors was also found on tumor-associated macrophages and fibroblasts, except that VEGFR-2 was not present on fibroblasts. Staining intensity for VEGFR-1 and VEGFR-2 was significantly higher in tumor cells and macrophages than in VECs stained for the same receptor. Both cultured HNSCC cell lines demonstrated expression of all 3 receptors.

CONCLUSIONS

This represents the first report of all 3 VEGFRs being expressed by HNSCC cells. These findings indicate that VEGF may be an autocrine regulator of tumor cell activity in addition to its known angiogenic effects on VECs. The presence of VEGFRs on tumor-associated macrophages and fibroblasts contributes to the complexity of the VEGF/VEGFR system in human cancer.

BACKGROUND

Lipotoxicity is a key feature of the pathogenesis of diabetic kidney disease, and is attributed to excessive lipid accumulation (hyperlipidemia). Increasing evidence suggests that <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>21</em> has a crucial role in lipid metabolism under diabetic conditions.

OBJECTIVE

The present study investigated whether FGF<em>21</em> can prevent hyperlipidemia- or diabetes-induced renal damage, and if so, the possible mechanism.

METHODS

Mice were injected with free fatty acids (FFAs, 10 mg/10 g body weight) or streptozotocin (150 mg/kg) to establish a lipotoxic model or type 1 diabetic model, respectively. Simultaneously the mice were treated with FGF<em>21</em> (100 µg/kg) for 10 or 80 days. The kidney weight-to-tibia length ratio and renal function were assessed. Systematic and renal lipid levels were detected by ELISA and Oil Red O staining. Renal apoptosis was examined by TUNEL assay. Inflammation, oxidative stress, and fibrosis were assessed by Western blot.

RESULTS

Acute FFA administration and chronic diabetes were associated with lower kidney-to-tibia length ratio, higher lipid levels, severe renal apoptosis and renal dysfunction. Obvious inflammation, oxidative stress and fibrosis also observed in the kidney of both mice models. Deletion of the fgf<em>21</em> gene further enhanced the above pathological changes, which were significantly prevented by administration of exogenous FGF<em>21</em>.

CONCLUSIONS

These results suggest that FFA administration and diabetes induced renal damage, which was further enhanced in FGF<em>21</em> knock-out mice. Administration of FGF<em>21</em> significantly prevented both FFA- and diabetes-induced renal damage partially by decreasing renal lipid accumulation and suppressing inflammation, oxidative stress, and fibrosis.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a recently discovered metabolic regulator. Interestingly, FGF<em>21</em> is also known to inhibit Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) signaling from the GH receptor in the liver, where FGF<em>21</em> mRNA is predominantly expressed. In this study, we tested the hypothesis that FGF<em>21</em> gene expression in the liver is controlled by GH through STAT5. We found that GH injection to cattle increased FGF<em>21</em> mRNA expression in the liver. Mapped by a 5'-rapid amplification of cDNA ends assay, transcription of the FGF<em>21</em> gene in the bovine liver was mainly initiated from a nucleotide 24 bp downstream of a TATA box. The bovine FGF<em>21</em> promoter contains three putative STAT5-binding sites. EMSA confirmed the ability of them to bind to liver STAT5 protein from GH-injected cattle. Chromatin immunoprecipitation assays demonstrated that GH administration increased the binding of STAT5 to the FGF<em>21</em> promoter in the liver. Cotransfection analyses showed that GH induced reporter gene expression from the FGF<em>21</em> promoter in a STAT5-dependent manner. GH also stimulated FGF<em>21</em> mRNA expression in cultured mouse hepatocytes. These data together indicate that GH directly stimulates FGF<em>21</em> gene transcription in the liver, at least in part, through STAT5. This finding, together with the fact that FGF<em>21</em> inhibits GH-induced JAK2-STAT5 signaling in the liver, suggests a novel negative feedback loop that prevents excessive JAK2-STAT5 signaling from the GH receptor in the liver.

Autophagy is essential for cellular survival and energy homeostasis under nutrient deprivation. Despite the emerging importance of nuclear events in autophagy regulation, epigenetic control of autophagy gene transcription remains unclear. Here, we report fasting-induced <em>Fibroblast</em> <em>Growth</em> <em>Factor</em>-<em>21</em> (FGF<em>21</em>) signaling activates hepatic autophagy and lipid degradation via Jumonji-D3 (JMJD3/KDM6B) histone demethylase. Upon FGF<em>21</em> signaling, JMJD3 epigenetically upregulates global autophagy-network genes, including Tfeb, Atg7, Atgl, and Fgf<em>21</em>, through demethylation of histone H3K27-me3, resulting in autophagy-mediated lipid degradation. Mechanistically, phosphorylation of JMJD3 at Thr-1044 by FGF<em>21</em> signal-activated PKA increases its nuclear localization and interaction with the nuclear receptor PPARα to transcriptionally activate autophagy. Administration of FGF<em>21</em> in obese mice improves defective autophagy and hepatosteatosis in a JMJD3-dependent manner. Remarkably, in non-alcoholic fatty liver disease patients, hepatic expression of JMJD3, ATG7, LC3, and ULK1 is substantially decreased. These findings demonstrate that FGF<em>21</em>-JMJD3 signaling epigenetically links nutrient deprivation with hepatic autophagy and lipid degradation in mammals.

Diabetes and associated metabolic conditions have reached pandemic proportions worldwide, and there is a clear unmet medical need for effective and safe therapies. <em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>21</em> is an atypical member of the FGF family. The ability of FGF<em>21</em> to normalize glucose, lipid and energy homeostasis has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. Many different engineering approaches have successfully improved the plasma half life, protein stability and solubility, as well as 'manufacturability' of FGF<em>21</em>. Novel approaches such as agonist antibodies to FGF<em>21</em> receptor complexes have opened new opportunities previously unavailable. This review summarizes recent advances in understanding the functions, target tissues and receptors for FGF<em>21</em>. Furthermore, it provides an up-to-date appraisal of the approaches on therapeutic development targeting this pathway.

The conversion of white adipose to the highly thermogenic beige adipose tissue has been proposed as a potential strategy to counter the unfavorable consequences of obesity. Three regulators of this conversion have recently emerged but information regarding their control is limited, and contradictory. We present two studies examining the control of these regulators. Study 1: In 10 young men, the plasma concentrations of irisin and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) were determined prior to and during activation of the sympathetic nervous system via hypoxic gas breathing (FIO2 = 0.11). The measurements were performed twice, once with and once without prior/concurrent sympathetic inhibition via transdermal clonidine administration. FGF<em>21</em> was unaffected by basal sympathetic inhibition (338±113 vs. 295±80 pg/mL; P = 0.43; mean±SE), but was increased during hypoxia mediated sympathetic activation (368±135); this response was abrogated (P = 0.035) with clonidine (269±93). Irisin was unaffected by sympathetic inhibition and/or hypoxia (P>0.<em>21</em>). Study 2: The plasma concentration of irisin and FGF<em>21</em>, and the skeletal muscle protein content of fibronectin type III domain containing 5 (FNDC5) was determined in 19 young adults prior to and following three weeks of sprint interval training (SIT). SIT decreased FGF<em>21</em> (338±78 vs. 251±36; P = 0.046) but did not affect FNDC5 (P = 0.79). Irisin was decreased in males (127±18 vs. 90±23 ng/mL; P = 0.045) and increased in females (139±14 vs. 170±18). Collectively, these data suggest a potential regulatory role of acute sympathetic activation pertaining to the browning of white adipose; further, there appears to be a sexual dimorphic response of irisin to SIT.

Mesenchymal stem cells (MSCs) are used clinically in tissue engineering and regenerative medicine. The proliferation and osteogenic differentiation potential of MSCs vary according to factors such as tissue source and cell population heterogeneity. Dental tissue has received attention as an easily accessible source of high-quality stem cells. In this study, we compared the in vitro characteristics of dental pulp stem cells from deciduous teeth (SHED), human dental pulp stem cells (hDPSCs), and human bone marrow mesenchymal stem cells (hBMSCs).

SEHD and hDPSCs were isolated from dental pulp and analyzed in comparison with human bone marrow (hBM)MSCs. Proliferative capacity of cultured cells was analyzed using a bromodeoxyuridine immunoassay and cell counting. Alkaline phosphatase (ALP) levels were monitored to assess osteogenic differentiation. Mineralization was evaluated by alizarin red staining. Levels of bone marker mRNA were examined by real-time PCR analysis.

SHED were highly proliferative compared with hDPSCs and hBMSCs. SHED, hDPSCs, and hBMSCs exhibited dark alizarin red staining on day 21 after induction of osteogenic differentiation, and staining of hBMSCs was significantly higher than that of SHED and hDPSCs by spectrophotometry. ALP staining was stronger in hBMSCs compared with SHED and hDPSCs, and ALP activity was significantly higher in hBMSCs compared with SHED or hDPSCs. SHED showed significantly higher expression of the Runx2 and ALP genes compared with hBMSCs, based on real-time PCR analysis. In bFGF, SHED showed significantly higher expression of the basic fibroblast growth factor (bFGF) gene compared with hDPSCs and hBMSCs.

SHED exhibited higher proliferative activity and levels of bFGF and BMP-2 gene expression compared with BMMSCs and DPSCs. The ease of harvesting cells and ability to avoid invasive surgical procedures suggest that SHED may be a useful cell source for application in bone regeneration treatments.

We tested the hypothesis that early vascularization of the embryonic heart is enhanced after bolus injections of vascular, endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) into the vitelline vein before the onset of myocardial vasculogenesis (3.5 days, stage <em>21</em>). Electron and light microscopy were utilized to obtain morphometric data. At stages 29 and 31, myocardial vessel volume or numerical density were higher in embryos injected with 50 ng bFGF than in the saline-injected controls. A VEGF injection increased vascular volume density at stage 29 and both volume and numerical, density at stage 31, bFGF, but not VEGF, was associated with an enhancement of the sinusoidal system (spongy layer of the ventricle) at stage 29. This effect disappeared by stage 31. In conclusion, 1) enhancement of bFGF or VEGF before myocardial vascularization increases vascular <em>growth</em>, but the initial effect of bFGF is greater; 2) the effects of these <em>growth</em> <em>factors</em> on vascular volume and numerical density are temporally dependent; and 3) bFGF, in addition to its effects on the coronary vasculature, influences ventricular modeling by apparently acting on myocytes as well as endothelial cells.

BACKGROUND

Liraglutide is a glucagon-like peptide-1 analogue that stimulates insulin secretion and improves β-cell function. However, it is not clear whether liraglutide achieves its glucose lowering effect only by its known effects or whether other as yet unknown mechanisms are involved. The aim of this study was to examine the effects of liraglutide on <em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF-<em>21</em>) activity in High-fat diet (HFD) fed ApoE(-/-) mice with adiponectin (Acrp30) knockdown.

METHODS

HFD-fed ApoE(-/-) mice were treated with adenovirus vectors expressing shAcrp30 to produce insulin resistance. Hyperinsulinemic-euglycemic clamp studies were performed to evaluate insulin sensitivity of the mouse model. QRT-PCR and Western blot were used to measure the mRNA and protein expression of the target genes.

RESULTS

The combination of HFD, ApoE deficiency, and hypoadiponectinemia resulted in an additive effect on insulin resistance. FGF-<em>21</em> mRNA expressions in both liver and adipose tissues were significantly increased while FGF-<em>21</em> receptor 1 (FGFR-1) and β-Klotho mRNA levels in adipose tissue, as well as FGFR-1-3 and β-Klotho mRNA levels in liver were significantly decreased in this model. Liraglutide treatment markedly improved insulin resistance and increased FGF-<em>21</em> expression in liver and FGFR-3 in adipose tissue, restored β-Klotho mRNA expression in adipose tissue as well as FGFR-1-3, β-Klotho levels and phosphorylation of FGFR1 up to the levels observed in control mice in liver. Liraglutide treatment also further increased FGF-<em>21</em> proteins in liver and plasma. In addition, as shown by hyperinsulinemic-euglycemic clamp, liraglutide treatment also markedly improved glucose metabolism and insulin sensitivity in these animals.

CONCLUSIONS

These findings demonstrate an additive effect of HFD, ApoE deficiency, and adiponectin knockdown on insulin resistance and unveil that the regulation of glucose metabolism and insulin sensitivity by liraglutide may be partly mediated via increased FGF-<em>21</em> and its receptors action.

The increase in the amount of airway smooth muscle in the bronchial wall associated with asthma is partly due to hyperplasia. It is therefore important to determine which <em>factors</em> regulate <em>growth</em> and especially proliferation. In this study, we describe the effect of interleukin-4 (IL-4), a mast cell- and T lymphocyte-derived cytokine, on human airway smooth muscle proliferation as determined by [3H]thymidine uptake in the presence of fetal bovine serum (FBS), platelet-derived <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and thrombin. IL-4 (5, 15, 50, and 150 ng/ml) significantly decreased 10% FBS-induced proliferation by 50, 73, 43, and 46%, respectively. The proliferative responses to platelet-derived <em>growth</em> <em>factor</em> (20 and 40 ng/ml), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (30 ng/ml), and thrombin (1 and 10 U/ml) were significantly reduced by 19, <em>21</em>, 37, 36, and 57% respectively in the presence of 50 ng/ml of IL-4. We investigated the effect of IL-4 and other known inhibitors of smooth muscle proliferation, namely PGE2, heparin, and forskolin, on intracellular cAMP concentrations. IL-4 (50 ng/ml) and heparin (100 U/ml) did not alter intracellular cAMP levels when cells were treated with 1 or 10% FBS. PGE2 (1 microM) and forskolin (10 microM) significantly increased cAMP concentration above the control value in nonproliferating cells (1% FBS treated) by 7- and 37-fold, respectively. The effect of IL-4 (50 ng/ml), PGE2 (1 microM), and forskolin (10 microM) on cyclin D1 protein expression in 10% FBS-stimulated human airway smooth muscle cells was also examined. PGE2 and forskolin did not significantly inhibit cyclin D1 expression. However, IL-4 decreased cyclin D1 expression by <em>21</em>%. These results provide evidence that IL-4 decreases human airway smooth muscle cell proliferation via a mechanism that is cAMP independent and mediated, in part, by a decrease in cyclin D1 protein expression.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroids, pancreatic islets, and anterior pituitary. We previously reported a basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF)-like substance in the plasma of subjects with MEN1. In the present study we used a novel sensitive specific 2-site immunoradiometric assay to test for bFGF in plasma. The assay employs immobilized affinity-purified N-terminal-specific anti-bFGF antibodies (antigen capture) and high affinity binding to radioiodinated heparin. bFGF-like immunoreactivity was undetectable (< 0.2 ng/mL) in normal subjects and in most unaffected relatives of MEN1 subjects. We found detectable bFGF ranging from 0.24-1.28 ng/mL in <em>21</em> of 50 subjects with MEN1. Seven of 8 MEN1 subjects with untreated pituitary tumors had detectable plasma bFGF-like immunoreactivity. Plasma bFGF-like immunoreactivity decreased after surgery for pituitary tumor in 4 patients and after initiation of bromocryptine therapy in 4 patients. bFGF was increased in the plasma of several subjects with sporadic endocrine disorders, including 3 with untreated or persistent acromegaly. We conclude that pituitary tumor is a possible source of high circulating bFGF immunoreactivity in MEN1 plasma.

Primary isolates of chondrocytes and mesenchymal stem cells are often insufficient for cell-based autologous grafting procedures, necessitating in vitro expansion of cell populations. However, the potential for expansion is limited by cellular senescence, a form of irreversible cell cycle arrest regulated by intrinsic and extrinsic <em>factors</em>. Intrinsic mechanisms common to most somatic cells enforce senescence at the so-called "Hayflick limit" of 60 population doublings. Termed "replicative senescence", this mechanism prevents cellular immortalization and suppresses oncogenesis. Although it is possible to overcome the Hayflick limit by genetically modifying cells, such manipulations are regarded as prohibitively dangerous in the context of tissue engineering. On the other hand, senescence associated with extrinsic <em>factors</em>, often called "stress-induced" senescence, can be avoided simply by modifying culture conditions. Because stress-induced senescence is "premature" in the sense that it can halt <em>growth</em> well before the Hayflick limit is reached, <em>growth</em> potential can be significantly enhanced by minimizing culture related stress. Standard culture techniques were originally developed to optimize the <em>growth</em> of <em>fibroblasts</em> but these conditions are inherently stressful to many other cell types. In particular, the <em>21</em>% oxygen levels used in standard incubators, though well tolerated by <em>fibroblasts</em>, appear to induce oxidative stress in other cells. We reasoned that chondrocytes and MSCs, which are adapted to relatively low oxygen levels in vivo, might be sensitive to this form of stress. To test this hypothesis we compared the <em>growth</em> of MSC and chondrocyte strains in <em>21</em>% and 5% oxygen. We found that incubation in <em>21</em>% oxygen significantly attenuated <em>growth</em> and was associated with increased oxidant production. These findings indicated that sub-optimal standard culture conditions sharply limited the expansion of MSC and chondrocyte populations and suggest that cultures for grafting purposes should be maintained in a low-oxygen environment.

Neurons can be specifically induced from bone marrow stromal cells (MSCs) with extremely high efficiency using gene transfection of the Notch intracellular domain and subsequent treatment with basic-<em>fibroblast</em> <em>growth</em> <em>factor</em>, forskolin, and ciliary neurotrophic <em>factor</em>. We investigated the behavioral and histologic efficacy of such bone marrow stromal cell-derived neuronal cell (MSDNC) transplantation into a focal cerebral infarction model in rats. A left middle cerebral artery occlusion (MCAO) was performed on adult Wistar rats. MSDNC transplantation into the ipsilateral hemisphere was performed on day 7 after MCAO. The behavioral analyses were conducted on days 14, <em>21</em>, 28, 35, and 36-40, and a histologic evaluation was performed on day 41. MSDNC-transplanted rats showed significant recovery compared with controls (MCAO without cell transplantation) in beam balance, limb placing, and Morris water maze tests. Histologically, transplanted cells migrated from the injection site into the ischemic boundary area, including the cortex, corpus callosum, striatum, and hippocampus. Transplanted MSDNCs were positive for MAP-2 (84% +/- 8.11%), whereas only a small number of cells were positive for GFAP (1.0% +/- 0.23%). The survival rates of MSDNCs and MSCs 1 month after transplantation were approximately 45% and 10%, respectively. These results suggest that use of MSDNCs may be a promising therapeutic strategy for cerebral infarction.