Our previous report has shown that FGF21 has anti-inflammatory properties in a collagen-induced arthritis (CIA) model. In this study, the underlying molecular mechanisms of action were also investigated using RAW 264.7 cells, a murine monocyte-macrophage. RAW 264.7 cells were pre-incubated with various concentrations (2000, 500, 100ng/ml) of FGF21 and stimulated with LPS to induce oxidative stress and inflammation. The result of flow cytometry showed that β-Klotho, FGF21 specific receptor, was expressed in murine splenic macrophages and RAW 264.7. In vitro, FGF21 reduced the expression of TNF-α, IL-1β, IL-6 and IFN-γ and increased the level of IL-10 in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophages. FGF21 also suppressed profound elevation of ROS production and oxidative stress, as evidenced by an increase of the MDA level and depletion of the intracellular GSH level, and restored the activities of antioxidant enzymes SOD and GSH-Px in LPS-stimulated RAW 264.7 macrophages. Moreover, FGF21 inhibited LPS-induced nuclear factor-κB (NF-κB) activation, including degradation of I-κB and nuclear translocation of p65. In addition, the result of Western blot and real-time PCR showed that FGF21 induced heme oxygenase-1 (HO-1) expression and increased the nuclear transcription factor-E2-related factor 2 (Nrf2) levels in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophages. In conclusion, the results suggest that macrophages are the targets for the anti-inflammatory effects of FGF21, and FGF21 exerted an anti-inflammatory effect mainly via enhancing Nrf2-mediated anti-oxidant capacity and suppressing NF-κB signaling pathway.

OBJECTIVE

Since fibroblast growth factor 19 (FGF-19) is a potent metabolic regulator that influences glucose and lipid homeostasis, our aim was to develop an ELISA assay for measuring FGF-19 in human serum and to investigate its concentrations in healthy volunteers and patients suffering from metabolic syndrome.

METHODS

A sandwich ELISA method was developed for quantitative determination of human FGF-19 in serum samples. Blood pressure, waist circumference, FGF-21 serum levels, serum cholesterol, triacylglycerols, HDL-cholesterol, LDL-cholesterol, insulin, glucose, adiponectin, uric acid, creatinine, hs-CRP and calculated BMI and Quicki insulin sensitivity index were measured in 153 healthy volunteers and 66 persons with metabolic syndrome.

RESULTS

Neither sex nor age influenced FGF-19 serum concentration in the healthy volunteers. Probands with metabolic syndrome had 65 % lower FGF-19 serum values than the healthy ones (medians 158.6 versus 242.4 ng/L; p<0.01). FGF-19 correlated with glucose (r = -0.35, p<0.01), HDL (r = 0.24, p = 0.045), triacylglycerols (r = -0.19, p = 0.05) and with a number of other risk factors for metabolic syndrome (r = -0.28, p = 0.01). When adjusted to the concentrations of triacylglycerols, BMI and glucose, and finally to all data pertinent to FGF-19 (according to correlation analysis), our data indicate that FGF-19 is an independent marker of metabolic syndrome.

CONCLUSIONS

The present study demonstrates the analytical properties of the ELISA FGF-19 assay and its usefulness when studying the metabolic syndrome. Serum concentrations of FGF-19 could be new key predictors of metabolic syndrome and thereby even a new negative risk factor of atherosclerosis.

Increased tonic contractile activity from exercise or electrical stimulation induces a variety of changes in skeletal muscle, including vascular <em>growth</em>, myoblast proliferation, and fast to slow fiber type conversion. Little is known about the cellular control of such changes, but pleiotropic biochemical modulators such as <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) may be involved in this response and thus may be regulated in response to such stimuli. We examined the regulation of FGF expression in an in vivo model of exercise conditioning previously shown to exhibit vascular <em>growth</em> and fast to slow fiber conversion. FGFs were extracted by heparin-affinity chromatography from extensor digitorum longus muscles of adult rabbits subjected to chronic motor nerve stimulation at 10 Hz. <em>Growth</em> <em>factor</em> activity (expressed in <em>growth</em> <em>factor</em> units [GFUs]) of muscle stimulated for 3 and <em>21</em> d was assayed by [3H]thymidine incorporation in 3T3 <em>fibroblasts</em> and compared with that present in the contralateral unstimulated muscle. A small increase in heparin-binding mitogenic activity was observed as early as 3 d of stimulation, and by <em>21</em> d mitogenic activity increased significantly when normalized to either wet weight (stimulated, 287 +/- 61 GFU/g; unstimulated, 145 +/- 39 GFU/g) or to protein (stimulated, 5.3 +/- 1.1 GFU/mg; unstimulated, 2.2 +/- 0.6 GFU/mg) (+/- SE, P less than 0.05). Western analysis demonstrated increased amounts of peptides with immunological identity to acidic and basic FGFs in stimulated muscle. The increase in FGF content observed in this study is synchronous with neovascularization, myoblast proliferation, and fast to slow fiber type conversion previously shown in this model. These results demonstrate that increased expression of FGFs is associated with motor nerve stimulation and increased tonic contractile activity of skeletal muscle, and suggests that these proteins may play a regulatory role in the cellular changes that occur during exercise conditioning.

<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 pleiotropic effects on regulating lipid and glucose metabolism. Its expression is increased in skeletal muscle of mice and humans with mitochondrial disorders. However, the effects of FGF<em>21</em> on skeletal muscle in response to mitochondrial respiratory chain deficiency are largely unknown. Here we demonstrate that the increased expression of FGF<em>21</em> is a compensatory response to respiratory chain deficiency. The mRNA and protein levels of FGF<em>21</em> were robustly raised in skeletal muscle from patients with mitochondrial myopathy or MELAS. The mammalian target of rapamycin (mTOR) phosphorylation levels and its downstream targets, Yin Yang 1 (YY1) and peroxisome proliferator-activated receptor γ, coactivator 1α (PGC-1α), were increased by FGF<em>21</em> treatment in C2C12 myoblasts. Activation of the mTOR-YY1-PGC1α pathway by FGF<em>21</em> in myoblasts regulated energy homeostasis as demonstrated by significant increases in intracellular ATP synthesis, oxygen consumption rate, activity of citrate synthase, glycolysis, mitochondrial DNA copy number, and induction of the expression of key energy metabolic genes. The effects of FGF<em>21</em> on mitochondrial function required phosphoinositide 3-kinase (PI3K), which activates mTOR. Inhibition of PI3K, mTOR, YY1, and PGC-1α activities attenuated the stimulating effects of FGF<em>21</em> on intracellular ATP levels and mitochondrial gene expression. Our findings revealed that mitochondrial respiratory chain deficiency elicited a compensatory response in skeletal muscle by increasing the FGF<em>21</em> expression levels in muscle, which resulted in enhanced mitochondrial function through an mTOR-YY1-PGC1α-dependent pathway in skeletal muscle.

Cultured skin substitutes have been used as adjunctive therapies in the treatment of burns and chronic wounds, but they are limited by lack of a vascular plexus. This deficiency leads to greater time for vascularization compared with native skin autografts and contributes to graft failure. Genetic modification of cultured skin substitutes to enhance vascularization could hypothetically lead to improved wound healing. To address this hypothesis, human keratinocytes were genetically modified by transduction with a replication incompetent retrovirus to overexpress vascular endothelial <em>growth</em> <em>factor</em>, a specific and potent mitogen for endothelial cells. Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates inoculated with human <em>fibroblasts</em> and either vascular endothelial <em>growth</em> <em>factor</em>-modified or control keratinocytes were prepared, and were cultured in vitro for <em>21</em> d. Northern blot analysis demonstrated enhanced expression of vascular endothelial <em>growth</em> <em>factor</em> mRNA in genetically modified keratinocytes and in cultured skin substitutes prepared with modified cells. Furthermore, the vascular endothelial <em>growth</em> <em>factor</em>-modified cultured skin substitutes secreted greatly elevated levels of vascular endothelial <em>growth</em> <em>factor</em> protein throughout the entire culture period. The bioactivity of vascular endothelial <em>growth</em> <em>factor</em> protein secreted by the genetically modified cultured skin substitutes was demonstrated using a microvascular endothelial cell <em>growth</em> assay. Vascular endothelial <em>growth</em> <em>factor</em>-modified and control cultured skin substitutes were grafted to full-thickness wounds on athymic mice, and elevated vascular endothelial <em>growth</em> <em>factor</em> mRNA expression was detected in the modified grafts for at least 2 wk after surgery. Vascular endothelial <em>growth</em> <em>factor</em>-modified grafts exhibited increased numbers of dermal blood vessels and decreased time to vascularization compared with controls. These results indicate that genetic modification of keratinocytes in cultured skin substitutes can lead to increased vascular endothelial <em>growth</em> <em>factor</em> expression, which could prospectively improve vascularization of cultured skin substitutes for wound healing applications.

OBJECTIVE

Glucagon reduces body weight by modifying food intake, glucose/lipid metabolism and energy expenditure. All these physiological processes are also controlled by <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>), a circulating hepatokine that improves the metabolic profile in obesity and type 2 diabetes. Animal experiments have suggested a possible interaction between glucagon and FGF-<em>21</em> however, the metabolic consequences of this crosstalk are not understood.

METHODS

The effects of exogenous glucagon on plasma FGF-<em>21</em> levels and lipolysis were evaluated in healthy volunteers and humans with type 1 diabetes, as well as in rodents with streptozotocin (STZ)-induced insulinopenic diabetes. In vitro, the role of glucagon on FGF-<em>21</em> secretion and lipolysis was studied using isolated primary rat hepatocytes and adipocytes. Fgf-<em>21</em> expression in differentiated rat pre-adipocytes was suppressed by small interfering RNA and released FGF-<em>21</em> was immunoneutralised by polyclonal antibodies.

RESULTS

Glucagon induced lipolysis in healthy human volunteers, patients with type 1 diabetes, mice and rats with STZ-induced insulinopenic diabetes, and in adipocytes isolated from diabetic and non-diabetic animals. In addition, glucagon increased circulating FGF-<em>21</em> in healthy humans and rodents, as well as in patients with type 1 diabetes, and insulinopenic rodents. Glucagon stimulated FGF-<em>21</em> secretion from isolated primary hepatocytes and adipocytes derived from animals with insulinopenic diabetes. Furthermore, FGF-<em>21</em> stimulated lipolysis in primary adipocytes isolated from non-diabetic and diabetic rats. Reduction of Fgf-<em>21</em> expression (by approximately 66%) or immunoneutralisation of released FGF-<em>21</em> markedly attenuated glucagon-stimulated lipolysis in adipocytes.

CONCLUSIONS

These results indicate that glucagon increases circulating FGF-<em>21</em> independently of endogenous insulin levels. FGF-<em>21</em> participates in glucagon-induced stimulation of lipolysis.

Angora is an autosomal recessive mouse mutation caused by a deletion of approximately 2 kilobases in the <em>fibroblast</em> <em>growth</em> <em>factor</em> 5 (Fgf5) gene. Phenotypically, homozygous angora (Fgf5go/Fgf5go) mice have excessively long truncal hair and can be differentiated from heterozygous (+/Fgf5go) and wild-type (+/+) littermates by <em>21</em> days of age. Abnormal hair length is due to a prolongation of the anagen phase of the hair cycle of approximately 3 days. In addition, widely scattered hair follicles produce structurally defective hair shafts that twist within the follicle, presumably causing secondary hyperplasia of the outer root sheath and epidermis adjacent to the follicle. These follicular abnormalities were accentuated by immunohistochemical detection of mouse specific keratin 6, a nonspecific marker of epidermal hyperplasia. These abnormalities could be identified from birth throughout life in angora mice genotyped by polymerase chain reaction techniques. Moreover, the long truncal hair phenotype was maintained in skin grafted onto C.B-17/Sz-scid/scid mice that had normal pelage hairs and hair cycles, suggesting that circulating or diffusible humoral <em>factors</em> regulating the mouse hair cycle are not involved in this mutation. The angora mutation provides another useful mouse model for studying the hair cycle and its modulation.

Histone deacetylase (HDAC) 6 exists exclusively in cytoplasm and deacetylates cytoplasmic proteins such as α-tubulin. HDAC6 dysfunction is associated with several pathological conditions in the central nervous system. This study investigated the beneficial effects of tubastatin A (TubA), a novel specific HDAC6 inhibitor, in a rat model of transient middle cerebral artery occlusion (MCAO) and an in vitro model of excitotoxicity. Post-ischemic TubA treatment robustly improved functional outcomes, reduced brain infarction, and ameliorated neuronal cell death in MCAO rats. These beneficial effects lasted at least three days after MCAO. Notably, when given at 24 hours after MCAO, TubA still exhibited significant protection. Levels of acetylated α-tubulin were decreased in the ischemic hemisphere on Days 1 and 3 after MCAO, and were significantly restored by TubA. MCAO markedly downregulated <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF-<em>21</em>) and TubA significantly reversed this downregulation. TubA also mitigated impaired FGF-<em>21</em> signaling in the ischemic hemisphere, including up-regulating β-Klotho, and activating ERK and Akt/GSK-3β signaling pathways. In addition, both TubA and exogenous FGF-<em>21</em> conferred neuroprotection and restored mitochondrial trafficking in rat cortical neurons against glutamate-induced excitotoxicity. Our findings suggest that the neuroprotective effects of TubA likely involve HDAC6 inhibition and the subsequent up-regulation of acetylated α-tubulin and FGF-<em>21</em>.

BACKGROUND

Elevated <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) levels have been suggested, from cross-sectional studies, as an indicator of subclinical diabetic nephropathy. We investigated whether serum FGF<em>21</em> was predictive of the development of diabetic nephropathy.

METHODS

Baseline serum FGF<em>21</em> levels were measured in 1136 Chinese type 2 diabetic subjects recruited from the Hong Kong West Diabetes Registry. The role of serum FGF<em>21</em> in predicting decline in estimated glomerular filtration rate (eGFR) over a median follow-up of 4 years was analyzed using Cox regression analysis.

RESULTS

At baseline, serum FGF<em>21</em> levels increased progressively with eGFR category (P for trend <.001). Among 1071 subjects with baseline eGFR ≥ 30 mL/min/1.73 m(2), serum FGF<em>21</em> levels were significantly higher in those with eGFR decline during follow-up (n = 171) than those without decline (n = 900) (P < .001). In multivariable Cox regression analysis, baseline serum FGF<em>21</em> was independently associated with eGFR decline (hazard ratio, 1.<em>21</em>; 95% confidence interval [CI], 1.01-1.43; P = .036), even after adjustment for baseline eGFR. In a subgroup of 559 subjects with baseline eGFR ≥ 60 mL/min/1.73 m(2) and normoalbuminuria, serum FGF<em>21</em> level remained an independent predictor of eGFR decline (hazard ratio, 1.36; 95% CI, 1.06-1.76; P = .016). Integrated discrimination improvement (IDI) suggested that the inclusion of baseline serum FGF<em>21</em> significantly improved the prediction of eGFR decline (IDI, 1%; 95% CI, 0.1-3.0; P = .013) in this subgroup, but not in the initial cohort involving all subjects.

CONCLUSIONS

Elevated serum FGF<em>21</em> levels may be a useful biomarker for predicting kidney disease progression, especially in the early stages of diabetic nephropathy.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a PPARα-regulated gene elucidated in the liver of PPARα-deficient mice or PPARα agonist-treated mice. Mice globally lacking adipose triglyceride lipase (ATGL) exhibit a marked defect in TG catabolism associated with impaired PPARα-activated gene expression in the heart and liver, including a drastic reduction in hepatic FGF<em>21</em> mRNA expression. Here we show that FGF<em>21</em> mRNA expression is markedly increased in the heart of ATGL-deficient mice accompanied by elevated expression of endoplasmic reticulum (ER) stress markers, which can be reversed by reconstitution of ATGL expression in cardiac muscle. In line with this assumption, the induction of ER stress increases FGF<em>21</em> mRNA expression in H9C2 cardiomyotubes. Cardiac FGF<em>21</em> expression was also induced upon fasting of healthy mice, implicating a role of FGF<em>21</em> in cardiac energy metabolism. To address this question, we generated and characterized mice with cardiac-specific overexpression of FGF<em>21</em> (CM-Fgf<em>21</em>). FGF<em>21</em> was efficiently secreted from cardiomyocytes of CM-Fgf<em>21</em> mice, which moderately affected cardiac TG homeostasis, indicating a role for FGF<em>21</em> in cardiac energy metabolism. Together, our results show that FGF<em>21</em> expression is activated upon cardiac ER stress linked to defective lipolysis and that a persistent increase in circulating FGF<em>21</em> levels interferes with cardiac and whole body energy homeostasis.

Ectopic lipid accumulation in the liver is an almost universal feature of human and rodent models of generalized lipodystrophy and is also a common feature of type 2 diabetes, obesity, and metabolic syndrome. Here we explore the progression of fatty liver disease using a mouse model of lipodystrophy created by a fat-specific knockout of the insulin receptor (F-IRKO) or both IR and insulin-like <em>growth</em> <em>factor</em> 1 receptor (F-IR/IGFRKO). These mice develop severe lipodystrophy, diabetes, hyperlipidemia, and fatty liver disease within the first weeks of life. By 12 weeks of age, liver demonstrated increased reactive oxygen species, lipid peroxidation, histological evidence of balloon degeneration, and elevated serum alanine aminotransferase and aspartate aminotransferase levels. In these lipodystrophic mice, stored liver lipids can be used for energy production, as indicated by a marked decrease in liver weight with fasting and increased liver <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> expression and intact ketogenesis. By 52 weeks of age, liver accounted for 25% of body weight and showed continued balloon degeneration in addition to inflammation, fibrosis, and highly dysplastic liver nodules. Progression of liver disease was associated with improvement in blood glucose levels, with evidence of altered expression of gluconeogenic and glycolytic enzymes. However, these mice were able to mobilize stored glycogen in response to glucagon. Feeding F-IRKO and F-IR/IGFRKO mice a high-fat diet for 12 weeks accelerated the liver injury and normalization of blood glucose levels. Thus, severe fatty liver disease develops early in lipodystrophic mice and progresses to advanced nonalcoholic steatohepatitis with highly dysplastic liver nodules. The liver injury is propagated by lipotoxicity and is associated with improved blood glucose levels.

Cholestasis induces adaptive mechanisms protecting the liver against bile acids (BA) toxicity including modulation of BA synthesis. Whether <em>fibroblast</em> <em>growth</em> <em>factor</em> 19 (FGF19) or farnesoid X receptor (FXR) dependent signaling are involved in the regulation of BA homeostasis in primary biliary cirrhosis (PBC) remains unknown. Here we analyzed hepatic expression of FGF19 and other genes relevant to the adaptive response to cholestasis in tissues from non-cirrhotic (n = 24) and cirrhotic (n = <em>21</em>) patients along with control tissues (n = <em>21</em>). Moreover we searched for relationships between serum FGF19 and laboratory/clinical findings in 51 patients. Hepatic FGF19 mRNA expression was increased in non-cirrhotic and cirrhotic tissues (9-fold,p = 0.01; 69-fold,p < 0.0001, respectively). Protein levels of FGF19, FGF receptor 4, FXR and short heterodimer partner were increased in cirrhotic livers (9-fold, p < 0.001; 3.5-fold,p = 0.007; 2.4-fold,p < 0.0001; 2.8-fold,p < 0.0001 vs controls, respectively) which was accompanied by down-regulation of CYP7A1 (50% reduction, p = 0.006). Serum and liver levels of FGF19 correlated with worse liver biochemistry, BAs, quality of life and Mayo Risk Score. Serum FGF19 was elevated in UDCA non-responders. We conclude that PBC induces characteristic changes in liver expression of BAs synthesis regulatory molecules. FGF19 correlates with severity of liver disease and can potentially serve as an indicator of chronic cholestatic liver injury.

The protein tyrosine phosphatase 1B (PTP1B) modulates tyrosine kinase receptors, among which is the vascular endothelial <em>growth</em> <em>factor</em> receptor type 2 (VEGFR2), a key component of angiogenesis. Because PTP1B deficiency in mice improves left ventricular (LV) function 2 mo after myocardial infarction (MI), we hypothesized that enhanced angiogenesis early after MI via activated VEGFR2 contributes to this improvement. At 3 d after MI, capillary density was increased at the infarct border of PTP1B(-/-) mice [+7±2% vs. wild-type (WT), P = 0.05]. This was associated with increased extracellular signal-regulated kinase 2 phosphorylation and VEGFR2 activation (i.e., phosphorylated-Src/Src/VEGFR2 and dissociation of endothelial VEGFR2/VE-cadherin), together with higher infiltration of proangiogenic M2 macrophages within unchanged overall infiltration. In vitro, we showed that PTP1B inhibition or silencing using RNA interference increased VEGF-induced migration and proliferation of mouse heart microvascular endothelial cells as well as <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-induced proliferation of rat aortic smooth muscle cells. At 8 d after MI in PTP1B(-/-) mice, increased LV capillary density (+<em>21</em>±3% vs. WT; P<0.05) and an increased number of small diameter arteries (15-50 μm) were likely to participate in increased LV perfusion assessed by magnetic resonance imaging and improved LV compliance, indicating reduced diastolic dysfunction. In conclusion, PTP1B deficiency reduces MI-induced heart failure promptly after ischemia by enhancing angiogenesis, myocardial perfusion, and diastolic function.

<AbstractText>Obesity and type 2 diabetes mellitus (T2DM) are risk <em>factors</em> for nonalcoholic fatty liver disease, including nonalcoholic steatohepatitis. This study assessed pegbelfermin (BMS-986036), recombinant PEGylated human <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), in patients with obesity and T2DM predisposed to fatty liver.</AbstractText><p><div><b>METHODS</b></div>In this randomized, double-blind, placebo-controlled study, patients with T2DM and BMI of 30 to 50 kg/m² received subcutaneous pegbelfermin (1, 5, or 20 mg daily or 20 mg weekly; n = 96) or placebo (n = 24) for 12 weeks. Primary end points were safety, tolerability, and change in HbA1c. Additional end points included insulin sensitivity, lipids, adiponectin, and disease progression biomarkers.</p><AbstractText>There were no significant effects of pegbelfermin versus placebo on HbA1c. Pegbelfermin 20 mg/d significantly improved high-density lipoprotein cholesterol (P = 0.015) and triglycerides (P = 0.037). All pegbelfermin regimens significantly increased adiponectin levels; 20-mg daily and weekly regimens decreased serum PRO-C3. Most adverse events were mild; the most frequent adverse events were injection-site bruising and diarrhea.</AbstractText><AbstractText>Twelve-week pegbelfermin treatment did not impact HbA1c concentrations, but QW and higher daily doses were associated with improved metabolic parameters and fibrosis biomarkers in patients with obesity and T2DM predisposed to fatty liver. These results support evaluation of pegbelfermin in patients with obesity-related metabolic diseases (e.g., nonalcoholic steatohepatitis).</AbstractText>

BACKGROUND

Circulating FGF21 levels are commonly elevated in disease states. There is limited information regarding concentrations of circulating FGF21 in the absence of disease, as well as age-related differences in body composition that may contribute to FGF21 regulation across groups.

OBJECTIVE

The objectives of this study were to assess FGF21 levels across age groups (childhood to elder adulthood), and investigate whether body composition indices are associated with age-related differences in circulating FGF21.

METHODS

We cross-sectionally analyzed serum concentrations of FGF21 in 184 healthy subjects aged 5-80y (45% male). Multiple linear regression was performed to assess the independent association of categorical age (children: 5-12y, young adults: 20-29y, adults: 30-50y, older adults: 55-64y, elder adults: 65-80y) with FGF21 concentration taking into account DXA-measured body composition indices [bone mineral density (BMD) and percent lean, trunk, and fat mass]. We also stratified analysis by tertile of FGF21.

RESULTS

Incremental increases in FGF21 levels were observed across age groups (youngest to highest). Age group was positively associated with FGF21 level independent of body composition indices (age group variable: β=0.25, 0.24, 0.24, 0.23, all P<0.0001, controlling for percent lean, BMD, percent fat, and percent trunk fat, respectively). By FGF21 tertile, age group was associated with FGF21 in the lowest tertile only (β=13.1, 0.19, 0.18, all P≤0.01, accounting for percent lean, fat and trunk fat, respectively), but not when accounting for BMD.

CONCLUSIONS

Our findings in a healthy population display an age-related increase in serum FGF21, highlighting a potential age effect in response to metabolic demand over the lifecourse. FGF21 levels increase with age independently of body composition. At lower levels of FGF21, BMD, but not other body composition parameters, attenuates the association between FGF21 level and age, suggesting the metabolic demand of the skeleton may provide a link between FGF21 and energy metabolism.

The molecular mechanisms and metabolic pathways whereby the citrus flavonoid, naringenin, reduces dyslipidemia and improves glucose tolerance were investigated in C57BL6/J wild-type mice and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) null (Fgf<em>21</em>(-/-)) mice. FGF<em>21</em> regulates energy homeostasis and the metabolic adaptation to fasting. One avenue of this regulation is through induction of peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc1a), a regulator of hepatic fatty acid oxidation and ketogenesis. Because naringenin is a potent activator of hepatic FA oxidation, we hypothesized that induction of FGF<em>21</em> might be an integral part of naringenin's mechanism of action. Furthermore, we predicted that FGF<em>21</em> deficiency would potentiate high-fat diet (HFD)-induced metabolic dysregulation and compromise metabolic protection by naringenin. The absence of FGF<em>21</em> exacerbated the response to a HFD. Interestingly, naringenin supplementation to the HFD robustly prevented obesity in both genotypes. Gene expression analysis suggested that naringenin was not primarily targeting fatty acid metabolism in white adipose tissue. Naringenin corrected hepatic triglyceride concentrations and normalized hepatic expression of Pgc1a, Cpt1a, and Srebf1c in both wild-type and Fgf<em>21</em>(-/-) mice. HFD-fed Fgf<em>21</em>(-/-) mice displayed greater muscle triglyceride deposition, hyperinsulinemia, and impaired glucose tolerance as compared with wild-type mice, confirming the role of FGF<em>21</em> in insulin sensitivity; however, naringenin supplementation improved these metabolic parameters in both genotypes. We conclude that FGF<em>21</em> deficiency exacerbates HFD-induced obesity, hepatic steatosis, and insulin resistance. Furthermore, FGF<em>21</em> is not required for naringenin to protect mice from HFD-induced metabolic dysregulation. Collectively these studies support the concept that naringenin has potent lipid-lowering effects and may act as an insulin sensitizer in vivo.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family. It actually functions as endocrine hormones but does not regulate cell <em>growth</em> and differentiation. It is demonstrated that FGF<em>21</em> acts on multiple tissue to coordinate carbohydrate and lipid metabolism, including enhancing insulin sensitivity, decreasing triglyceride concentrations, causing weight loss, ameliorating obesity-associated hyperglycemia and hyperlipidemia. Moreover, FGF<em>21</em> also plays important roles in some physiological processes, such as fasting and feeding, <em>growth</em> hormone axis and thermogenic function of brown adipose tissue. Clinical relevance of FGF<em>21</em> in humans is still unclear, and the basis and consequences of increased FGF<em>21</em> in metabolic disease remain to be determined. Both the pharmacological actions and physiological roles make FGF<em>21</em> attractive drug candidates for treating metabolic disease, but some questions remain to be answered. This article concentrates on recent advances in our understanding of FGF<em>21</em>.

Despite best evidence-based treatment including statins, residual cardiovascular risk poses a major challenge for clinicians in the twenty first century. Atherogenic dyslipidaemia, in particular elevated triglycerides, a marker for increased triglyceride-rich lipoproteins and their remnants, is an important contributor to lipid-related residual risk, especially in insulin resistant conditions such as type 2 diabetes mellitus. Current therapeutic options include peroxisome proliferator-activated receptor alpha (PPARα) agonists, (fibrates), but these have low potency and limited selectivity for PPARα. Modulating the unique receptor-co<em>factor</em> binding profile to identify the most potent molecules that induce PPARα-mediated beneficial effects, while at the same time avoiding unwanted side effects, offers a new therapeutic approach and provides the rationale for development of pemafibrate (K-877, Parmodia™), a novel selective PPARα modulator (SPPARMα). In clinical trials, pemafibrate either as monotherapy or as add-on to statin therapy was effective in managing atherogenic dyslipidaemia, with marked reduction of triglycerides, remnant cholesterol and apolipoprotein CIII. Pemafibrate also increased serum <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>, implicated in metabolic homeostasis. There were no clinically meaningful adverse effects on hepatic or renal function, including no relevant serum creatinine elevation. A major outcomes study, PROMINENT, will provide definitive evaluation of the role of pemafibrate for management of residual cardiovascular risk in type 2 diabetes patients with atherogenic dyslipidaemia despite statin therapy.

We previously demonstrated that the neural cell adhesion molecule (N-CAM) inhibited the proliferation of cultured rat hippocampal progenitor cells and increased the number of neurons generated. We demonstrate here that the continued presence of <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 along with N-CAM or brain-derived neurotrophic <em>factor</em> over 12 days of culture greatly increased the number of both progenitors and neurons. These progenitor-derived neurons expressed neurotransmitters, neurotransmitter receptors, and synaptic proteins in vitro consistent with those expressed in the mature hippocampus. Progenitor cells cultured on microelectrode plates formed elaborate neural networks that exhibited spontaneously generated action potentials after <em>21</em> days. This activity was observed only in cultures grown in the presence of <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 and either N-CAM or brain-derived neurotrophic <em>factor</em>. Analysis of neuronal activity after various pharmacological treatments indicated that the networks formed functional GABAergic and glutamatergic synapses. We conclude that mitogenic <em>growth</em> <em>factors</em> can synergize with N-CAM or neurotrophins to generate spontaneously active neural networks from neural progenitors.

In this study we demonstrate the potential for combining biocompatible polymers with genetically engineered cells to elicit axon re<em>growth</em> across tissue defects in the injured CNS. Eighteen- to <em>21</em>-day-old rats received implants of poly N-(2-hydroxypropyl)-methacrylamide (HPMA) hydrogels containing RGD peptide sequences that had been infiltrated with control (untransfected) <em>fibroblasts</em> (n = 8), <em>fibroblasts</em> engineered to express brain-derived neurotrophic <em>factor</em> (BDNF) (n = 5), ciliary neurotrophic <em>factor</em> (CNTF) (n = 5), or a mixture of BDNF and CNTF expressing <em>fibroblasts</em> (n = 11). <em>Fibroblasts</em> were prelabeled with Hoechst 33342. Cell/polymer constructs were inserted into cavities made in the left optic tract, between thalamus and superior colliculus. After 4-8 weeks, retinal projections were analyzed by injecting right eyes with cholera toxin (B-subunit). Rats were perfused 24 h later and sections were immunoreacted to visualize retinal axons, other axons (RT97 antibody), host astrocytes and macrophages, donor <em>fibroblasts</em>, and extracellular matrix molecules. The volume fraction (VF) of each gel that was occupied by RT97(+) axons was quantified. RT-PCR confirmed expression of the transgenes prior to, and 5 weeks after, transplantation. Compared to control rats (mean VF = 0.02 +/- 0.01% SEM) there was increased in<em>growth</em> of RT97(+) axons into implants in CNTF (mean VF = 0.33 +/- 0.19%) and BDNF (mean VF = 0.62 +/-0.19%) groups. Axon <em>growth</em> into hydrogels in the mixed BDNF/CNTF group (mean VF = 3.58 +/- 0.92%) was significantly greater (P < 0.05) than in the BDNF or CNTF <em>fibroblast</em> groups. Retinal axons exhibited a complex branching pattern within gels containing BDNF or BDNF/CNTF <em>fibroblasts</em>; however, they regrew the greatest distances within implants containing both BDNF and CNTF expressing cells.

BACKGROUND

Fibroblast growth factor 2 (FGF2) is a growth factor that is immediately released after cartilage injury and plays a pivotal role in cartilage homeostasis. In human adult articular cartilage, FGF2 mediates anti-anabolic and potentially catabolic effects via the suppression of proteoglycan (PG) production along with the upregulation of matrix-degrading enzyme activity. The aim of the present study was to determine the biological effects of FGF2 in spine disc cells and to elucidate the complex biochemical pathways utilized by FGF2 in bovine intervertebral disc (IVD) cells in an attempt to further understand the pathophysiologic processes involved in disc degeneration.

METHODS

We studied the effect of FGF2 on IVD tissue homeostasis by assessing MMP-13 expression (potent matrix-degrading enzyme), PG accumulation, and PG synthesis in the bovine spine IVD, as well as evaluating whether FGF2 counteracts known anabolic factors such as BMP7. To understand the molecular mechanisms by which FGF2 antagonizes BMP7 activity, we also investigated the signaling pathways utilized by FGF2 in bovine disc tissue.

RESULTS

The primary receptor expressed in bovine nucleus pulposus cartilage is FGFR1, and this receptor is upregulated in degenerative human IVD tissue compared with normal IVD tissue. Stimulation of bovine nucleus pulposus cells cultured in monolayer with FGF2 augmented the production of MMP-13 at the transcriptional and translational level in a dose-dependent manner. Stimulation of bovine nucleus pulposus cells cultured in alginate beads for 21 days with FGF2 resulted in a dose-dependent decrease in PG accumulation, due at least in part to the inhibition of PG synthesis. Further studies demonstrate that FGF2 (10 ng/ml) antagonizes BMP7-mediated acceleration of PG production in bovine nucleus pulposus cells via the upregulation of noggin, an inhibitor of the transforming growth factor beta/bone morphogenetic protein signaling pathway. Chemical inhibitor studies showed that FGF2 utilizes the mitogen-activated protein kinase and NF-kappaB pathways to upregulate noggin, serving as one potential mechanism for its anti-anabolic effects.

CONCLUSIONS

FGF2 is anti-anabolic in bovine spine disc cells, revealing the potential of FGF2 antagonists as unique biologic treatments for both prevention and reversal of IVD degeneration.

Chronic hepatic regeneration constitutes an important part of the cirrhotic process. The <em>factors</em> regulating chronic hepatic regeneration, however, remain unclear. We therefore analyzed the intrahepatic messenger RNA (mRNA) expression of <em>growth</em> <em>factors</em> (epidermal <em>growth</em> <em>factor</em> [EGF], basic <em>fibroblast</em> <em>growth</em> <em>factor</em> [bFGF], hepatocyte <em>growth</em> <em>factor</em> [HGF], transforming <em>growth</em> <em>factor</em> [TGF]-alpha, and TGF-beta) at progressive time points (postoperative days 2, 7, 14, and <em>21</em>) in a rat bile duct-ligated (BDL) model of cirrhosis versus sham controls. Intrahepatic <em>growth</em> <em>factor</em> mRNA expression was quantitatively assessed by polymerase chain reaction (PCR) using a dot-blot hybridization technique. Cirrhosis was associated with statistically significant (P < .05) progressive increases in the intrahepatic mRNA expression of bFGF (80-fold), EGF (25-fold), and TGF-beta (fourfold) in BDL animals versus controls. Furthermore, immunohistochemistry of hepatic sections showed a progressive up-regulation of bFGF protein in areas of bile duct proliferation. These areas also showed a dramatic increase in the number of hepatic stellate cells (HSC). In contrast, the intrahepatic expression of hepatocyte <em>growth</em> <em>factor</em> (HGF) mRNA was only significantly increased at postoperative days 7 and 14 in BDL animals before returning to control levels as cirrhosis developed. There were no significant differences found at any timepoint in the expression of TGF-alpha in BDL animals versus controls. In conclusion, the development of cirrhosis in this BDL rat model was associated with a progressive increase in the intrahepatic expression of EGF, bFGF, and TGF-beta. Early increased expression of HGF was not maintained in established cirrhosis. The findings suggest that these <em>growth</em> <em>factors</em> may play important roles in the pathogenesis of chronic hepatic regeneration in cirrhosis.

BACKGROUND

The development of organ fibrosis after injury requires activation of transforming growth factor β(1) which regulates the transcription of profibrotic genes. The systemic administration of a proteasomal inhibitor has been reported to prevent the development of fibrosis in the liver, kidney and bone marrow. It is hypothesised that proteasomal inhibition would prevent lung and skin fibrosis after injury by inhibiting TGF-β(1)-mediated transcription.

METHODS

Bortezomib, a small molecule proteasome inhibitor in widespread clinical use, was administered to mice beginning 7 days after the intratracheal or intradermal administration of bleomycin and lung and skin fibrosis was measured after 21 or 40 days, respectively. To examine the mechanism of this protection, bortezomib was administered to primary normal lung fibroblasts and primary lung and skin fibroblasts obtained from patients with idiopathic pulmonary fibrosis and scleroderma, respectively.

RESULTS

Bortezomib promoted normal repair and prevented lung and skin fibrosis when administered beginning 7 days after the initiation of bleomycin. In primary human lung fibroblasts from normal individuals and patients with idiopathic pulmonary fibrosis and in skin fibroblasts from a patient with scleroderma, bortezomib inhibited TGF-β(1)-mediated target gene expression by inhibiting transcription induced by activated Smads. An increase in the abundance and activity of the nuclear hormone receptor PPARγ, a repressor of Smad-mediated transcription, contributed to this response.

CONCLUSIONS

Proteasomal inhibition prevents lung and skin fibrosis after injury in part by increasing the abundance and activity of PPARγ. Proteasomal inhibition may offer a novel therapeutic alternative in patients with dysregulated tissue repair and fibrosis.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has been shown to stimulate wound healing. However, consistent delivery of bFGF has been problematic. We studied the stability of bFGF incorporated into a chitosan film as a delivery vehicle for providing sustained release of bFGF. The therapeutic effect of this system on wound healing in genetically diabetic mice was determined as a model for treating clinically impaired wound healing. A chitosan film was prepared by freeze-drying hydroxypropylchitosan (a water-soluble derivative of chitosan) acetate buffer solution. <em>Growth</em> <em>factor</em> was incorporated into films before drying by mixing bFGF solution with the hydroxypropylchitosan solution. bFGF activity remained stable for <em>21</em> days at 5 degrees C, and 86.2% of activity remained with storage at 25 degrees C. Full-thickness wounds were created on the backs of diabetic mice, and chitosan film or bFGF-chitosan film was applied to the wound. The wound was smaller in after 5 days in both groups, but the wound was smaller on day 20 only in the bFGF-chitosan group. Proliferation of <em>fibroblasts</em> and an increase in the number of capillaries were observed in both groups, but granulation tissue was more abundant in the bFGF-chitosan group. These results suggest that chitosan itself facilitates wound repair and that bFGF incorporated into chitosan film is a stabile delivery vehicle for accelerating wound healing.