OBJECTIVE

Obesity is characterized by chronic oxidative stress. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has recently been identified as a novel hormone that regulates metabolism. NFE2-related <em>factor</em> 2 (Nrf2) is a transcription <em>factor</em> that orchestrates the expression of a battery of antioxidant and detoxification genes under both basal and stress conditions. The current study investigated the role of Nrf2 in a mouse model of long-term high-fat diet (HFD)-induced obesity and characterized its crosstalk to FGF<em>21</em> in this process.

METHODS

Wild-type (WT) and Nrf2 knockout (Nrf2-KO) mice were fed an HFD for 180 days. During this period, food consumption and body weights were measured. Glucose metabolism was assessed by an intraperitoneal glucose tolerance test and intraperitoneal insulin tolerance test. Total RNA was prepared from liver and adipose tissue and was used for quantitative real-time RT-PCR. Fasting plasma was collected and analyzed for blood chemistries. The ST-2 cell line was used for transfection studies.

RESULTS

Nrf2-KO mice were partially protected from HFD-induced obesity and developed a less insulin-resistant phenotype. Importantly, Nrf2-KO mice had higher plasma FGF<em>21</em> levels and higher FGF<em>21</em> mRNA levels in liver and white adipose tissue than WT mice. Thus, the altered metabolic phenotype of Nrf2-KO mice under HFD was associated with higher expression and abundance of FGF<em>21</em>. Consistently, the overexpression of Nrf2 in ST-2 cells resulted in decreased FGF<em>21</em> mRNA levels as well as in suppressed activity of a FGF<em>21</em> promoter luciferase reporter.

CONCLUSIONS

The identification of Nrf2 as a novel regulator of FGF<em>21</em> expands our understanding of the crosstalk between metabolism and stress defense.

BACKGROUND

Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression.

OBJECTIVE

To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC).

METHODS

A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription-quantitative polymerase chain reaction. The two most frequent mutations were investigated, using a SNaPshot assay, in an independent set of 184 non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo and <em>21</em> mo, respectively). Voided urine from patients with suspicion of incident UBC (n=174), or under surveillance after diagnosis of non-muscle-invasive UBC (n=194), was tested using a SNaPshot assay.

METHODS

Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease-specific survival, and overall survival.

CONCLUSIONS

In the two series, 78 of 111 (70%) and 283 of 357 (79%) tumors harbored TERT mutations, C228T being the most frequent substitution (83% for both series). TERT mutations were not associated with clinical or pathologic parameters, but were more frequent among FGFR3 mutant tumors (p=0.0002). There was no association between TERT mutations and mRNA expression (p=0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC patients. The sensitivity was 62% in incident and 42% in recurrent UBC. A limitation of the study is its retrospective nature.

CONCLUSIONS

Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may help to detect UBC recurrences.

OBJECTIVE

Disturbances in mineral metabolism define an increased cardiovascular risk in patients with chronic kidney disease. Fibroblast growth factor-23 (FGF23) is a circulating regulator of phosphate and vitamin D metabolism and has recently been implicated as a putative pathogenic factor in cardiovascular disease. Because other members of the FGF family play a role in lipid and glucose metabolism, we hypothesized that FGF23 would associate with metabolic factors that predispose to an increased cardiovascular risk. The goal of this study was to investigate the relationship between FGF23 and metabolic cardiovascular risk factors in the community.

RESULTS

Relationships between serum FGF23 and body mass index (BMI), waist circumference, waist-to-hip ratio, serum lipids, and fat mass were examined in 2 community-based, cross-sectional cohorts of elderly whites (Osteoporotic Fractures in Men Study: 964 men aged 75±3.2; Prospective Investigation of the Vasculature in Uppsala Seniors study: 946 men and women aged 70). In both cohorts, FGF23 associated negatively with high-density lipoprotein and apolipoprotein A1 (7% to 21% decrease per 1-SD increase in log FGF23; P<0.01) and positively with triglycerides (11% to 14% per 1-SD increase in log FGF23; P<0.01). A 1-SD increase in log FGF23 was associated with a 7% to 20% increase in BMI, waist circumference, and waist-to-hip ratio and a 7% to 18% increase in trunk and total body fat mass (P<0.01) as determined by whole-body dual x-ray absorptiometry. FGF23 levels were higher in subjects with the metabolic syndrome compared with those without (46.4 versus 41.2 pg/mL; P<0.05) and associated with an increased risk of having the metabolic syndrome (OR per 1-SD increase in log FGF23, 1.21; 95% CI, 1.04 to 1.40; P<0.05).

CONCLUSIONS

We report for the first time on associations between circulating FGF23, fat mass, and adverse lipid metabolism resembling the metabolic syndrome, potentially representing a novel pathway(s) linking high FGF23 to an increased cardiovascular risk.

Given the high prevalence and rising incidence of non-alcoholic fatty liver disease (NAFLD), the absence of approved therapies is striking. Although the mainstay of treatment of NAFLD is weight loss, it is hard to maintain, prompting the need for pharmacotherapy as well. A greater understanding of disease pathogenesis in recent years was followed by development of new classes of medications, as well as potential repurposing of currently available agents. NAFLD therapies target four main pathways. The dominant approach is targeting hepatic fat accumulation and the resultant metabolic stress. Medications in this group include peroxisome proliferator-activator receptor agonists (eg, pioglitazone, elafibranor, saroglitazar), medications targeting the bile acid-farnesoid X receptor axis (obeticholic acid), inhibitors of de novo lipogenesis (aramchol, NDI-010976), incretins (liraglutide) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>21</em> or FGF-19 analogues. A second approach is targeting the oxidative stress, inflammation and injury that follow the metabolic stress. Medications from this group include antioxidants (vitamin E), medications with a target in the tumour necrosis <em>factor</em> α pathway (emricasan, pentoxifylline) and immune modulators (amlexanox, cenicriviroc). A third group has a target in the gut, including antiobesity agents such as orlistat or gut microbiome modulators (IMM-124e, faecal microbial transplant, solithromycin). Finally, as the ongoing injury leads to fibrosis, the harbinger of liver-related morbidity and mortality, antifibrotics (simtuzumab and GR-MD-02) will be an important element of therapy. It is very likely that in the next few years several medications will be available to clinicians treating patients with NAFLD across the entire spectrum of disease.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em>, a novel member of the FGF family, plays a role in a variety of endocrine functions, including regulation of glucose and lipid metabolism. The role of FGF<em>21</em> in skeletal muscle is currently not known.

METHODS

Serum levels and skeletal muscle mRNA of FGF<em>21</em> were determined in normal glucose tolerant (n = 40) and type 2 diabetic (T2D; n = 40) subjects. We determined whether FGF<em>21</em> has direct effects on glucose metabolism in cultured myotubes (n = 8) and extensor digitorum longus skeletal muscle.

RESULTS

Serum FGF<em>21</em> levels increased 20% in T2D versus normal glucose tolerant subjects (p < 0.05), whereas skeletal muscle mRNA expression was unaltered. Fasting insulin, homeostatic model assessment of insulin resistance (HOMA-IR), waist circumference, and body mass index (BMI) significantly correlated with serum FGF<em>21</em> levels in T2D (p < 0.01), but not in normal glucose tolerant subjects. Serum FGF<em>21</em> concentrations were greater in T2D patients in the highest tertile of fasting insulin (p < 0.05) and BMI (p < 0.05). Stepwise regression analysis identified BMI as the strongest independent variable correlating with FGF<em>21</em>. FGF<em>21</em> exposure increased basal and insulin-stimulated glucose uptake in human myotubes, coincident with increased glucose transporter 1 mRNA, and enhanced glucose transporter 1 abundance at the plasma membrane. In isolated extensor digitorum longus muscle, FGF<em>21</em> potentiated insulin-stimulated glucose transport, without altering phosphorylation of Akt or AMP-activated protein kinase.

CONCLUSIONS

Plasma FGF<em>21</em> is increased in T2D patients, and positively correlated with fasting insulin and BMI. However, FGF<em>21</em> has direct effects in enhancing skeletal muscle glucose uptake, providing additional points of regulation that may contribute to the beneficial effects of FGF<em>21</em> on glucose homeostasis. Whether increased plasma FGF<em>21</em> in T2D is a compensatory mechanism to increase glucose metabolism remains to be determined.

"Browning," the appearance and activation of brown-in-white (brite) adipose cells within inguinal white adipose tissue (iWAT), and induction of uncoupling protein 1 (UCP1) correlate with <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>)-induced weight loss and glucose homeostasis improvements. Therefore, antiobesity therapies targeting browning and brite adipocyte activation are currently being sought. To test the dependence of weight loss on browning, we examined whether this event was responsible for FGF<em>21</em>-Fc's beneficial effects. Lean and diet-induced obese mice housed at <em>21</em>°C or 30°C that received FGF<em>21</em>-Fc exhibited similar degrees of body weight reduction and glucose homeostasis improvement. Substantial browning of iWAT occurred only in FGF<em>21</em>-Fc-treated lean mice housed at <em>21</em>°C. Further, FGF<em>21</em>-Fc-treated Ucp1(-/-) mice showed robust improvements in body weight, glucose homeostasis, and plasma lipids, associated with increased energy expenditure and FGF<em>21</em>-Fc-induced Ppargc1 expression in iWAT. We conclude that FGF<em>21</em> requires neither UCP1 nor brite adipocytes to elicit weight loss and improve glucose homeostasis.

UCP1-Tg mice with ectopic expression of uncoupling protein 1 (UCP1) in skeletal muscle (SM) are a model of improved substrate metabolism and increased longevity. Analysis of myokine expression showed an induction of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) in SM, resulting in approximately fivefold elevated circulating FGF<em>21</em> in UCP1-Tg mice. Despite a reduced muscle mass, UCP1-Tg mice showed no evidence for a myopathy or muscle autophagy deficiency but an activation of integrated stress response (ISR; eIF2α/ATF4) in SM. Targeting mitochondrial function in vitro by treating C2C12 myoblasts with the uncoupler FCCP resulted in a dose-dependent activation of ISR, which was associated with increased expression of FGF<em>21</em>, which was also observed by treatment with respiratory chain inhibitors antimycin A and myxothiazol. The co<em>factor</em> required for FGF<em>21</em> action, β-klotho, was expressed in white adipose tissue (WAT) of UCP1-Tg mice, which showed an increased browning of WAT similar to what occurred in altered adipocyte morphology, increased brown adipocyte markers (UCP1, CIDEA), lipolysis (HSL phosphorylation), and respiratory capacity. Importantly, treatment of primary white adipocytes with serum of transgenic mice resulted in increased UCP1 expression. Additionally, UCP1-Tg mice showed reduced body length through the suppressed IGF-I-GH axis and decreased bone mass. We conclude that the induction of FGF<em>21</em> as a myokine is coupled to disturbance of mitochondrial function and ISR activation in SM. FGF<em>21</em> released from SM has endocrine effects leading to increased browning of WAT and can explain the healthy metabolic phenotype of UCP1-Tg mice. These results confirm muscle as an important endocrine regulator of whole body metabolism.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a pleiotropic hormone-like protein and a major metabolic regulator. However, several key aspects of FGF<em>21</em> biology remain poorly understood. Indeed, the list of controversies in the FGF<em>21</em> field spans a variety of topics, from basic matters such as the anatomic distribution of FGF<em>21</em> expression and the molecular composition of the FGF<em>21</em> receptor, to the ultimate question of therapeutic relevance of FGF<em>21</em>-dependent pathways in humans. In this paper, we focus on current challenges in the field in an attempt to provide a balanced overview of FGF<em>21</em> biology and guide future research into this exciting metabolic target.

Purpose No standard treatment exists for patients with cholangiocarcinoma for whom first-line gemcitabine-based therapy fails. <em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 ( FGFR2) fusions/translocations are present in 13% to 17% of intrahepatic cholangiocarcinomas. BGJ398, an orally bioavailable, selective pan-FGFR kinase inhibitor, has shown preliminary clinical activity against tumors with FGFR alterations. Methods A multicenter, open-label, phase II study ( ClinicalTrials.gov identifier: NCT0<em>21</em>50967) evaluated BGJ398 antitumor activity in patients age ≥ 18 years with advanced or metastatic cholangiocarcinoma containing FGFR2 fusions or other FGFR alterations whose disease had progressed while receiving prior therapy. Patients received BGJ398 125 mg once daily for <em>21</em> days, then 7 days off (28-day cycles). The primary end point was investigator-assessed overall response rate. Results Sixty-one patients (35 women; median age, 57 years) with FGFR2 fusion (n = 48), mutation (n = 8), or amplification (n = 3) participated. At the prespecified data cutoff (June 30, 2016), 50 patients had discontinued treatment. All responsive tumors contained FGFR2 fusions. The overall response rate was 14.8% (18.8% FGFR2 fusions only), disease control rate was 75.4% (83.3% FGFR2 fusions only), and estimated median progression-free survival was 5.8 months (95% CI, 4.3 to 7.6 months). Adverse events included hyperphosphatemia (72.1% all grade), fatigue (36.1%), stomatitis (29.5%), and alopecia (26.2%). Grade 3 or 4 treatment-related adverse events occurred in 25 patients (41%) and included hyperphosphatemia (16.4%), stomatitis (6.6%), and palmar-plantar erythrodysesthesia (4.9%). Conclusion BGJ398 is a first-in-class FGFR kinase inhibitor with manageable toxicities that shows meaningful clinical activity against chemotherapy-refractory cholangiocarcinoma containing FGFR2 fusions. This promising antitumor activity supports continued development of BGJ398 in this highly selected patient population.

Human tumors can constitutively express cytokines and <em>growth</em> <em>factors</em>, but the extent of this expression has not been investigated. Using 44 different probes to cytokines, <em>growth</em> <em>factors</em>, and their receptors, we tested <em>21</em> melanoma and 5 melanocyte cultures for RNA transcript expression by reverse transcriptase-polymerase chain reaction. With 30 amplification cycles, expression of the cytokines interleukin (IL)-1 beta, IL-6, leukemia inhibitory <em>factor</em> (LIF), IL-7, gro alpha, IL-8 and the p35 chain of IL-12 was detected in more than 60% of melanomas. Concomitant receptors for IL-6 and IL-7 were also detected. IL-1 alpha, IL-5, Rantes, IL-10, interferon (IFN)-beta, tumor-necrosis <em>factor</em> (TNF)-alpha, G-colony-stimulating <em>factor</em> (CSF) and GM-CSF were expressed at lower levels. Melanocytes showed greatly reduced cytokine RNA transcripts, and only gro alpha was consistently detected. No expression of IL-2, IL-3, IL-4, IL-9, the p40 chain of IL-12, IFN-alpha or IFN-gamma RNA transcripts was detected in melanomas or melanocytes. The <em>growth</em> <em>factors</em> expressed by melanomas and, after further signal amplification, by melanocytes were transforming <em>growth</em> <em>factor</em> (TGF)-alpha, epidermal <em>growth</em> <em>factor</em> (EGF), TGF-beta, endothelial-cell <em>growth</em> <em>factor</em> (ECGF), basic-<em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), nerve <em>growth</em> <em>factor</em> (NGF) and steel. The receptors EGFR, FGFR, NGFRp70 and c-kit were also expressed by melanomas and melanocytes. These results point to new possible autocrine and paracrine pathways in melanoma biology.

The prototype members of the heparin-binding <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family, acidic FGF (FGF-1) and basic FGF (FGF-2), are among the <em>growth</em> <em>factors</em> that act directly on vascular cells to induce endothelial cell <em>growth</em> and angiogenesis. In vivo, the role of the FGF prototypes in vascular pathology has been difficult to determine. We report here the introduction, by direct gene transfer into porcine arteries, of a eukaryotic expression vector encoding a secreted form of FGF-1. This somatic transgenic model defines gene function in the arterial wall in vivo. FGF-1 expression induced intimal thickening in porcine arteries <em>21</em> days after gene transfer, in contrast to control arteries transduced with an Escherichia coli beta-galactosidase gene. Where there was substantial intimal hyperplasia, neocapillary formation was detected in the expanded intima. These findings suggest that FGF-1 induces intimal hyperplasia in the arterial wall in vivo and, through its ability to stimulate angiogenesis in the neointima, FGF-1 could stimulate neovascularization of atherosclerotic plaques. Potentially, gene transfer of FGF-1 could also be used as a genetic intervention to improve blood flow to ischaemic tissues in selected clinical settings.

BACKGROUND

The <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) hormonal pathway is a metabolic signalling cascade and has been recently identified as the master hormonal regulator of glucose, lipids and overall energy balance. In this observational, case-control study, we assayed serum levels of FGF<em>21</em> in patients with nonalcoholic fatty liver disease (NAFLD), a hepatic manifestation of the metabolic syndrome, and examined their association with clinical, biochemical and histological phenotypes.

METHODS

Serum levels of FGF<em>21</em> were assayed by ELISA in 82 patients with biopsy-proven NAFLD and 77 controls. We analysed associations between FGF<em>21</em> and the characteristics of patients with NAFLD by multiple linear regression analysis.

RESULTS

Levels of FGF<em>21</em> were significantly higher in patients with NAFLD (median 200 pg mL(-1) ; interquartile range: 87-410 pg mL(-1)) than in healthy controls (median 93 pg mL(-1) ; interquartile range: 70-180 pg mL(-1) , Mann-Whitney U-test, P<0·001). There was a stepwise increase in serum FGF<em>21</em> levels according to the liver steatosis score (median level in subjects with score 1: 170 pg mL(-1) ; score 2: 220 pg mL(-1) ; score 3: 280 pg mL(-1) , P for trend <0·01). After stepwise linear regression analysis, serum FGF<em>21</em> levels were the only independent predictor of hepatic steatosis scores in patients with NAFLD (β=0·26; t=2·659, P<0·01).

CONCLUSIONS

Serum FGF<em>21</em> levels are increased in patients with NAFLD regardless of potential confounders and represent an independent predictor of liver steatosis. These findings support further investigation of this molecule in metabolic liver diseases.

BACKGROUND

Idiopathic pulmonary fibrosis is a progressive diffuse parenchymal lung disorder of unknown etiology. Mesenchymal stem cell (MSC)-based therapy is a novel approach with great therapeutic potential for the treatment of lung diseases. Despite demonstration of MSC grafting, the populations of engrafted MSCs have been shown to decrease dramatically 24 hours post-transplantation due to exposure to harsh microenvironments. Hypoxia is known to induce expression of cytoprotective genes and also secretion of anti-inflammatory, anti-apoptotic and anti-fibrotic factors. Hypoxic preconditioning is thought to enhance the therapeutic potency and duration of survival of engrafted MSCs. In this work, we aimed to prolong the duration of survival of engrafted MSCs and to enhance the effectiveness of idiopathic pulmonary fibrosis transplantation therapy by the use of hypoxia-preconditioned MSCs.

METHODS

Hypoxic preconditioning was achieved in MSCs under an optimal hypoxic environment. The expression levels of cytoprotective factors and their biological effects on damaged alveolar epithelial cells or transforming growth factor-beta 1-treated fibroblast cells were studied in co-culture experiments in vitro. Furthermore, hypoxia-preconditioned MSCs (HP-MSCs) were intratracheally instilled into bleomycin-induced pulmonary fibrosis mice at day 3, and lung functions, cellular, molecular and pathological changes were assessed at 7 and 21 days after bleomycin administration.

RESULTS

The expression of genes for pro-survival, anti-apoptotic, anti-oxidant and growth factors was upregulated in MSCs under hypoxic conditions. In transforming growth factor-beta 1-treated MRC-5 fibroblast cells, hypoxia-preconditioned MSCs attenuated extracellular matrix production through paracrine effects. The pulmonary respiratory functions significantly improved for up to 18 days of hypoxia-preconditioned MSC treatment. Expression of inflammatory factors and fibrotic factor were all downregulated in the lung tissues of the hypoxia-preconditioned MSC-treated mice. Histopathologic examination observed a significant amelioration of the lung fibrosis. Several LacZ-labeled MSCs were observed within the lungs in the hypoxia-preconditioned MSC treatment groups at day 21, but no signals were detected in the normoxic MSC group. Our data further demonstrated that upregulation of hepatocyte growth factor possibly played an important role in mediating the therapeutic effects of transplanted hypoxia-preconditioned MSCs.

CONCLUSIONS

Transplantation of hypoxia-preconditioned MSCs exerted better therapeutic effects in bleomycin-induced pulmonary fibrotic mice and enhanced the survival rate of engrafted MSCs, partially due to the upregulation of hepatocyte growth factor.

METHODS

We collected the specimens of lumbar intervertebral disc (i.e., the symptomatic degenerative disc) from patients with discogenic low back pain to study the histopathologic features and growth factor expressions.

OBJECTIVE

To study the pathogenesis of disc degeneration, meanwhile discriminating between common disc degeneration (aging disc) (i.e., black asymptomatic disc, not clinically relevant) and painful disc degeneration (i.e., symptomatic disc, clinically relevant).

BACKGROUND

The pathogenesis of intervertebral disc degeneration is poorly understood, mainly because of the difficulty to establish the experimental model with good reproducibility. Recently, the popularity of spinal fusion leads to more opportunities to obtain disc specimens, which could be applied to explore the pathogenesis of disc degeneration with modern biologic techniques.

METHODS

There were 21 specimens of lumbar intervertebral discs from 15 patients with discogenic low back pain during posterior lumbar interbody fusion, 16 aging discs from patients without low back pain, and 10 normal discs as control collected for the study of their histopathologic features, as well as the expressions of basic fibroblast growth factor (bFGF) and its receptor (Flg), transforming growth factor-beta1 (TGF-beta1) and its receptor (TGF-betaRI) by immunohistochemistry. The distribution of macrophages and mast cells was also noted. Proliferating cell nuclear antigen was assessed to evaluate proliferating activities of disc cells.

RESULTS

The distinct histologic characteristic of the disc from the patient with discogenic low back pain was the ingrowth of vascularized granulation tissue along torn fissures, extending from the external layer of the anulus fibrosus into the nucleus pulposus. The immunohistochemical staining showed that there were strong expressions of bFGF and TGF-beta1 and their receptors, as well as a strong expression of proliferating cell nuclear antigen in the zones of granulation tissue in the painful discs. However, there were only weak expressions in the nongranulation tissue zones in the painful discs and aging discs, and no expression in the control discs. In addition, abundant macrophages and mast cells were found in the granulation tissue zones of painful discs but absent in the nongranulation tissue zones of painful discs or aging discs and the normal control discs.

CONCLUSIONS

The findings indicated that degeneration of the painful disc might originate from the injury and subsequent repair of anulus fibrosus. Growth factors, such as bFGF and TGF-beta1, macrophages and mast cells might play a key role in the repair of the injured anulus fibrosus and subsequent disc degeneration.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>) is a metabolic regulator with multiple beneficial effects on glucose homeostasis and lipid metabolism in animal models. The relationship between plasma levels of FGF-<em>21</em> and coronary heart disease (CHD) in unknown.

RESULTS

This study aimed to investigate the correlation of serum FGF-<em>21</em> levels and lipid metabolism in the patients with coronary heart disease. We performed a logistic regression analysis of the relation between serum levels of FGF-<em>21</em> and CHD patients with and without diabetes and hypertension. This study was conducted in the Departments of Endocrinology and Cardiovascular Diseases at two University Hospitals. Participants consisted of one hundred and thirty-five patients who have been diagnosed to have CHD and sixty-one control subjects. Serum FGF-<em>21</em> level and levels of fasting blood glucose; triglyceride; apolipoprotein B100; HOMA-IR; insulin; total cholesterol; HDL-cholesterol; LDL-cholesterol; and C-reactive protein were measured. We found that median serum FGF-<em>21</em> levels were significantly higher in CHD than that of control subjects (P<0.0001). Serum FGF-<em>21</em> levels in CHD patients with diabetes, hypertension, or both were higher than that of patients without these comorbidities. Serum FGF-<em>21</em> levels correlated positively with triglycerides, fasting blood glucose, apolipoprotein B100, insulin and HOMA-IR but negatively with HDL-C and apolipoprotein A1 after adjusting for BMI, diabetes and hypertension. Logistic regression analysis demonstrated that FGF-<em>21</em> showed an independent association with triglyceride and apolipoprotein A1.

CONCLUSIONS

High levels of FGF-<em>21</em> are associated with adverse lipid profiles in CHD patients. The paradoxical increase of serum FGF-<em>21</em> in CHD patients may indicate a compensatory response or resistance to FGF-<em>21</em>.

Currently available therapies for diabetes or obesity produce modest efficacy and are usually used in combination with agents targeting cardiovascular risk <em>factors</em>. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a circulating protein with pleiotropic metabolic actions; pharmacological doses of FGF<em>21</em> produce anti-diabetic, lipid-lowering, and weight-reducing effects in rodents. Several potential benefits have translated to non-human primates and obese humans with type 2 diabetes (T2D). Accumulating results point to a specific receptor complex and actions in adipose tissue, liver, and brain; several pathways lead to enhanced fatty acid oxidation, increased insulin sensitivity, and augmented energy expenditure. A range of strategies are being explored to derive potent, safe, and convenient therapies which could potentially represent novel approaches to prevent and treat a variety of metabolic disorders.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) acts as a hormonal regulator during fasting and is involved in lipid metabolism. Fgf<em>21</em> gene expression is regulated by peroxisome proliferator-activated receptor (PPAR)-dependent pathways, which are enhanced during pancreatitis. Therefore, the aim of this study was to investigate FGF<em>21</em>'s role in pancreatic injury.

METHODS

Fgf<em>21</em> expression was quantified during cerulein-induced pancreatitis (CIP) or following mechanical or thapsigargin-induced stress through Northern blot analysis, in situ hybridization, and quantitative reverse transcription polymerase chain reaction. FGF<em>21</em> protein was quantified by Western blot analysis. Isolated acinar cells or AR42J acinar cells were treated with recombinant FGF<em>21</em> protein, and extracellular regulated kinase 1/2 activation was examined. The severity of CIP was compared between wild-type mice and mice overexpressing FGF<em>21</em> (FGF<em>21</em>Tg) or harboring a targeted deletion of Fgf<em>21</em> (Fgf<em>21</em>(-/-)).

RESULTS

Acinar cell Fgf<em>21</em> expression markedly increased during CIP and following injury in vitro. Purified FGF<em>21</em> activated the extracellular regulated kinase 1/2 pathway in pancreatic acinar cells. The severity of CIP is inversely correlated to FGF<em>21</em> expression because FGF<em>21</em>Tg mice exhibited decreased serum amylase and decreased pancreatic stellate cell activation, whereas Fgf<em>21</em>(-/-) mice had increased serum amylase and tissue damage. The expression of Fgf<em>21</em> was also inversely correlated to expression of Early <em>growth</em> response 1, a proinflammatory and profibrotic transcription <em>factor</em>.

CONCLUSIONS

These studies suggest a novel function for Fgf<em>21</em> as an immediate response gene protecting pancreatic acini from overt damage.

To investigate the function of the Rb-related p107 gene, a null mutation in p107 was introduced into the germ line of mice and bred into a BALB/cJ genetic background. Mice lacking p107 were viable and fertile but displayed impaired <em>growth</em>, reaching about 50% of normal weight by <em>21</em> days of age. Mutant mice exhibited a diathetic myeloproliferative disorder characterized by ectopic myeloid hyperplasia in the spleen and liver. Embryonic p107(-/-) <em>fibroblasts</em> and primary myoblasts isolated from adult p107(-/-) mice displayed a striking twofold acceleration in doubling time. However, cell sort analysis indicated that the fraction of cells in G1, S, and G2 was unaltered, suggesting that the different phases of the cell cycle in p107(-/-) cells was uniformly reduced by a <em>factor</em> of 2. Western analysis of cyclin expression in synchronized p107(-/-) <em>fibroblasts</em> revealed that expression of cyclins E and A preceded that of D1. Mutant embryos expressed approximately twice the normal level of Rb, whereas p130 levels were unaltered. Lastly, mutant mice reverted to a wild-type phenotype following a single backcross with C57BL/6J mice, suggesting the existence of modifier genes that have potentially epistatic relationships with p107. Therefore, we conclude that p107 is an important player in negatively regulating the rate of progression of the cell cycle, but in a strain-dependent manner.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family signaling largely controls cellular homeostasis through short-range intercell paracrine communication. Recently FGF15/19, <em>21</em>, and 23 have been implicated in endocrine control of metabolic homeostasis. The identity and location of the FGF receptor isotypes that mediate these effects are unclear. The objective was to determine the role of FGFR4, an isotype that has been proposed to mediate an ileal FGF15/19 to hepatocyte FGFR4 axis in cholesterol homeostasis, in metabolic homeostasis in vivo.

METHODS

FGFR4(-/-) mice-mice overexpressing constitutively active hepatic FGFR4--and FGFR4(-/-) with constitutively active hepatic FGFR4 restored in the liver were subjected to a normal and a chronic high-fat diet sufficient to result in obesity. Systemic and liver-specific metabolic phenotypes were then characterized.

RESULTS

FGFR4-deficient mice on a normal diet exhibited features of metabolic syndrome that include increased mass of white adipose tissue, hyperlipidemia, glucose intolerance, and insulin resistance, in addition to hypercholesterolemia. Surprisingly, the FGFR4 deficiency alleviated high-fat diet-induced fatty liver in obese mice, which is also a correlate of metabolic syndrome. Restoration of FGFR4, specifically in hepatocytes of FGFR4-deficient mice, decreased plasma lipid levels and restored the high-fat diet-induced fatty liver but failed to restore glucose tolerance and sensitivity to insulin.

CONCLUSIONS

FGFR4 plays essential roles in systemic lipid and glucose homeostasis. FGFR4 activity in hepatocytes that normally serves to prevent systemic hyperlipidemia paradoxically underlies the fatty liver disease associated with chronic high-fat intake and obesity.

Syndecan-1 is a ubiquitous and multifunctional extracellular matrix proteoglycan,which mediates basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) binding and activity. Shedding of syndecan-1 ectodomain from the plasma membrane is highly regulated. We evaluated the influence of soluble syndecan-1 and serum bFGF determined by ELISA on outcome in 184 lung cancer patients (non-small cell lung cancer, n = 138; small cell lung cancer, n = 46). Serum syndecan-1 and bFGF levels were determined from sera taken before treatment. The median follow-up of the patients alive (n = <em>21</em>) was 8.1 years (range, 6.6-8.9 years). High serum syndecan-1 and bFGF levels tended to occur in the same patients (P = 0.044). When the serum values corresponding to the highest tertile were used as the cutoff value, the median survival time of the patients with a high serum syndecan-1 level (>59 ng/ml) was 4 months [95% confidence interval (CI), 3-6 months] as compared with 11 months (9-16 months) among those with lower serum levels (P = 0.0001), and the median survival time of the patients with a high bFGF level (>3.4 pg/ml) was 5 months (3-8 months) versus 11 months (8-14 months) in those with a lower level (P = 0.023). In general, the prognostic influence of both <em>factors</em> was independent of the histological subtype. Both serum syndecan-1 level (relative risk, 1.8; 95% CI, 1.1-3.1) and serum bFGF level (relative risk, 1.6; 95% CI, 1.0-2.7) had independent influence on survival in a multivariate survival analysis in non-small cell lung cancer. We conclude that high serum syndecan-1 and bFGF levels at diagnosis are associated with poor outcome in lung cancer.

OBJECTIVE

Nonalcoholic fatty liver disease is a common consequence of human and rodent obesity. Disruptions in lipid metabolism lead to accumulation of triglycerides and fatty acids, which can promote inflammation and fibrosis and lead to nonalcoholic steatohepatitis. Circulating levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>21</em> increase in patients with nonalcoholic fatty liver disease or nonalcoholic steatohepatitis; therefore, we assessed the role of FGF<em>21</em> in the progression of murine fatty liver disease, independent of obesity, caused by methionine and choline deficiency.

METHODS

C57BL/6 wild-type and FGF<em>21</em>-knockout (FGF<em>21</em>-KO) mice were placed on methionine- and choline-deficient (MCD), high-fat, or control diets for 8-16 weeks. Mice were weighed, and serum and liver tissues were collected and analyzed for histology, levels of malondialdehyde and liver enzymes, gene expression, and lipid content.

RESULTS

The MCD diet increased hepatic levels of FGF<em>21</em> messenger RNA more than 50-fold and serum levels 16-fold, compared with the control diet. FGF<em>21</em>-KO mice had more severe steatosis, fibrosis, inflammation, and peroxidative damage than wild-type C57BL/6 mice. FGF<em>21</em>-KO mice had reduced hepatic fatty acid activation and β-oxidation, resulting in increased levels of free fatty acid. FGF<em>21</em>-KO mice given continuous subcutaneous infusions of FGF<em>21</em> for 4 weeks while on an MCD diet had reduced steatosis and peroxidative damage, compared with mice not receiving FGF<em>21</em>. The expression of genes that regulate inflammation and fibrosis were reduced in FGF<em>21</em>-KO mice given FGF<em>21</em>, similar to those of wild-type mice.

CONCLUSIONS

FGF<em>21</em> regulates fatty acid activation and oxidation in livers of mice. In the absence of FGF<em>21</em>, accumulation of inactivated fatty acids results in lipotoxic damage and increased steatosis.

White adipose tissue is recognized as both a site of energy storage and an endocrine organ that produces a myriad of endocrine <em>factors</em> called adipokines. Brown adipose tissue (BAT) is the main site of nonshivering thermogenesis in mammals. The amount and activity of brown adipocytes are associated with protection against obesity and associated metabolic alterations. These effects of BAT are traditionally attributed to its capacity for the oxidation of fatty acids and glucose to sustain thermogenesis. However, recent data suggest that the beneficial effects of BAT could involve a previously unrecognized endocrine role through the release of endocrine <em>factors</em>. Several signaling molecules with endocrine properties have been found to be released by brown fat, especially under conditions of thermogenic activation. Moreover, experimental BAT transplantation has been shown to improve glucose tolerance and insulin sensitivity mainly by influencing hepatic and cardiac function. It has been proposed that these effects are due to the release of endocrine <em>factors</em> by brown fat, such as insulin-like <em>growth</em> <em>factor</em> I, interleukin-6, or <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em>. Further research is needed to determine whether brown fat plays an endocrine role and, if so, to comprehensively identify which endocrine <em>factors</em> are released by BAT. Such research may reveal novel clues for the observed association between brown adipocyte activity and a healthy metabolic profile, and it could also enlarge a current view of potential therapeutic tools for obesity and associated metabolic diseases.

OBJECTIVE

To determine the role and expression of the cytokine/receptor pair interleukin-<em>21</em> (IL-<em>21</em>)/IL-<em>21</em> receptor (IL-<em>21</em>R) in rheumatoid arthritis (RA).

METHODS

The expression of IL-<em>21</em>R and IL-<em>21</em> was analyzed by TaqMan real-time polymerase chain reaction (PCR) and in situ hybridization of synovial biopsy samples from patients with RA and osteoarthritis (OA). Double labeling by immunohistochemistry after in situ hybridization was performed with anti-CD68 antibodies. The expression of IL-<em>21</em>R at the protein level was confirmed by Western blotting. Stimulation experiments were performed with recombinant IL-1beta, tumor necrosis factor alpha (TNFalpha), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGFbeta). The role of IL-<em>21</em>R in cartilage destruction was analyzed in the SCID mouse coimplantation model of RA.

RESULTS

IL-<em>21</em>R was found in total RNA extracts and in synovial biopsy samples from RA patients, whereas no expression or only minimal expression was seen in samples from OA patients. Double labeling indicated that both synovial macrophages and synovial fibroblasts expressed IL-<em>21</em>R. Western blotting with anti-IL-<em>21</em>R antibodies confirmed the expression of IL-<em>21</em>R protein in RA synovial fibroblasts (RASFs). Of note, IL-<em>21</em> was not detectable by real-time PCR and in situ hybridization in the same samples in vivo as in vitro. The level of expression of IL-<em>21</em>R messenger RNA (mRNA) was not altered by stimulation with IL-1beta, TNFalpha, PDGF, or TGFbeta. Interestingly, in the SCID mouse coimplantation model, RASFs did not maintain their expression of IL-<em>21</em>R at sites of invasion into the cartilage. Similarly, IL-<em>21</em>R mRNA was not expressed at sites of invasion into cartilage and bone in RA synovium.

CONCLUSIONS

Our data demonstrate that IL-<em>21</em>R is expressed in RA synovium by RASFs and synovial macrophages. IL-<em>21</em>R is associated with the activated phenotype of RASFs independently of the major proinflammatory cytokines IL-1beta and TNFalpha, but correlates negatively with the destruction of articular cartilage and bone.

We established a continuous cell line, NCI-H295, from an invasive primary adrenocortical carcinoma. The cell line was established in a fully defined medium (HITES) and later could be adapted for <em>growth</em> in a simple medium supplemented only with selenium, insulin, and transferrin and devoid of serum, steroids, <em>fibroblast</em> <em>growth</em> <em>factor</em>, and a source of exogenous cholesterol. NCI-H295 cells had a relatively long population doubling time and were tumorigenic when inoculated s.c. into athymic nude mice. The cultured cells had ultrastructural features of steroid-secreting cells and contained complex cytogenetic abnormalities including the presence of multiple marker chromosomes. Steroid analyses (radioimmunoassays and mass spectrometry), performed 7 to 9 years after culture initiation, demonstrated secretion of more than 30 steroids characteristic of adrenocortical cells. Total unconjugated steroid secretion in serum-supplemented medium was 2.83 micrograms/10(6) cells/24 h and about 4-fold less in serum-free medium. The major pathway of pregnenolone metabolism in NCI-H295 cells is androgen synthesis, with formation of dehydroepiandrosterone, androstenedione, testotesterone, and at least three sulfated androgens, as well as estrogens. In addition, formation of cortisol, corticosterone, aldosterone, and 11 beta-hydroxyandrostenidione indicated the presence of 11 beta-hydroxylase. Thus, multiple pathways of steroidogenesis are expressed by NCI-H295 cells, including formation of corticosteroids, mineralocorticoids, androgens, and estrogens. Our findings indicate the presence in NCI-H295 cells of all of the major adrenocortical enzyme systems, including 11 beta-hydroxylase, desmolase, <em>21</em> alpha-hydroxylase, 17 alpha-hydroxylase, 18-hydroxylase, lyase, sulfokinase, and aromatase. The NCI-H295 cell line should prove of value in studying the regulation, metabolic pathways, and enzymes involved in steroid formation and secretion. In addition, it may provide insights into the biology and treatment of adrenocortical carcinoma.