<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>), a novel metabolic <em>factor</em> in obesity and fasting metabolism, has been shown to be regulated by supraphysiological levels of free fatty acids (FFAs) under hyperinsulinemic conditions. Interestingly, it is still unclear whether the observed effects of FFAs on FGF-<em>21</em> are relevant under physiological conditions, and the relative functions of FFAs and insulin within this context also need to be determined. Fourteen healthy men were studied in a randomized controlled crossover trial (RCT) using lipid heparin infusion (LHI) at a dose inducing physiological elevations of FFAs vs. saline heparin infusion. In a second randomized controlled trial, FGF-<em>21</em> was analyzed in 14 patients with type 1 diabetes (6 men, 8 women) during continuous insulin supply vs. discontinued insulin infusion and subsequently increased lipolysis and ketosis. Circulating FGF-<em>21</em> increased during physiologically elevated FFAs induced by LHI, which was accompanied by mild hyperinsulinemia. Interestingly, a mild elevation of FFAs resulting from complete insulin deficiency also increased FGF-<em>21</em> levels. These results from two independent human RCTs suggest that FFAs increase circulating FGF-<em>21</em>, while insulin is only of minor importance under physiological conditions. This mechanism might explain the apparent paradox of increased FGF-<em>21</em> levels in obesity, insulin resistance, and starvation.

OBJECTIVE

HIV-1-infected patients with lipodystrophy show insulin resistance, dyslipidemia and other signs of metabolic syndrome. <em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>) is a novel metabolic regulator that has been suggested to exert beneficial effects on metabolic homeostasis and insulin sensitivity. Our goal was to determine the relationship between FGF<em>21</em> levels and metabolic alterations in these patients.

METHODS

Serum FGF<em>21</em> levels were analyzed in 179 individuals belonging to four groups: HIV-1-infected, antiretroviral-treated patients that have developed lipodystrophy (n = 59); HIV-1-infected, antiretroviral-treated patients without lipodystrophy (n = 45); untreated (naive) HIV-1-infected patients (n = 41); and healthy control individuals (n = 34). Serum FGF<em>21</em> levels were correlated with parameters indicative of altered fat distribution, metabolic and cardiovascular risk, and in relation to HIV-1 infection and antiretroviral treatment regimens.

RESULTS

Serum FGF<em>21</em> levels were increased in all HIV-1-infected patients, but the increases were most marked in those with lipodystrophy. FGF<em>21</em> levels showed a strong positive correlation with indicators of lipodystrophy (trunk/apendicular fat ratio, waist-to-hip ratio), insulin resistance (fasting glucose, HOMA-R), dyslipidemia (low-density lipoprotein cholesterol), and liver injury (γ-glutamyltransferase).

CONCLUSIONS

FGF<em>21</em> levels are increased in HIV-1-infected patients, especially in those with lipodystrophy, and this increase is closely associated with insulin resistance, metabolic syndrome and makers of liver damage. Further research will be required to determine whether the increase in FGF<em>21</em> levels is caused by a compensatory response or resistance to FGF<em>21</em>, and to establish the potential of FGF<em>21</em> as a biomarker of altered metabolism in HIV-1-infected, antiretroviral-treated patients.

BACKGROUND

Despite the advent of biological therapies for the treatment of rheumatoid arthritis, there is a compelling need to develop alternative therapeutic targets for nonresponders to existing treatments. Soluble receptors occur naturally in vivo, such as the splice variant of the cell surface receptor for vascular endothelial growth factor (VEGF)--a key regulator of angiogenesis in rheumatoid arthritis. Bioinformatics analyses predict that the majority of human genes undergo alternative splicing, generating proteins--many of which may have regulatory functions. The objective of the present study was to identify alternative splice variants (ASV) from cell surface receptor genes, and to determine whether the novel proteins encoded exert therapeutic activity in an in vivo model of arthritis.

METHODS

To identify novel splice variants, we performed RT-PCR using an mRNA pool representing major human tissue types and tumors. Novel ASV were identified by alignment of each cloned sequence to its respective genomic sequence in comparison with full-length transcripts. To test whether these ASV have biologic activity, we characterized a subset of them for ligand binding, and for efficacy in an animal model of arthritis. The in vivo study was accomplished using adenoviruses expressing secreted ASV.

RESULTS

We cloned 60 novel human ASV from 21 genes, encoding cell surface receptors--many of which are known to be important in the regulation of angiogenesis. The ASV were characterized by exon extension, intron retention and alternative exon utilization. Efficient expression and secretion of selected ASV--corresponding to VEGF receptor type 1, VEGF receptor type 2, VEGF receptor type 3, angiopoietin receptor Tie1, Met (receptor for hepatocyte growth factor), colony-stimulating factor 1 receptor, platelet-derived growth factor receptor beta, fibroblast growth factor receptor 1, Kit, and RAGE--was demonstrated, together with binding to their cognate ligands. Importantly, ASV derived from VEGF receptor type 1 and Tie1, and to a lesser extent from VEGF receptor type 2 and fibroblast growth factor receptor 1, reduced clinical signs of arthritis in vivo. The reduction was paralleled by decreased joint inflammation and destruction.

CONCLUSIONS

The present study shows that unique ASV derived from receptors that play key roles in angiogenesis--namely, VEGF receptor type 1 and, for the first time, Tie1--can markedly reduce arthritis severity. More broadly, our results demonstrate that ASV are a source of novel proteins with therapeutic potential in diseases in which angiogenesis and cellular hyperplasia play a central role, such as rheumatoid arthritis.

Heparan sulfate (HS) sugar chains attached to core proteoglycans (PGs) termed HSPGs mediate an extensive range of cell-extracellular matrix (ECM) and <em>growth</em> <em>factor</em> interactions based upon their sulfation patterns. When compared with non-osteogenic (maintenance media) culture conditions, under established osteogenic culture conditions, MC3T3-E1 cells characteristically increase their osteogenic gene expression profile and switch their dominant <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) from FGFR1 (0.5-fold decrease) to FGFR3 (1.5-fold increase). The change in FGFR expression profile of the osteogenic-committed cultures was reflected by their inability to sustain an FGF-2 stimulus, but respond to BMP-2 at day 14 of culture. The osteogenic cultures decreased their chondroitin and dermatan sulfate PGs (biglycan, decorin, and versican), but increased levels of the HS core protein gene expression, in particular glypican-3. Commitment and progress through osteogenesis is accompanied by changes in FGFR expression, decreased GAG initiation but increased N- and O-sulfation and reduced remodeling of the ECM (decreased heparanase expression) resulting in the production of homogenous (<em>21</em> kDa) HS chain. With the HSPG glypican-3 expression strongly upregulated in these processes, siRNA was used to knockdown this gene to examine the effect on osteogenic commitment. Reduced glypican-3 abrogated the expression of Runx2, and thus differentiation. The reintroduction of this HSPG into Runx2-null cells allowed osteogenesis to proceed. These results demonstrate the dependence of osteogenesis on specific HS chains, in particular those associated with glypican-3.

The objective of this study was to develop and evaluate a hydrogel vehicle for sustained release of <em>growth</em> <em>factors</em> for wound healing applications. Hydrogels were fabricated using ultraviolet photo-crosslinking of acrylamide-functionalized nondegradable poly(vinyl alcohol) (PVA). Protein permeability was initially assessed using trypsin inhibitor (TI), a <em>21</em> 000 MW model protein drug. TI permeability was altered by changing the solids content of the gel and by adding hydrophilic PVA fillers. As the PVA content increased from 10% to 20%, protein flux decreased, with no TI permeating through 20% PVA hydrogels. Further increase in model drug release was achieved by incorporating hydrophilic PVA fillers into the hydrogel. As filler molecular weight increased, TI flux increased. The mechanism for this is most likely an alteration in protein/gel interactions and transient variations in water content. The percent protein released was also altered by varying protein loading concentration. Release studies conducted using <em>growth</em> <em>factor</em> in vehicles with hydrophilic filler showed sustained release of platelet-derived <em>growth</em> <em>factor</em> (PDGF-beta,beta) for up to 3 days compared with less than 24 hours in the controls. In vitro bioactivity was demonstrated by doubling of normal human dermal <em>fibroblast</em> numbers when exposed to <em>growth</em> <em>factor</em>-loaded vehicle compared to control. The release vehicle developed in this study uses a rapid and simple fabrication method, and protein release can be tailored by modifying solid content, incorporating biocompatible hydrophilic fillers, and varying protein loading concentration.

The new steroidal 5,7-diene, 3beta-hydroxyandrosta-5,7-diene-17beta-carboxylic acid (17-COOH-7DA), was synthesized from <em>21</em>-acetoxypregnenolone, with the oxidative cleavage of the side chain being dependent on the presence of oxygen. In human epidermal (HaCaT) keratinocytes, 17-COOH-7DA inhibited proliferation in a dose-dependent manner, starting at a dose as low as 10(-11) M. This inhibition was accompanied by decreased expression of epidermal <em>growth</em> <em>factor</em> receptor, bcl2 and cyclin E2 mRNAs and by increased expression of involucrin mRNA. Inhibition of proliferation was associated with slowing of the cell cycle in G1/G0 phases but not with cell death. 17-COOH-7DA was significantly more potent than pregnenolone, 17-COOH-pregnenolone, 17-COOCH(3)-7DA and calcitriol. 17-COOH-7DA also inhibited proliferation of normal human epidermal melanocytes and human and hamster melanoma lines, however, with lower potency than for keratinocytes. In normal human dermal <em>fibroblasts</em> 17-COOH-7DA stimulated proliferation in serum-free media but inhibited it in the presence of 5% serum. 17-COOH-7DA inhibited cell colony formation of human and hamster melanoma cells, and induced monocyte-like differentiation of human HL60 leukemia cells. Thus, the new steroidal 5,7-diene, 17-COOH-7DA, can serve as an inhibitor of proliferation of normal keratinocytes and normal and malignant melanocytes, as a condition-dependent regulator of <em>fibroblast</em> proliferation and a stimulator of leukemia cell differentiation.

The intracellular localization of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was studied in BHK-<em>21</em> cells transfected with an expression vector containing the complementary DNA (cDNA) of the human bFGF gene (pbFGF). The intracellular location of bFGF was determined using indirect immunofluorescence. The antibodies used were polyclonal antibodies directed against either recombinant human bFGF or recombinant Xenopus bFGF. The nuclei of transfected cells that produce bFGF, but not the nuclei of untransfected cells, were labeled strongly by the antibodies. The nuclear staining was totally abolished when anti-bFGF antibodies preadsorbed with bFGF were used. Several types of endothelial cells known to produce bFGF were also stained in their nuclei by the antibodies. Nuclear extracts prepared from transfected cells were found to contain bFGF as determined using heparin-sepharose affinity chromatography, followed by Western blot analysis of fractions, which stimulated the proliferation BHK-<em>21</em> cells. The mitogenic activity associated with the nuclei was not destroyed when isolated cell nuclei were digested by trypsin. It is therefore likely that the nucleus associated bFGF is intranuclear. These findings suggest that some biological activities of bFGF may be mediated by nuclear bFGF binding proteins or by the direct binding of bFGF to DNA.

Short-term low-frequency electrical stimulation (ESTIM) of proximal peripheral nerve stumps prior to end-to-end coaptation or tubular bridging of small distances has been reported to increase preferential motor reinnervation and functional motor recovery in animal models and human patients undergoing carpal tunnel release surgery. We investigated the effects of ESTIM on regeneration across rat sciatic nerve gaps, which exceed distances that allow spontaneous regeneration. Three different reconstruction approaches were combined with ESTIM in the experimental groups. Nerve gaps (13 mm) were bridged using (I) nerve autotransplantation, (II) transplantation of differentially filled silicone tubes, or (III) transplantation of tubular grafts containing <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 overexpressing Schwann cells (SCs) for gene therapy. The regeneration outcome was followed for up to 8 weeks, and functionally as well as histomorphometrically analyzed in comparison to non-stimulated control groups. Combining ESTIM with nerve autotransplantation significantly increased the nerve fiber density in the regenerated nerve, and the grade of functional recovery as detected by electrodiagnostic recordings from the gastrocnemius muscle. The combination of ESTIM with transplantation of naïve SCs increased the regeneration of gap-bridging nerve tissue. Although macroscopic tissue regeneration was not further improved after combining ESTIM with FGF-2(<em>21</em>/23-kD) gene therapy, the latter resulted in a high rate of regenerated nerves that functionally reconnected to the target muscle. Based on our results, brief ESTIM shows high potential to accelerate axonal as well as functional (motor and sensory) outcomes in the clinical setting of peripheral nerve gap reconstruction in human patients.

BACKGROUND

P450 oxidoreductase (POR) deficiency is a disorder of steroidogenesis affecting the microsomal P450 enzymes that use POR as an electron donor. The clinical presentation is variable; patients can be asymptomatic or can present with genital anomalies and the Antley-Bixler syndrome, characterized by craniosynostosis and other bony anomalies. Obligately heterozygous parents are normal. Combined POR and <em>21</em>-hydroxylase deficiencies have not been reported.

OBJECTIVE

The aim was to explore the manifestations of combined deficiencies of <em>21</em>-hydroxylase and POR and to search for lesions in apparent manifesting POR heterozygotes.

METHODS

A newborn female had craniosynostosis, severe salt wasting, minimal virilization, grossly elevated 17OH-progesterone, and minimally elevated androgens. DNA encoding <em>21</em>-hydroxylase, POR, and fibroblast growth factor receptor 2 was sequenced. For POR, the first untranslated exon (exon 1U), 5' flanking DNA, and most introns were sequenced in five apparent manifesting POR heterozygotes.

RESULTS

CYP<em>21</em>B mutations were found on both alleles, proving classical <em>21</em>-hydroxylase deficiency. Fibroblast growth factor receptor 2 exons 8 and 10 were normal. A POR mutation, A287P, was found only on the maternal allele. Five previously reported patients had POR mutations found on only one allele, but their clinical characteristics were indistinguishable from patients with mutations on both alleles. Sequencing of exon 1U, 274 bp of POR 5' flanking DNA, and 12 of the 15 POR introns did not identify additional mutations affecting gene expression or splicing.

CONCLUSIONS

Manifesting heterozygosity is a possible feature of POR deficiency and may ameliorate the findings in coexisting <em>21</em>-hydroxylase deficiency.

P450 oxidoreductase (POR) deficiency is an autosomal recessive disorder of steroidogenesis with multiple clinical manifestations. POR is the electron donor for all microsomal P450 enzymes, including the three steroidogenic enzymes P450c17 (17alpha-hydroxylase/17,20-lyase), P450c<em>21</em> (<em>21</em>-hydroxylase), and P450aro (aromatase). Since the first description of POR mutations in 2004, about 50 patients have been reported. Serum steroid profiles indicate partial deficiencies in <em>21</em>-hydroxylase, 17alpha-hydroxylase and 17,20-lyase. The 17-OH progesterone levels are elevated, as in <em>21</em>-hydroxylase deficiency, while androgen levels are low; cortisol may be normal but is poorly responsive to adrenocorticotropic hormone. Most patients also have associated skeletal malfor- mations (craniosynostosis, radio-ulnar synostosis, midface hypoplasia, bowed femora) termed Antley-Bixler syndrome. Antley-Bixler syndrome with normal steroidogenesis is caused by autosomal dominant gain-of-function mutations in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2. Males with POR deficiency are often undervirilized, while females can be virilized. The prognosis for patients with POR deficiency appears to depend on the severity of the bony malformations and their timely treatment. The potential impact of POR mutations on drug metabolism by other hepatic P450 enzymes requires further investigation. Given the varied physical and biochemical phenotype of POR deficiency and the risk of adrenal insufficiency, clinicians should be alert to this potential diagnosis.

The mechanism by which replacement of some dietary carbohydrates with protein during weight loss favors lipid metabolism remains obscure. In this study, we investigated the effect of an energy-restricted, high-protein/low-carbohydrate diet on lipid metabolism in obese rats. High-sucrose-induced obese rats were assigned randomly to one of two energy-restricted dietary interventions: a carbohydrate-based control diet (CD) or a high-protein diet (HPD). Lean rats of the same age were assigned as normal control. There was significantly greater improvement in fatty liver and hypertriglyceridemia with the HPD diet relative to the CD diet. Expression of genes regulated by <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>) and involved in liver lipolysis and lipid utilitization, such as lipase and acyl-CoA oxidase, increased in obese rats fed the HPD. Furthermore, there was an inverse correlation between levels of FGF<em>21</em> gene expression (regulated by glucagon/insulin balance) and increased triglyceride concentrations in liver from obese rats. Expression of hepatic stearoyl-CoA desaturase-1 (SCD1), regulated primarily by the dietary carbohydrate, was also markedly reduced in the HPD group (similar to plasma triglyceride levels in fasting animals) relative to the CD group. In conclusion, a hypocaloric high-protein diet improves fatty liver and hypertriglyceridemia effectively relative to a carbohydrate diet. The two cellular pathways at work behind these benefits include stimulation of hepatic lipolysis and lipid utilization mediated by FGF<em>21</em> and reduction of hepatic VLDL-TG production by SCD1 regulation.

BACKGROUND

Microvascular proliferation, a prominent feature of tumors of the central nervous system, is a prime target for anti-cancer therapy.

METHODS

Because basic fibroblast growth factor (bFGF) plays a key role in the regulation of angiogenesis, surgical specimens from 52 human brain tumors were examined by immunocytochemical studies with a murine monoclonal antibody to bFGF. Sections from these tumors also were incubated with Ki-67 monoclonal antibody to measure the growth fraction.

RESULTS

Immunostaining for bFGF was observed in 45 of 52 (87%) neoplasms, reacting with 97% of the malignant brain tumors and 67% of benign tumors (P < 0.01). The nonreactive tumors were a medulloblastoma and 7 of 21 (33%) benign, noninvasive, slow-growing neoplasms (1 acoustic schwannoma, 3 meningiomas, 2 pituitary adenomas, and 1 cholesteatoma). The indices of proliferation (Ki-67 labeling) were lower for the 21 benign tumors (1.2 +/- 1.1%) than the 31 malignant tumors (10.3 +/- 10.5%; P < 0.001). The bFGF was immunolocalized in the tumor cell nuclei in 23 of 52 tumors (44%) and in the cytoplasm of 8 of 52 (15%) tumors. Immunostaining to bFGF was prominent in the microvascular endothelial compartment in 84% of the malignant tumors and only 52% of benign tumors (P < 0.01). Immunostaining was not present after preabsorption of the antibody with pure human recombinant bFGF.

CONCLUSIONS

The presence of bFGF predominantly within the tumor microvasculature indicates a cellular depot for this potent growth factor that mediates angiogenesis and tumorigenesis. These data support a role for bFGF in the transition from the benign to the malignant phenotype.

Berberine, a compound from rhizome coptidis, is traditionally used to treat gastrointestinal infections, such as bacterial diarrhoea. Recently, berberine was shown to have hypoglycaemic and hypolipidaemic effects. We investigated the mechanisms by which berberine regulates hepatic lipid metabolism and energy expenditure in mice.

Liver-specific SIRT1 knockout mice and their wild-type littermates were fed a high-fat, high-sucrose (HFHS) diet and treated with berberine by i.p. injection for five weeks. Mouse primary hepatocytes and human HepG2 cells were treated with berberine and then subjected to immunoblotting analysis and Oil Red O staining.

Berberine attenuated hepatic steatosis and controlled energy balance in mice by inducing autophagy and FGF21. These beneficial effects of berberine on autophagy and hepatic steatosis were abolished by a deficiency of the nutrient sensor SIRT1 in the liver of HFHS diet-fed obese mice and in mouse primary hepatocytes. SIRT1 is essential for berberine to potentiate autophagy and inhibit lipid storage in mouse livers in response to fasting. Mechanistically, the berberine stimulates SIRT1 deacetylation activity and induces autophagy in an autophagy protein 5-dependent manner. Moreover, the administration of berberine was shown to promote hepatic gene expression and circulating levels of FGF21 and ketone bodies in mice in a SIRT1-dependent manner.

Berberine acts in the liver to regulate lipid utilization and maintain whole-body energy metabolism by mediating autophagy and FGF21 activation. Hence, it has therapeutic potential for treating metabolic defects under nutritional overload, such as fatty liver diseases, type 2 diabetes and obesity.

Lactation is associated with significant alterations in both body composition and bone mass. Systemic and local skeletal <em>factors</em> such as receptor activator of nuclear <em>factor</em> κ-B ligand (RANKL), PTHrP, calcitonin, and estrogen are known to regulate bone remodeling during and after lactation. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>) may function as an endocrine <em>factor</em> to regulate body composition changes during lactation by inducing gluconeogenesis and fatty acid oxidation. In this study, we hypothesized that the metabolic changes during lactation were due in part to increased circulating FGF-<em>21</em>, which in turn could accentuate bone loss. We longitudinally characterized body composition in C57BL/6J (B6) mice during (day 7 and day <em>21</em> of lactation) and after normal lactation (day <em>21</em> postlactation). At day 7 of lactation, areal bone density declined by 10% (P < .001), bone resorption increased (P < .0001), percent fat decreased by 20%, energy expenditure increased (P < .01), and markers of brown-like adipogenesis were suppressed in the inguinal depot and in preformed brown adipose tissue. At day 7 of lactation there was a 2.4-fold increase in serum FGF-<em>21</em> vs baseline (P < .0001), a 8-fold increase in hepatic FGF-<em>21</em> mRNA (P < .03), a 2-fold increase in undercarboxylated osteocalcin (Glu13 OCn) (P < .01), and enhanced insulin sensitivity. Recovery of total areal bone density was noted at day <em>21</em> of lactation, whereas the femoral trabecular bone volume fraction was still reduced (P < .01). Because FGF-<em>21</em> levels rose rapidly at day 7 of lactation in B6 lactating mice, we next examined lactating mice with a deletion in the Fgf<em>21</em> gene. Trabecular and cortical bone masses were maintained throughout lactation in FGF-<em>21</em>(-/-) mice, and pup <em>growth</em> was normal. Compared with lactating control mice, lactating FGF-<em>21</em>(-/-) mice exhibited an increase in bone formation, but no change in bone resorption. In conclusion, in addition to changes in calciotropic hormones, systemic FGF-<em>21</em> plays a role in skeletal remodeling and changes in body composition during lactation in B6 mice.

OBJECTIVE

Complex repertoires of IgG autoantibodies have been detected against ocular antigens in patients with glaucoma. The goal was to identify and characterize the IgG autoantibody repertoires in sera of patients with pseudoexfoliation glaucoma (PXFG) with protein macroarrays.

METHODS

Serum samples of <em>21</em> patients with PXFG and 19 age- and sex-matched control subjects were profiled on high-density colony protein macroarrays expressing His-tagged recombinant human proteins derived from a human fetal brain cDNA library. Statistically prevalent expression clones in the PXFG group were sequenced. mRNA expression of identified antigens was examined in the rat ganglion cell line RGC-5 and in human brain and optic nerve cDNA. The IgG immunoreactivity of the sera of 20 control and 26 PXFG patients to purified C6orf129 was analyzed in a reverse enzyme-linked immunosorbent assay.

RESULTS

An increased prevalence was detected among the PXFG patients of serum antibodies to seven proteins: C6orf129; stathmin-like 4; transmembrane protein 9 domain family, member B; fibroblast growth factor receptor 3; cleft lip and palate transmembrane protein 1; EH-domain-containing protein 1; and eukaryotic translation elongation factor 2. All antigens were expressed in the RGC-5 cells and in cDNA from human brain and optic nerve, with the exception of stathmin-like 4, which was not expressed in the RGC-5 cells. The patients with PXFG had increased anti-C6orf129 IgG immunoreactivity compared with that in the control subjects (P < 0.05).

CONCLUSIONS

Screening high-density protein arrays identifies unique antibody profiles that may discriminate between patients with and without PXFG. Characterization of the autoantibody repertoire may provide new insights into the pathophysiology of PXFG.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>21</em> has been recently characterized as a potent metabolic regulator, but the physiological role of FGF-<em>21</em> remains poorly understood. This study was designed to investigate serum FGF-<em>21</em> levels in type 2 diabetes mellitus (T2DM) patients and explore the correlations between its serum levels and various cardiometabolic parameters in humans.

METHODS

Fifty-four newly diagnosed patients with T2DM (NDDM), 54 subjects with long duration of T2DM (LDDM), that is, more than 5 years, and 35 normal controls participated in this study. Fasting serum FGF-<em>21</em> levels were measured using ELISA kits. The anthropometric parameters, glucose, glycosylated hemoglobin A1c, lipids, insulin, and high-sensitive C-reactive protein (hsCRP) levels were also measured in fasting status.

RESULTS

Serum FGF-<em>21</em> levels were significantly higher in patients with NDDM and LDDM than in controls (361.80 ± 140.28 pg/mL and 351.17 ± 133.44 pg/mL vs. 185.89 ± 59.29 pg/mL, both p < 0.01), but there was no difference in serum FGF-<em>21</em> levels between NDDM and LDDM. Fasting serum FGF-<em>21</em> was found to correlate positively and significantly with age, waist-to-hip ratio, systolic blood pressure, fasting plasma glucose, glycosylated hemoglobin A1c, homeostasis model assessment (HOMA) of insulin resistance, and hsCRP, but negatively with HOMA of β-cell insulin secretion. Furthermore, multiple regression analyses showed that fasting insulin, HOMA of insulin resistance, low-density lipoprotein cholesterol, and hsCRP were independent <em>factor</em>s influencing serum FGF-<em>21</em> levels. Logistic regression analyses demonstrated that serum FGF-<em>21</em> was significantly associated with T2DM.

CONCLUSIONS

Serum FGF-<em>21</em> levels were significantly increased in patients with T2DM. They may play a role in the pathogenesis of T2DM.

Adipocyte browning is a promising strategy for obesity prevention. Using onion-peel-derived extracts and their bioactive compounds, we demonstrate that onion peel, a by-product of onion, can change the characteristics of white adipocytes to those of brown-like adipocytes in the white adipose tissue of mice and 3T3-L1 cells. The expression of the following brown adipose tissue-specific genes was increased in the retroperitoneal and subcutaneous adipose tissues of 0.5% onion-peel-extract-fed mice: PR domain-containing 16, peroxisome proliferator-activated receptor gamma coactivator 1α, uncoupling protein 1, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> and cell death-inducing DFFA-like effector. In 3T3-L1 adipocytes, onion peel extract induced the expression of brown adipose tissue-specific genes and increased the expression of carnitine palmitoyltransferase 1α. This effect was supported by decreased lipid levels and multiple small-sized lipid droplets. The ethyl acetate fraction of the onion peel extract that contained the highest proportion of hydrophobic molecules showed the same browning effect in 3T3-L1 adipocytes. A high-performance liquid chromatography analysis further identified quercetin as a functional compound in the browning effect of onion peel. The quercetin-associated browning effect was mediated in part by the activation of AMP-activated protein kinase. In summary, our study provides the first demonstration of the browning effects of onion peel and quercetin using both animal and cell models. This result indicates that onion peel has the potential to remodel the characteristics of white adipocytes to those of brown-like adipocytes.

MSCs from non-cartilaginous knee joint tissues such as the infrapatellar fat pad (IFP) and synovium possess significant chondrogenic potential and provide a readily available and clinically feasible source of chondroprogenitor cells. <em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) has been shown to be a potent mitotic stimulator during ex vivo expansion of MSCs, as well as regulating their subsequent differentiation potential. The objective of this study was to investigate the longer term effects of FGF-2 expansion on the functional development of cartilaginous tissues engineered using MSCs derived from the IFP. IFP MSCs were isolated and expanded to passage 2 in a standard media formulation with or without FGF-2 (5 ng/ml) supplementation. Expanded cells were encapsulated in agarose hydrogels, maintained in chondrogenic media for 42 days and analysed to determine their mechanical properties and biochemical composition. Culture media, collected at each feed, was also analysed for biochemical constituents. MSCs expanded in the presence of FGF-2 proliferated more rapidly, with higher cell yields and lower population doubling times. FGF-2 expanded MSCs generated the most mechanically functional tissue. Matrix accumulation was dramatically higher after <em>21</em> days for FGF-2 expanded MSCs, but decreased between day <em>21</em> and 42. By day 42, FGF-2 expanded MSCs had still accumulated ∼1.4 fold higher sGAG and ∼1.7 fold higher collagen compared to control groups. The total amount of sGAG synthesised (retained in hydrogels and released into the media) was ∼2.4 fold higher for FGF-2 expanded MSCs, with only ∼25% of the total amount generated being retained within the constructs. Further studies are required to investigate whether IFP derived MSCs have a diminished capacity to synthesise other matrix components important in the aggregation, assembly and retention of proteoglycans. In conclusion, expanding MSCs in the presence of FGF-2 rapidly accelerates chondrogenesis in 3D agarose cultures resulting in superior mechanical functionality.

Prolyl hydroxylase domain 2 (PHD2) has been implicated in several pathways of cell signaling, most notably in its regulation of hypoxia-inducible <em>factor</em> (HIF)-1α stability. In normoxia, PHD2 hydroxylates proline residues on HIF-1α, rendering it inactive. However, in hypoxia, PHD2 is inactive, HIF-1α is stabilized and downstream effectors such as vascular endothelial <em>growth</em> <em>factor</em> and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 are produced to promote angiogenesis. In the present study we utilize RNA interference to PHD2 to promote therapeutic angiogenesis in a diabetic wound model, presumably by the stabilization of HIF-1α. Stented wounds were created on the dorsum of diabetic Lepr db/db mice. Mice were treated with PHD2 small interfering RNA (siRNA) or nonsense siRNA. Wounds were measured photometrically on days 0-28. Wounds were harvested for histology, protein, and RNA analysis. Diabetic wounds treated with siRNA closed within <em>21</em>±1.2 days; sham-treated closed in 28±1.5 days. By day 7, Western blot revealed near complete suppression of PHD protein and corresponding increased HIF-1α. Angiogenic mediators vascular endothelial <em>growth</em> <em>factor</em> and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 were elevated, corresponding to increased CD31 staining in the treated groups. siRNA-mediated silencing of PHD2 increases HIF-1α and several mediators of angiogenesis. This corresponded to improved time to closure in diabetic wounds compared with sham-treated wounds. These findings suggest that impaired wound healing in diabetes can be ameliorated with therapeutic angiogenesis.

Excess ethanol consumption has serious pathologic consequences. In humans, repeated episodes of binge drinking can lead to liver damage and have adverse effects on other organs such as pancreas and brain. Long term chronic consumption of ethanol can also result in progressive alcoholic liver disease and cirrhosis. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a metabolic regulator with multiple physiologic functions. FGF<em>21</em> is a novel biomarker for non-alcoholic fatty liver disease (NAFLD) in humans and limits hepatotoxicity in mice. Therefore, we explored the possibility that FGF<em>21</em> plays a role in response to ethanol consumption in both humans and mice.

We used a binge drinking paradigm in humans to examine the effect of acute ethanol consumption on circulating FGF<em>21</em>. We adapted this paradigm to evaluate the acute response to ethanol in mice. We then examined the role of FGF<em>21</em> on liver pathology in two models of chronic ethanol consumption in both wild type (WT) mice and mice lacking FGF<em>21</em> (FGF<em>21</em>-KO).

Acute ethanol consumption resulted in a robust induction of serum FGF<em>21</em> after 6 h in both humans and mice. Serum ethanol peaked at 1 h in both species and was cleared by 6 h. Ethanol clearance was the same in WT and FGF<em>21</em>-KO mice, indicating that FGF<em>21</em> does not play a major role in ethanol metabolism in a binge paradigm. When FGF<em>21</em>-KO mice were fed the Lieber-DeCarli diet, a high fat diet supplemented with ethanol, a higher mortality was observed compared to WT mice after 16 days on the diet. When FGF<em>21</em>-KO mice consumed 30% ethanol in drinking water, along with a normal chow diet, there was no mortality observed even after 16 weeks, but the FGF<em>21</em>-KO mice had significant liver pathology compared to WT mice.

Acute or binge ethanol consumption significantly increases circulating FGF<em>21</em> levels in both humans and mice. However, FGF<em>21</em> does not play a role in acute ethanol clearance. In contrast, chronic ethanol consumption in the absence of FGF<em>21</em> is associated with significant liver pathology alone or in combination with excess mortality, depending on the type of diet consumed with ethanol. This suggests that FGF<em>21</em> protects against long term ethanol induced hepatic damage and may attenuate progression of alcoholic liver disease. Further study is required to assess the therapeutic potential of FGF<em>21</em> in the treatment of alcoholic liver disease.

The authors have investigated gene expression of ST2 in the lung tissue of a bleomycin (BLM)-induced lung fibrosis model in vivo and in a human lung <em>fibroblast</em> cell line, WI38, and a human type II alveolar epithelial cell line, A549, reacting to proinflammatory and type 2 helper T cell (Th2)-type cytokine stimuli in vitro. The lung mRNA expression of interleukin (IL)-4, IL-5, IL-1beta, and tumor necrosis <em>factor</em> (TNF)-alpha increased significantly at day 7 after instillation of BLM, whereas interferon (IFN)-gamma mRNA expression did not increase. ST2 and transforming <em>growth</em> <em>factor</em> (TGF)-beta1 mRNA expression of the lung increased significantly between days 7 and <em>21</em>, and increased to maximal levels at day 14 post-BLM challenge. ST2 mRNA expression statistically correlated with TGF-beta 1 mRNA expression. In addition, the combination of IL-1 beta, TNF-alpha, and IL-4 had an additive effect on ST2 mRNA expression from A549 cells and WI38 cells. These findings suggest that soluble ST2 gene may increase, possibly reflecting the development of the inflammatory process and the Th2-type immune response in the fibrotic lung tissue, and may modulate a process of pulmonary fibrosis.

Pharmacological manipulation of opioid receptors alters feeding behavior. However, the individual contributions of each opioid receptor subtype on energy balance remain largely unknown. Herein, we investigated whether genetic disruption of the δ-opioid receptor (DOR) also controls energy homeostasis. Mice lacking DOR and wild-type mice were fed with standard diet and high-energy diet (HED). Mice were analyzed in vivo with the indirect calorimetry system, and tissues were analyzed by real-time PCR and Western blot analysis. DOR-knockout (KO) mice gained less weight (P<0.01) and had lower fat mass (P<0.01) when compared to WT mice fed an HED. Although DOR-KO mice were hyperphagic, they showed higher energy expenditure (P<0.05), which was the result of an increased activation of the thermogenic program in brown adipose tissue. The increased nonshivering thermogenesis involved the stimulation of uncoupling protein 1 (UCP1; P<0.01), peroxisome proliferator-activated receptor γ coactivator (PGC1α; P<0.05), and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>; P<0.01). DOR deficiency also led to an attenuation of triglyceride content in the liver (P<0.05) in response to an HED. These findings reveal a novel role of DOR in the control of thermogenic markers and energy expenditure, and they provide a potential new therapeutic approach for the treatment of obesity.

Rather than a traditional <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>) is considered to be a metabolic hormone. In the current study, we investigated serum FGF<em>21</em> levels in the self-contained population of Sorbs. Serum FGF<em>21</em> concentrations were quantified by ELISA and correlated with IGF1 as well as metabolic, renal, hepatic, inflammatory, and cardiovascular parameters in 913 Sorbs from Germany. Moreover, human IGF1 protein secretion was investigated in FGF<em>21</em>-stimulated HepG2 cells. Median FGF<em>21</em> serum concentrations were 2.1-fold higher in subjects with type 2 diabetes mellitus (141.8 ng/l) compared with controls (66.7 ng/l). Furthermore, nondiabetic subjects with FGF<em>21</em> levels below the detection limit of the ELISA showed a more beneficial metabolic profile compared with subjects with measurable FGF<em>21</em>. Moreover, FGF<em>21</em> was significantly lower in female compared with male subjects after adjustment for age and BMI. In multiple regression analyses, circulating FGF<em>21</em> concentrations remained independently and positively associated with gender, systolic blood pressure, triglycerides, and γ glutamyl transferase whereas a negative association was observed with IGF1 in nondiabetic subjects. Notably, FGF<em>21</em> significantly inhibited IGF1 secretion into HepG2 cell culture supernatants in preliminary in vitro experiments. FGF<em>21</em> serum concentrations are associated with facets of the metabolic syndrome, hepatocellular function, as well as GH status.

OBJECTIVE

The review summarizes recent findings examining the effects of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em> on carbohydrate and lipid metabolism with emphasis on publications from 2010.

RESULTS

FGF<em>21</em> is considered to be a metabolic hormone rather than a traditional <em>growth</em> <em>factor</em>. Recent studies identified the important role of FGF<em>21</em> in regulation of energy balance. Thus, the protein regulates hepatic metabolism and improves insulin sensitivity. Furthermore, central FGF<em>21</em> action in the brain increases energy expenditure and insulin sensitivity in rodents. Interestingly, FGF<em>21</em> expression is elevated in the adaptive response to fasting but also regulated by feeding-induced mechanisms. Moreover, FGF<em>21</em> levels are elevated in obese animals and positively correlate with BMI in humans suggesting obesity as a FGF<em>21</em>-resistant state.

CONCLUSIONS

FGF<em>21</em> is a metabolic hormone that is regulated by nutritional status and influences glucose and lipid metabolism by central and peripheral mechanisms. Future research is needed to expand our understanding of the diagnostic and therapeutic relevance of FGF<em>21</em>-dependent pathways in humans.