Malignant gliomas are highly angiogenic and aggressive tumors. IFN-beta has been used for the treatment of patients with malignant glioma; however, its antitumor mechanism in vivo remains unclear. To understand the in vivo antitumor effect and mechanism of recombinant human IFN-beta (rhIFN-beta) depending on the stages of tumor development or progression, we used orthotopic xenograft brain tumors generated by stereotactic intracerebral implantation of U-87 human glioma cells in nude mice. Mice bearing tumors 7 days (group 1) and <em>21</em> days (group 2) postimplant were treated with 2 x 10(5) IU/day of rhIFN-beta or saline i.p. for 15 days, respectively. Tumor <em>growth</em> was suppressed by 69.6% in group 1 and 10.8% in group 2 compared with tumors of each control group treated with saline. rhIFN-beta-treated group 1 animals showed 38% reduction in vascularization along with a 2.5-fold increase of the apoptotic index and no change in the proliferative index as compared with untreated tumors. The expression level of vascular endothelial cell <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> was not affected by rhIFN-beta treatment. rhIFN-beta showed inhibitory activity on proliferation of U-87 cells, human umbilical vein endothelial cells, and PAM <em>21</em>2 murine keratinocytes in vitro. Our results indicate that the in vivo antitumor effect of rhIFN-beta on malignant gliomas may be mediated, at least in part, via angiogenesis inhibition rather than antiproliferative activity and that rhIFN-beta may be more effective for the treatment of malignant glioma patients at an early stage with minimal or microscopic tumor burdens rather than at an advanced stage of tumor development.

OBJECTIVE

To identify changes in growth factor expression in miniature pig retinas following retinal laser photocoagulation.

METHODS

Pigs were sacrificed at different times (15 minutes to 42 days) post-laser and the retinas were immunolabelled for basic fibroblast growth factor, insulin-like growth factor I, transforming growth factor beta, epidermal growth factor, transforming growth factor alpha, platelet derived growth factor, vascular endothelial growth factor, and epidermal growth factor receptor. Total mRNA levels were also determined.

RESULTS

With the exception of vascular endothelial growth factor, immunoreactivity for all other growth factors studied and epidermal growth factor receptor was observed throughout normal non-lasered control retina, generally being high in the retinal pigment epithelium and low in the neural retina. Changes in growth factor expression following laser photocoagulation were observed only in burn areas and changes were mainly confined to the retinal pigment epithelium and outer nuclear layer. The immunoreactivity within retinal pigment epithelial cells in burn areas was either absent or decreased following laser treatment but returned to normal by 21 days. The immunoreactivity was increased within the outer nuclear layer of burn areas during the healing process but returned to normal by 42 days. Vascular endothelial growth factor immunoreactivity was weak/absent in the normal retina and remained unchanged following laser photocoagulation. Change of total mRNA levels in burn areas during time post-laser was confined to retinal pigment epithelial cells, being low immediately following photocoagulation and returning to normal by 42 days.

CONCLUSIONS

These results demonstrate a temporal alteration in growth factor expression and transcriptional activity in the retina following laser photocoagulation.

FGF21 is a novel secreted protein with robust anti-diabetic, anti-obesity, and anti-atherogenic activities in preclinical species. In the current study, we investigated the signal transduction pathways downstream of FGF21 following acute administration of the growth factor to mice. Focusing on adipose tissues, we identified FGF21-mediated downstream signaling events and target engagement biomarkers. Specifically, RNA profiling of adipose tissues and phosphoproteomic profiling of adipocytes, following FGF21 treatment revealed several specific changes in gene expression and post-translational modifications, specifically phosphorylation, in several relevant proteins. Affymetrix microarray analysis of white adipose tissues isolated from both C57BL/6 (fed either regular chow or HFD) and db/db mice identified over 150 robust potential RNA transcripts and over 50 potential secreted proteins that were changed greater than 1.5 fold by FGF21 acutely. Phosphoprofiling analysis identified over 130 phosphoproteins that were modulated greater than 1.5 fold by FGF21 in 3T3-L1 adipocytes. Bioinformatic analysis of the combined gene and phosphoprotein profiling data identified a number of known metabolic pathways such as glucose uptake, insulin receptor signaling, Erk/Mapk signaling cascades, and lipid metabolism. Moreover, a number of novel events with hitherto unknown links to FGF21 signaling were observed at both the transcription and protein phosphorylation levels following treatment. We conclude that such a combined "omics" approach can be used not only to identify robust biomarkers for novel therapeutics but can also enhance our understanding of downstream signaling pathways; in the example presented here, novel FGF21-mediated signaling events in adipose tissue have been revealed that warrant further investigation.

OBJECTIVE

Intracisternal basic fibroblast growth factor (bFGF) enhances sensorimotor recovery after focal cerebral infarction in rats. One possible mechanism is stimulation of endogenous progenitor cells in brain. We investigated the effects of intracisternal bFGF on brain progenitor cells after stroke.

METHODS

Proliferating brain cells were labeled with bromodeoxyuridine (BrdU) before middle cerebral artery (MCA) occlusion or sham surgery in rats. bFGF (0.5 microg) or vehicle was administered intracisternally at 24 and 48 hours after MCA occlusion, and rats were killed at 7, 14, or 21 days after stroke. Immunohistochemistry for BrdU and neuron- or astrocyte-specific markers was used to characterize progenitor cells and their progeny in the subventricular zone and dentate gyrus of the hippocampus.

RESULTS

Infarct size did not differ among rats with or without bFGF treatment. MCA occlusion alone increased the number of BrdU-labeled cells in the ipsilateral subventricular zone at days 7 to 21, and there was a trend toward increased cell proliferation with bFGF treatment. In the dentate gyrus, the number of BrdU-labeled cells was increased bilaterally after MCA occlusion (peak at day 7). This increase was greater after bFGF treatment. In the subventricular zone, 30% of BrdU-labeled cells were immunopositive for the immature neuron-specific marker doublecortin at day 7, and their number declined to 2% at day 21. In the dentate gyrus, the majority of BrdU-labeled cells colabeled with doublecortin at day 7, becoming NeuN positive at day 21.

CONCLUSIONS

Stroke produces significant changes in progenitor cells in brain that are augmented by bFGF treatment.

BACKGROUND

<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, and it is still unclear whether FGF<em>21</em> is related to atherosclerosis. Here, we explored the potential link between FGF<em>21</em> and lower extremity atherosclerotic disease (LEAD) in type 2 diabetic patients.

METHODS

A cross-sectional study was conducted on 504 type 2 diabetic patients (283 men, 2<em>21</em> women). LEAD was defined by Ankle-brachial index (ABI) <0.9 and lower extremity arterial plaque evaluated by color Doppler ultrasound. Serum FGF<em>21</em> concentrations were quantified by a sandwich enzyme-linked immunosorbent assay.

RESULTS

The total FGF<em>21</em> levels of male and female patients had no significant differenence ((299.14(177.31-534.49) vs 362.50(<em>21</em>4.01-578.73), P=0.516). Serum FGF<em>21</em> levels in LEAD group were significantly higher than non-LEAD group in females (385.34(243.89-661.54) vs 313.13(156.38-485.79), P=0.006), while not in male patients (295.52(177.09-549.64) vs 342.09 (198.70-549.87), P=0.613). In diabetic women, subjects with LEAD had significantly higher serum FGF<em>21</em> regardless of non-alcoholic fatty liver disease (NAFLD) (P < 0.05). And serum FGF<em>21</em> levels were positively correlated with waist circumference and systolic blood pressure after adjusted for age and BMI (r=0.198, P=0.004; r=0.152, P=0.027; respectively). Moreover, FGF<em>21</em> was independently tied to femoral intima-media thickness (FIMT) (β=0.208, P=0.031). After adjusted for other LEAD risk <em>factor</em>s, FGF<em>21</em> was demonstrated to be an independent risk <em>factor</em> for LEAD in type 2 diabetic women (OR, 1.106; 95%CI 1.008-1.223; P=0.028). In addition, FGF<em>21</em> was negatively correlated with estradiol in premenopausal diabetic women (r=-0.368, P=0.009). After adjusted for estradiol, serum FGF<em>21</em> levels were still positively associated with FIMT in premenopausal diabetic women (r=0.381, P=0.007). In diabetic men, serum FGF<em>21</em> levels were correlated with triglyceride and C-reactive protein even after adjusted for age and BMI (r=0.204, P=0.001; r=0.312, P < 0.001; respectively). However, serum FGF<em>21</em> was not an independent impact <em>factor</em> for LEAD in men (P>> 0.05).

CONCLUSIONS

Serum FGF<em>21</em> level independently and positively links LEAD in Chinese women with type 2 diabetes. The gender difference may be due to different estrogen levels.

BACKGROUND

Ticks are obligate hematophagous ectoparasites that suppress the host's immune and inflammatory responses by secreting immuno-modulatory and anti-inflammatory molecules in their saliva. In previous studies we have shown that tick salivary gland extract (SGE) and saliva from Dermacentor variabilis have distinct effects on platelet-derived <em>growth</em> <em>factor</em> (PDGF)-stimulated IC-<em>21</em> macrophage and NIH3T3-L1 <em>fibroblast</em> migration. Since tick saliva contains a high concentration of prostaglandin E2 (PGE2), a potent modulator of inflammation, we used a PGE2 receptor antagonist to evaluate the role of PGE2 in the different migratory responses induced by saliva and its impact on macrophage cytokine profile.

METHODS

Adult ticks were fed on female New Zealand white rabbits for 5-8 days. Female ticks were stimulated with dopamine/theophylline to induce salivation and saliva was pooled. Competitive enzyme immunoassays (EIA) were used to measure saliva PGE2 content and the changes in macrophage intracellular cyclic adenosine monophosphate (cAMP) levels. The effects of tick saliva on macrophage and fibroblast migration were assessed in the absence and presence of the PGE2 receptor antagonist, AH 6809, using blind well chamber assays. A cytokine antibody array was used to examine the effects of tick saliva on macrophage cytokine secretion. Statistical significance was determined by one-way ANOVA; Student Newman-Kuels post-test was used for multiple comparisons.

RESULTS

The saliva-induced increase in PDGF-stimulated macrophage migration was reversed by AH 6809. The inhibition of PDGF-stimulated fibroblast migration by saliva was also antagonist-sensitive. Tick saliva induced macrophages to secrete copious amounts of PGE2, and conditioned medium from these cells caused an AH 6809-sensitive inhibition of stimulated fibroblast migration, showing that macrophages can regulate fibroblast activity. We show that tick saliva decreased the secretion of the pro-inflammatory cytokines regulated and normal T cell expressed and secreted (RANTES/CCL5), tumor necrosis factor-alpha (TNF-α), and soluble TNF receptor I (sTNFRI) through a PGE2-dependent mechanism mediated by cAMP. Saliva had similar effects on lipopolysaccharide (LPS) stimulated macrophages.

CONCLUSIONS

Our data show that ticks utilize salivary PGE2 to subvert the ability of macrophages to secrete pro-inflammatory mediators and recruit fibroblasts to the feeding lesion, therefore inhibiting wound healing.

We have shown previously (Villalonga, P., López- Alcalá, C., Bosch, M., Chiloeches, A., Rocamora, N., Gil, J., Marais, R., Marshall, C. J., Bachs, O., and Agell, N. (2001) Mol. Cell. Biol. <em>21</em>, 7345-7354) that calmodulin negatively regulates Ras activation in <em>fibroblasts</em>. Hence, anti-calmodulin drugs (such as W13, trifluoroperazine, or W7) are able to induce Ras/ERK pathway activation under low levels of <em>growth</em> <em>factors</em>. We show here that cell treatment with protein kinase C (PKC) inhibitors abolishes W13-induced activation of Ras, Raf-1, and ERK. Consequently, PKC activity is essential for achieving the synergism between calmodulin inhibition and <em>growth</em> <em>factors</em> to activate Ras. Furthermore, whereas the activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) does not induce Ras activation in 3T3 cells, activation is observed if calmodulin is simultaneously inhibited. This indicates that calmodulin is preventing Ras activation by PKC. Treatment of cells with epidermal <em>growth</em> <em>factor</em> receptor or platelet-derived <em>growth</em> <em>factor</em> receptor tyrosine kinase inhibitors does not abrogate the activation of Ras by calmodulin inhibition. This implies that epidermal <em>growth</em> <em>factor</em> receptor and platelet-derived <em>growth</em> <em>factor</em> receptor tyrosine kinase activities are dispensable for the activation of Ras by TPA plus W13, and, therefore, Ras activation is not a consequence of the transactivation of those receptors by the combination of the anti-calmodulin drug plus TPA. Furthermore, K-Ras, the isoform previously shown to bind to calmodulin, is the only one activated by TPA when calmodulin is inhibited. These data suggest that direct interaction between K-Ras and calmodulin may account for the inability of PKC to activate Ras in 3T3 <em>fibroblasts</em>. In vitro experiments showed that the phosphorylation of K-Ras by PKC was inhibited by calmodulin, suggesting that calmodulin-dependent modulation of K-Ras phosphorylation by PKC could be the mechanism underlying K-Ras activation in <em>fibroblasts</em> treated with TPA plus W13.

Our understanding of the mechanisms underlying process in Alzheimer's disease (AD) is far from completion and new therapeutic targets are urgently needed. Recently, the link between dementia and diabetes mellitus (DM) prompted us to search for new therapeutic strategies from glucose metabolism regulators for neurodegeneration. Previous studies have indicated that <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), an attractive and potential therapeutic treatment for DM, may exert diverse effects in the central nervous system. However, the specific biological function and mechanisms of FGF<em>21</em> on AD is still largely unknown. We report here a study in vivo and in vitro of the neuroprotective effects of FGF<em>21</em> on cell apoptosis, tau hyperphosphorylation and oxidative stress induced by amyloid β-peptide 25-35. In the present study, the results also further provided evidence for molecular mechanisms by which FGF<em>21</em> exerted its beneficial effects in neuron and suggested that the regulation of protein phosphatase 2A / mitogen-activated protein kinases / hypoxia-inducible <em>factor</em>-1α pathway may play a key role in mediating the neuroprotective effects of FGF<em>21</em> against AD-like pathologies.

Methionine restriction (MR) extends lifespan across different species. The main responses of rodent models to MR are well-documented in adipose tissue (AT) and liver, which have reduced mass and improved insulin sensitivity, respectively. Recently, molecular mechanisms that improve healthspan have been identified in both organs during MR. In fat, MR induced a futile lipid cycle concomitant with beige AT accumulation, producing elevated energy expenditure. In liver, MR upregulated <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> and improved glucose metabolism in aged mice and in response to a high-fat diet. Furthermore, MR also reduces mitochondrial oxidative stress in various organs such as liver, heart, kidneys, and brain. Other effects of MR have also been reported in such areas as cardiac function in response to hyperhomocysteinemia (HHcy), identification of molecular mechanisms in bone development, and enhanced epithelial tight junction. In addition, rodent models of cancer responded positively to MR, as has been reported in colon, prostate, and breast cancer studies. The beneficial effects of MR have also been documented in a number of invertebrate model organisms, including yeast, nematodes, and fruit flies. MR not only promotes extended longevity in these organisms, but in the case of yeast has also been shown to improve stress tolerance. In addition, expression analyses of yeast and Drosophila undergoing MR have identified multiple candidate mediators of the beneficial effects of MR in these models. In this review, we emphasize other in vivo effects of MR such as in cardiovascular function, bone development, epithelial tight junction, and cancer. We also discuss the effects of MR in invertebrates.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>) is a new metabolic regulator with beneficial effects on lipid and glucose metabolism in animal models of diabetes mellitus. The aim of this study was to explore the relationship between FGF-<em>21</em> and diabetic nephropathy in humans. Serum FGF-<em>21</em> levels were determined in groups of control (n = 50) and type 2 diabetes mellitus (T2DM) patients with normoalbuminuria (n = 158), microalbuminuria (n = 68), and macroalbuminuria (n = 38) using enzyme-linked immunosorbent assay. Multiple linear regression models were used to analyze the associations between FGF-<em>21</em> or other biomedical indices and urinary albumin excretion (UAE). Median serum FGF-<em>21</em> levels were increased in T2DM patients compared with nondiabetic controls and were significantly higher in patients of higher UAE group. In groups of control and T2DM patients with normoalbuminuria, microalbuminuria, and macroalbuminuria, median serum (interquartile range) FGF-<em>21</em> levels were 467.89 (294.59-519.56), 492.30 (354.59-640.42), 595.01 (480.49-792.31), and 665.20 (448.68-829.75) ng/L (P < .001), respectively. After adjustment for the confounders, FGF-<em>21</em>, fasting plasma glucose, and high-density lipoprotein cholesterol levels were found to be independently associated with UAE in diabetic patients. Serum FGF-<em>21</em> level is independently correlated with UAE in T2DM patients, indicating that circulating FGF-<em>21</em> may be involved in diabetic nephropathy.

We previously reported that rat oligodendrocyte progenitors (OLP) express receptors for the pituitary adenylyl cyclase-activating peptide (PACAP) in vivo and in vitro. Addition of PACAP to cultured OLP triggered a potent elevation in intracellular cAMP contents, a dose-dependent stimulation of proliferation, and a delay in myelinogenesis (Lee M, Lelievre V, Zhao P, Torres M, Rodriguez W, Byun JY, Doshi S, Ioffe Y, Gupta G, de los Monteros AE, de Vellis J, Waschek J. Pituitary adenylyl cyclase-activating polypeptide stimulates DNA synthesis but delays maturation of oligodendrocyte progenitors. J Neurosci. 2001 <em>21</em>:3849-59.). In an attempt to understand how PACAP might interact with <em>growth</em> <em>factors</em> known to stimulate OLP proliferation, we investigated PACAP actions on OLP proliferation in the presence of <em>Fibroblast</em> <em>Growth</em> <em>Factor</em>-2 (FGF-2) and PDGF. Multiple PACAP receptor subtype mRNAs and splice variants were detected in these cultures. PACAP by itself potently stimulated OLP proliferation and enhanced the ability of FGF-2 to stimulate DNA synthesis. In contrast, this peptide strongly antagonized the mitogenic effects of PDGF in association with a reduction of PDGFalpha receptor gene expression. Additionally, we investigated the interaction of PACAP with the morphogenetic <em>factor</em> sonic hedgehog (Shh), which recently was shown to be crucial for oligodendrocyte generation. OLP cultures were found to express mRNAs for both ptc1 (Shh receptor) and gli1 (Shh target gene) and responded to Shh treatment with an increase in proliferation. PACAP antagonized the ability of Shh to stimulate OLP proliferation. Moreover, transcriptional targets of Shh signaling were also reduced by this treatment, suggesting that PACAP directly antagonized Shh signaling. These studies reveal complex in vitro interactions of PACAP with other <em>factors</em> involved in OLP development.

Adipose tissue is known to secrete hormones actively and produces many biologically active proteins called adipocytokines. Typically, obesity is followed by low-grade inflammation, which is characterized by increased circulating levels of pro-inflammatory cytokines. Macrophages play a role in the inflammatory process by secreting many cytokines such as tumor necrosis <em>factor</em> alpha, interleukin-6, resistin, and retinol binding protein-4. These cytokines and chemokines participate in low-grade pro-inflammatory processes leading to insulin resistance, metabolic impairment, and cardiovascular diseases. More metabolic regulators, such as <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>21</em>, FGF19, FGF1, vaspin, and visfatin have now been discovered but their exact roles in human diseases are still unclear. This review focuses on recent research regarding the role of adipokines and new metabolic <em>factors</em> in metabolic derangement or cardiovascular disease.

Mesenchymal stem cells (MSCs) have the potential to be the source for cell-based therapies. However, MSCs can undergo malignant transformation in a tumor microenvironment where a high level of interleukin (IL)-6 is present. In this study, we investigated the role of IL-6 and signal transducer and activator of transcription 3 (STAT3) signaling in malignant transformation of MSCs. Rat MSCs were isolated and indirectly cocultured with C6 glioma cells. Coculture of MSCs with astrocytes was used as a control. After 7 days of culture, the cells were assessed for malignant transformation using MTT assay and immunofluorescence staining. The levels of hepatocyte <em>growth</em> <em>factor</em>, IL-6, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and the expression of STAT3 and soluble IL-6 receptor in the cultured cells and conditioned media were measured using RT-PCR, ELISA, and Western blot analysis. The expression levels of STAT3 downstream targets, CyclinD1 and Bcl-xl, were determined as well. Our data showed that almost all of the MSCs became phenotypically malignant after indirect coculture with glioma cells, which was confirmed by tumor formation assays when these cells were injected into nude mice. The expression of IL-6 was significantly increased in MSCs cocultured with glioma cells, which was associated with significantly increased expressions of soluble IL-6 receptor, transmembrane glycoprotein GP130, STAT3, phosphorylated STAT3, CyclinD1, and Bcl-xl. Similar results were obtained when the MSCs were treated with IL-6. Treatment of the cocultured MSCs and glioma cells with STA-<em>21</em>, to block the constitutive STAT3 signaling, reduced the risk of MSC tumor-like transformation in the tumor microenvironment. These data suggest that IL-6 plays a critical role in malignant transformation of rat MSCs, which is associated with an enhancement of the STAT3 signaling pathway in the tumor microenvironment.

To assess the safety, tolerability, pharmacokinetics and pharmacodynamics of PF-05231023, a long-acting <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) analogue, in obese people with hypertriglyceridaemia on atorvastatin, with or without type 2 diabetes.

Participants received PF-05231023 or placebo intravenously once weekly for 4 weeks. Safety (12-lead ECGs, vital signs, adverse events [AEs], laboratory tests) and longitudinal weight assessments were performed. Blood samples were collected for pharmacokinetic and pharmacodynamic analyses. Cardiovascular safety studies were also conducted in telemetered rats and monkeys. Blood pressure (BP; mean, systolic and diastolic) and ECGs were monitored.

A total of 107 people were randomized. PF-05231023 significantly decreased mean placebo-adjusted fasting triglycerides (day 25, 33%-43%) and increased HDL cholesterol (day 25, 15.7%-28.6%) and adiponectin (day 25, 1574 to 3272 ng/mL) across all doses, without significant changes in body weight (day 25, -0.45% to -1.<em>21</em>%). Modest decreases from baseline were observed for N-terminal propeptides of type 1 collagen (P1NP) on day 25, although C-telopeptide cross-linking of type 1 collagen (CTX-1) increased minimally. Systolic, diastolic BP, and pulse rate increased in a dose- and time-related manner. There were 5 serious AEs (one treatment-related) and no deaths. Three participants discontinued because of AEs. The majority of AEs were gastrointestinal. PF-05231023 increased BP and heart rate in rats, but not in monkeys.

Once-weekly PF-05231023 lowered triglycerides markedly in the absence of weight loss, with modest changes in markers of bone homeostasis. This is the first report showing increases in BP and pulse rate in humans and rats after pharmacological administration of a long-acting FGF<em>21</em> molecule.

Although the pharmacological effects of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) are well-documented, uncertainty about its role in regulating excessive energy intake remains. Here, we show that FGF<em>21</em> improves systemic insulin sensitivity by promoting the healthy expansion of subcutaneous adipose tissue (SAT). Serum FGF<em>21</em> levels positively correlate with the SAT area in insulin-sensitive obese individuals. FGF<em>21</em> knockout mice (FGF<em>21</em>KO) show less SAT mass and are more insulin-resistant when fed a high-fat diet. Replenishment of recombinant FGF<em>21</em> to a level equivalent to that in obesity restores SAT mass and reverses insulin resistance in FGF<em>21</em>KO, but not in adipose-specific βklotho knockout mice. Moreover, transplantation of SAT from wild-type to FGF<em>21</em>KO mice improves insulin sensitivity in the recipients. Mechanistically, circulating FGF<em>21</em> upregulates adiponectin in SAT, accompanied by an increase of M2 macrophage polarization. We propose that elevated levels of endogenous FGF<em>21</em> in obesity serve as a defense mechanism to protect against systemic insulin resistance.

The cochlear implant (CI) involves the introduction of alloplastic materials into the cochlea. While current implants interact with cochlear neurons at a distance, direct interactions between spiral ganglion (SG) neurites and implants could be fostered by appropriate treatment with neurotrophic <em>factors</em>. The interactions of <em>fibroblasts</em> and osteoblasts with alloplastic materials have been well studied in vitro and in vivo. However, interactions of inner ear neurons with such alloplastic materials have yet to be described. To investigate survival and <em>growth</em> behavior of SG neurons on different materials, SG explants from post-natal day 5 rat SG were cultured for 72 h in the presence of neurotrophin-3 (10 ng/ml) on titanium, gold, stainless steel, platinum, silicone and plastic surfaces that had been coated with laminin and poly-L-lysine. Neurite out<em>growth</em> was investigated after immunohistological staining for neurofilament, by image analysis to determine neurite extension and directional changes. Neurite morphology and adhesion to the alloplastic material were also evaluated by scanning electron microscopy (SEM). On titanium, SG neurites reached the highest extent of out<em>growth</em>, with an average length of 662 microm and a mean of 31 neurites per explant, compared to 568 microm and <em>21</em> neurites on gold, 574 microm and 24 neurites on stainless steel, 509 microm and 16 neurites on platinum, 281 microm and 12 neurites on silicone and 483 microm and 31 neurites on plastic. SEM revealed details of adhesion of neurites and interaction with non-neuronal cells. The results of this study indicate that the <em>growth</em> of SG neurons in vitro is strongly influenced by alloplastic materials, with titanium exhibiting the highest degree of biocompatibility with respect to neurite extension. The knowledge of neurite interaction with different alloplastic materials is of clinical interest, as development in CI technology leads to closer contact of implanted electrodes with surviving inner ear neurons.

Diets deficient in protein often increase food consumption, body weight and fat mass; however, the underlying mechanisms remain poorly understood. We compared the effects of diets varying in protein concentrations on energy balance in obesity-prone rats. We demonstrate that protein-free (0% protein calories) diets decreased energy intake and increased energy expenditure, very low protein (5% protein) diets increased energy intake and expenditure, whereas moderately low protein (10% protein) diets increased energy intake without altering expenditure, relative to control diet (15% protein). These diet-induced alterations in energy expenditure are in part mediated through enhanced serotonergic and β-adrenergic signaling coupled with upregulation of key thermogenic markers in brown fat and skeletal muscle. The protein-free and very low protein diets decreased plasma concentrations of multiple essential amino acids, anorexigenic and metabolic hormones, but these diets increased the tissue expression and plasma concentrations of <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em>. Protein-free and very low protein diets induced fatty liver, reduced energy digestibility, and decreased lean mass and body weight that persisted beyond the restriction period. In contrast, moderately low protein diets promoted gain in body weight and adiposity following the period of protein restriction. Together, our findings demonstrate that low protein diets produce divergent effects on energy balance.

MicroRNAs (miRs) are small conserved RNA that regulate gene expression. Bioinformatic analysis of miRNA profiles during mouse lung development indicated a role for multiple miRNA, including miRNA-489. miR-489 increased on completion of alveolar septation [postnatal day 42 (P42)], associated with decreases in its conserved target genes insulin-like <em>growth</em> <em>factor</em>-1 (Igf1) and tenascin C (Tnc). We hypothesized that dysregulation of miR-489 and its target genes Igf1 and Tnc contribute to hyperoxia-induced abnormal lung development. C57BL/6 mice were exposed to normoxia (<em>21</em>%) or hyperoxia (85% O2) from P4 to P14, in combination with intranasal locked nucleic acid against miR-489 to inhibit miR-489, cytomegalovirus promoter (pCMV)-miR-489 to overexpress miR-489, or empty vector. Hyperoxia reduced miR-489 and increased Igf1 and Tnc. Locked nucleic acid against miR-489 improved lung development during hyperoxia and did not alter it during normoxia, whereas miR-489 overexpression inhibited lung development during normoxia. The 3' untranslated region in vitro reporter studies confirmed Igf1 and Tnc as targets of miR-489. While miR-489 was of epithelial origin and present in exosomes, its targets Igf1 and Tnc were produced by <em>fibroblasts</em>. Infants with bronchopulmonary dysplasia (BPD) had reduced lung miR-489 and increased Igf1 and Tnc compared with normal preterm or term infants. These results suggest increased miR-489 is an inhibitor of alveolar septation. During hyperoxia or BPD, reduced miR-489 and increased Igf1 and Tnc may be inadequate attempts at compensation. Further inhibition of miR-489 may permit alveolar septation to proceed. The use of specific miRNA antagonists or agonists may be a therapeutic strategy for inhibited alveolarization, such as in BPD.

Blood-brain barrier (BBB) disruption and dysfunction result in brain edema, which is responsible for more than half of all deaths after severe traumatic brain injury (TBI). <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has a potential neuroprotective function in the brain. However, the effects and underlying possible mechanism of action on BBB integrity following TBI remain unknown. The purpose of the current study was to determine the effects of FGF<em>21</em> on BBB protection and TBI treatment. The effects of recombinant human FGF<em>21</em> (rhFGF<em>21</em>) on BBB integrity and on tight junction (TJ) and adhesion junction (AJ) proteins were investigated both in a TBI mouse model and an in vitro BBB disruption model established with tumor necrosis <em>factor</em> alpha (TNF-α)-induced human brain microvascular endothelial cells (HBMECs). The ability of rhFGF<em>21</em> to form an FGF<em>21</em>/FGFR1/β-klotho complex was confirmed by in vitro β-klotho small interfering RNA (siRNA) transfection and FGFR1 co-immunoprecipitation. In addition, the specific FGFR1 and peroxisome proliferator-activated receptor gamma (PPARγ) inhibitors PD173074 and GW9662, respectively, were applied to further explore the possible mechanism of rhFGF<em>21</em> in BBB maintenance after TBI. rhFGF<em>21</em> markedly reduced neurofunctional behavior deficits and cerebral edema degree, preserved BBB integrity, and recued brain tissue loss and neuron apoptosis in the mouse model after TBI. Both in vivo and in vitro, rhFGF<em>21</em> upregulated TJ and AJ proteins, thereby preserving the BBB. Moreover, rhFGF<em>21</em> activated PPARγ in TNF-α-induced HBMECs through formation of an FGF<em>21</em>/FGFR1/β-klotho complex. rhFGF<em>21</em> protected the BBB through FGF<em>21</em>/FGFR1/β-klotho complex formation and PPARγ activation, which upregulated TJ and AJ proteins.

Transcriptional regulation of metabolic genes in the liver is the key to maintaining systemic energy homeostasis during starvation. The membrane-bound transcription <em>factor</em> cAMP-responsive element-binding protein 3-like 3 (CREB3L3) has been reported to be activated during fasting and to regulate triglyceride metabolism. Here, we show that CREB3L3 confers a wide spectrum of metabolic responses to starvation in vivo. Adenoviral and transgenic overexpression of nuclear CREB3L3 induced systemic lipolysis, hepatic ketogenesis, and insulin sensitivity with increased energy expenditure, leading to marked reduction in body weight, plasma lipid levels, and glucose levels. CREB3L3 overexpression activated gene expression levels and plasma levels of antidiabetic hormones, including <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> and IGF-binding protein 2. Amelioration of diabetes by hepatic activation of CREB3L3 was also observed in several types of diabetic obese mice. Nuclear CREB3L3 mutually activates the peroxisome proliferator-activated receptor (PPAR) α promoter in an autoloop fashion and is crucial for the ligand transactivation of PPARα by interacting with its transcriptional regulator, peroxisome proliferator-activated receptor gamma coactivator-1α. CREB3L3 directly and indirectly controls <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> expression and its plasma level, which contributes at least partially to the catabolic effects of CREB3L3 on systemic energy homeostasis in the entire body. Therefore, CREB3L3 is a therapeutic target for obesity and diabetes.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), a hormone implicated in the regulation of glucose homoeostasis, insulin sensitivity, lipid metabolism and body weight, is considered to be a promising therapeutic target for the treatment of metabolic disorders. Despite observations that FGF<em>21</em> is rapidly proteolysed in circulation rending it potentially inactive, little is known regarding mechanisms by which FGF<em>21</em> protein levels are regulated. We systematically investigated human FGF<em>21</em> protein processing using mass spectrometry. In agreement with previous reports, circulating human FGF<em>21</em> was found to be cleaved primarily after three proline residues at positions 2, 4 and 171. The extent of FGF<em>21</em> processing was quantified in a small cohort of healthy human volunteers. Relative abundance of FGF<em>21</em> proteins cleaved after Pro-2, Pro-4 and Pro-171 ranged from 16 to 30%, 10 to 25% and 10 to 34%, respectively. Dipeptidyl peptidase IV (DPP-IV) was found to be the primary protease responsible for N-terminal cleavages after residues Pro-2 and Pro-4. Importantly, <em>fibroblast</em> activation protein (FAP) was implicated as the protease responsible for C-terminal cleavage after Pro-171, rendering the protein inactive. The requirement of FAP for FGF<em>21</em> proteolysis at the C-terminus was independently demonstrated by in vitro digestion, immunodepletion of FAP in human plasma, administration of an FAP-specific inhibitor and by human FGF<em>21</em> protein processing patterns in FAP knockout mouse plasma. The discovery that FAP is responsible for FGF<em>21</em> inactivation extends the FGF<em>21</em> signalling pathway and may enable novel approaches to augment FGF<em>21</em> actions for therapeutic applications.

Transcription <em>factor</em> Nrf2 (nuclear <em>factor</em> E2-related <em>factor</em> 2) is a master regulator of cellular defense system against oxidative and electrophilic stresses and is negatively regulated by an adaptor protein Keap1 (Kelch-like ECH-associated protein 1). Nrf2 also plays a pivotal role in metabolic homeostasis, such as lipid metabolism and energy expenditure as well as redox homeostasis. FGF<em>21</em> (<em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>) is known as a key mediator of glucose and lipid metabolism. Here, we found that Nrf2 is involved in FGF<em>21</em> regulation in diabetic model mice. Nrf2 induction by genetic knockdown of Keap1 increased plasma FGF<em>21</em> level and hepatic Fgf<em>21</em> expression in diabetic db/db mice and high-calorie-diet-induced obesity model mice. Administration of CDDO-Im (oleanolic triterpenoid 1-[2-cyano-3,12-dioxooleane-1, 9(11)-dien-28-oyl] imidazole), a potent Nrf2 inducer, up-regulated plasma FGF<em>21</em> level and hepatic Fgf<em>21</em> expression in db/db mice, whereas CDDO-Im did not induce FGF<em>21</em> in db/db mice with Nrf2 knockout background. Furthermore, in Keap1-knockdown db/db mice, Nrf2 enhanced expression of glucose- and lipid-metabolism-related genes in adipose tissues, which improved plasma lipid profiles. These results show that Nrf2 positively regulates FGF<em>21</em> expression in diabetic mice. We propose that FGF<em>21</em> is a potential efficacy biomarker that mediates metabolic regulation by the Keap1-Nrf2 system.

<em>Fibroblast</em>-<em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is thought to be important in metabolic regulation. Recently, low protein diets have been shown to increase circulating FGF<em>21</em> levels. However, when energy contribution from dietary protein is lowered, other macronutrients, such as carbohydrates, must be increased to meet eucaloric balance. This raises the possibility that intake of a diet rich in carbohydrates may induce an increase in plasma FGF<em>21</em> levels per se. Here we studied the role of dietary carbohydrates on the levels of circulating FGF<em>21</em> and concomitant physiologic effects by feeding healthy men a carbohydrate rich diet without reducing protein intake.

A diet enriched in carbohydrates (80 E% carbohydrate; CHO) and a eucaloric control diet (CON) were provided to nine healthy men for three days. The energy intake during the CHO diet was increased (+75% energy) to ensure similar dietary protein intake in CHO and CON. To control for the effect of caloric surplus, we similarly overfed (+75% energy) the same subjects for three days with a fat-rich diet (78 E% fat; FAT), consisting of primarily unsaturated fatty acids. The three diets were provided in random order.

After CHO, plasma FGF<em>21</em> concentration increased 8-fold compared to CON (329 ± 99 vs. 39 ± 9 pg ml-1, p < 0.05). In contrast, after FAT only a non-significant tendency (p = 0.073) to an increase in plasma FGF<em>21</em> concentration was found. The increase in FGF<em>21</em> concentration after CHO correlated closely (r = 0.88, p < 0.01) with increased leg glucose uptake (62%, p < 0.05) and increased hepatic glucose production (17%, p < 0.01), indicating increased glucose turnover. Plasma fatty acid (FA) concentration was decreased by 68% (p < 0.01), supported by reduced subcutaneous adipose tissue HSL Ser660 phosphorylation (p < 0.01) and perilipin 1 protein content (p < 0.01), pointing to a suppression of adipose tissue lipolysis. Concomitantly, a 146% increase in the plasma marker of hepatic de novo lipogenesis C16:1 n-7 FA (p < 0.01) was observed together with 101% increased plasma TG concentration (p < 0.001) in association with CHO intake and increased plasma FGF<em>21</em> concentration.

Excess dietary carbohydrate, but not fat, led to markedly increased FGF<em>21</em> secretion in humans, notably without protein restriction, and affected glucose and lipid homeostais.

<AbstractText>Skeletal muscle is a plastic tissue that adapts to changes in exercise, nutrition, and stress by secreting myokines and myometabolites. These muscle-secreted <em>factors</em> have autocrine, paracrine, and endocrine effects, contributing to whole body homeostasis. Muscle dysfunction in aging sarcopenia, cancer cachexia, and diabetes is tightly correlated with the disruption of the physiological homeostasis at the whole body level. The expression levels of the myokine <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) are very low in normal healthy muscles. However, fasting, ER stress, mitochondrial myopathies, and metabolic disorders induce its release from muscles. Although our understanding of the systemic effects of muscle-derived FGF<em>21</em> is exponentially increasing, the direct contribution of FGF<em>21</em> to muscle function has not been investigated yet.</AbstractText><AbstractText>Muscle-specific FGF<em>21</em> knockout mice were generated to investigate the consequences of FGF<em>21</em> deletion concerning skeletal muscle mass and force. To identify the mechanisms underlying FGF<em>21</em>-dependent adaptations in skeletal muscle during starvation, the study was performed on muscles collected from both fed and fasted adult mice. In vivo overexpression of FGF<em>21</em> was performed in skeletal muscle to assess whether FGF<em>21</em> is sufficient per se to induce muscle atrophy.</AbstractText><AbstractText>We show that FGF<em>21</em> does not contribute to muscle homeostasis in basal conditions in terms of fibre type distribution, fibre size, and muscle force. In contrast, FGF<em>21</em> is required for fasting-induced muscle atrophy and weakness. The mass of isolated muscles from control-fasted mice was reduced by 15-25% (P < 0.05) compared with fed control mice. FGF<em>21</em>-null muscles, however, were significantly protected from muscle loss and weakness during fasting. Such important protection is due to the maintenance of protein synthesis rate in knockout muscles during fasting compared with a 70% reduction in control-fasted muscles (P < 0.01), together with a significant reduction of the mitophagy flux via the regulation of the mitochondrial protein Bnip3. The contribution of FGF<em>21</em> to the atrophy programme was supported by in vivo FGF<em>21</em> overexpression in muscles, which was sufficient to induce autophagy and muscle loss by 15% (P < 0.05). Bnip3 inhibition protected against FGF<em>21</em>-dependent muscle wasting in adult animals (P < 0.05).</AbstractText><AbstractText>FGF<em>21</em> is a novel player in the regulation of muscle mass that requires the mitophagy protein Bnip3.</AbstractText>