BACKGROUND

Advanced hepatocellular carcinoma (HCC) in humans is characterized by hypervascularity. In the present study, the expressions of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2) and endostatin were analyzed in patients with chronic liver disease to clarify the effect of these major angiogenic factors.

METHODS

Serum concentrations of VEGF, FGF-2 and endostatin in 24 patients with HCC, 16 patients with liver cirrhosis (LC) and 13 healthy volunteers were measured by enzyme-linked immunosorbent assay. The expression of VEGF in 21 surgically resected HCC samples was analyzed by immunohistochemistry, and that of VEGF isoforms in 15 HCC samples was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS

Serum VEGF, FGF-2 and endostatin concentrations were significantly elevated in patients with HCC compared with healthy volunteers; but there was no significant difference between patients with HCC and those with non-HCC liver disease. Immunohistochemical analysis showed that VEGF protein was strongly expressed in both well-differentiated HCC cells and non-cancerous hepatocytes, whereas in moderately and poorly differentiated HCC the expression was stronger in the endothelial cells (EC) lining intratumor vessels than in the cancer cells. On RT-PCR for VEGF isoforms it was found that VEGF-121, VEGF-165 and VEGF-189 were expressed in all but one of the HCC samples and in all corresponding non-HCC samples.

CONCLUSIONS

The results suggest that VEGF, FGF-2, and endostatin concentrations are elevated prior to the emergence of HCC and that the distribution of VEGF changes dynamically during the development of HCC.

The ability of newborns and immature animals to reossify calvarial defects has been well described. This capacity is generally lost in children greater than 2 years of age and in mature animals. The dura mater has been implicated as a regulator of calvarial reossification. To date, however, few studies have attempted to identify biomolecular differences in the dura mater that enable immature, but not mature, dura to induce osteogenesis. The purpose of these studies was to analyze metabolic characteristics, protein/gene expression, and capacity to form mineralized bone nodules of cells derived from immature and mature dura mater. Transforming <em>growth</em> <em>factor</em> beta-1, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, collagen type IalphaI, osteocalcin, and alkaline phosphatase are critical <em>growth</em> <em>factors</em> and extracellular matrix proteins essential for successful osteogenesis. In this study, we have characterized the proliferation rates of immature (6-day-old rats, n = 40) and mature (adult rats, n = 10) dura cell cultures. In addition, we analyzed the expression of transforming <em>growth</em> <em>factor</em> beta-1, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, proliferating cell nuclear antigen, and alkaline phosphatase. Our in vitro findings were corroborated with Northern blot analysis of mRNA expression in total cellular RNA isolated from snap-frozen age-matched dural tissues (6-day-old rats, n = 60; adult rats, n = 10). Finally, the capacity of cultured dural cells to form mineralized bone nodules was assessed. We demonstrated that immature dural cells proliferate significantly faster and produce significantly more proliferating cell nuclear antigen than mature dural cells (p < 0.01). Additionally, immature dural cells produce significantly greater amounts of transforming <em>growth</em> <em>factor</em> beta-1, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, and alkaline phosphatase (p < 0.01). Furthermore, Northern blot analysis of RNA isolated from immature and mature dural tissues demonstrated a greater than 9-fold, 8-fold, and <em>21</em>-fold increase in transforming <em>growth</em> <em>factor</em> beta-1, osteocalcin, and collagen IalphaI gene expression, respectively, in immature as compared with mature dura mater. Finally, in keeping with their in vivo phenotype, immature dural cells formed large calcified bone nodules in vitro, whereas mature dural cells failed to form bone nodules even with extended culture. These studies suggest that differential expression of <em>growth</em> <em>factors</em> and extracellular matrix molecules may be a critical difference between the osteoinductive capacity of immature and mature dura mater. Finally, we believe that the biomolecular bone- and matrix-inducing phenotype of immature dura mater regulates the ability of young children and immature animals to heal calvarial defects.

Mounting evidence connects non-alcoholic fatty liver disease (NAFLD) to chronic kidney disease (CKD). We review emerging mechanistic links between NAFLD and CKD, including altered activation of angiotensin converting enzyme (ACE)-2, nutrient/energy sensors sirtuin-1 and AMP-activated kinase, as well as impaired antioxidant defense mediated by nuclear <em>factor</em> erythroid 2-related <em>factor</em>-2 (Nrf2). Dietary fructose excess may also contribute to NAFLD and CKD. NAFLD affects renal injury through lipoprotein dysmetabolism and altered secretion of the hepatokines <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em>, fetuin-A, insulin-like <em>growth</em> <em>factor</em>-1, and syndecan-1. CKD may mutually aggravate NAFLD and associated metabolic disturbances through altered intestinal barrier function and microbiota composition, the accumulation of uremic toxic metabolites, and alterations in pre-receptor glucocorticoid metabolism. We conclude by discussing the implications of these findings for the treatment of NAFLD and CKD.

The major risk <em>factors</em> for non-alcoholic fatty liver disease (NAFLD) are obesity, insulin resistance and dyslipidemia. The cause for progression from the steatosis stage to the inflammatory condition (non-alcoholic steatohepatitis (NASH)) remains elusive at present. Aim of this study was to test whether the different stages of NAFLD as well as the associated metabolic abnormalities can be recreated in time in an overfed mouse model and study the mechanisms underlying the transition from steatosis to NASH. Male C57Bl/6J mice were subjected to continuous intragastric overfeeding with a high-fat liquid diet (HFLD) for different time periods. Mice fed a solid high-fat diet (HFD) ad libitum served as controls. Liver histology and metabolic characteristics of liver, white adipose tisue (WAT) and plasma were studied. Both HFD-fed and HFLD-overfed mice initially developed liver steatosis, but only the latter progressed in time to NASH. NASH coincided with obesity, hyperinsulinemia, loss of liver glycogen and hepatic endoplasmatic reticulum stress. Peroxisome proliferator-activated receptor γ (Pparγ), <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (Fgf<em>21</em>), fatty acid binding protein (Fabp) and fatty acid translocase (CD36) were induced exclusively in the livers of the HFLD-overfed mice. Inflammation, reduced adiponectin expression and altered expression of genes that influence adipogenic capacity were only observed in WAT of HFLD-overfed mice.

CONCLUSIONS

this dietary mouse model displays the different stages and the metabolic settings often found in human NAFLD. Lipotoxicity due to compromised adipose tissue function is likely associated with the progression to NASH, but whether this is cause or consequence remains to be established.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is an endocrine hormone that exhibits anti-obesity and anti-diabetes effects. Because metformin is widely used as a glucose-lowering agent in patients with type 2 diabetes (T2D), we investigated whether metformin modulates FGF<em>21</em> expression in cell lines, and in mice or human subjects. We found that metformin increased the expression and release of FGF<em>21</em> in a diverse set of cell types, including rat hepatoma FaO, primary mouse hepatocytes, and mouse embryonic <em>fibroblasts</em> (MEFs). Intriguingly, AMP-activated protein kinase (AMPK) was dispensable for the induction of FGF<em>21</em> by metformin. Mammalian target of rapamycin complex 1 (mTORC1) and peroxisome proliferator-activated receptor α (PPARα), which are additional targets of metformin, were not involved in metformin-induced FGF<em>21</em> expression. Importantly, inhibition of mitochondrial complex I activity by metformin resulted in FGF<em>21</em> induction through PKR-like ER kinase (PERK)-eukaryotic translation <em>factor</em> 2α (eIF2α)-activating transcription <em>factor</em> 4 (ATF4). We showed that metformin activated ATF4 and increased FGF<em>21</em> expression in the livers of mice, which led to increased serum levels of FGF<em>21</em>. We also found that serum FGF<em>21</em> level was increased in human subjects with T2D after metformin therapy for 6 months. In conclusion, our results indicate that metformin induced expression of FGF<em>21</em> through an ATF4-dependent mechanism by inhibiting mitochondrial respiration independently of AMPK. Therefore, FGF<em>21</em> induction by metformin might explain a portion of the beneficial metabolic effects of metformin.

BACKGROUND

Rosiglitazone and pioglitazone are high-affinity peroxisome proliferator-activated receptor (PPAR)-gamma agonists with potent anti-diabetic properties and potential anti-inflammatory effects. We compared the ability of a range of oral doses of these thiazolidinediones, including those sufficient to restore insulin sensitization, to inhibit the pathogenesis of adjuvant-induced arthritis (AIA).

METHODS

AIA was induced in Lewis rats by a subcutaneous injection of 1 mg of complete Freund's adjuvant. Rats were treated orally for <em>21</em> days with pioglitazone 3, 10 or 30 mg/kg/day, rosiglitazone 3 or 10 mg/kg/day, or with vehicle only. The time course of AIA was evaluated by biotelemetry to monitor body temperature and locomotor activity, by clinical score and plethysmographic measurement of hindpaw oedema. At necropsy, RT-PCR analysis was performed on synovium, liver and subcutaneous fat. Changes in cartilage were evaluated by histological examination of ankle joints, radiolabelled sulphate incorporation (proteoglycan synthesis), glycosaminoglycan content (proteoglycan turnover) and aggrecan expression in patellar cartilage. Whole-body bone mineral content was measured by dual-energy X-ray absorptiometry.

RESULTS

The highest doses of rosiglitazone (10 mg/kg/day) or pioglitazone (30 mg/kg/day) were required to reduce fever peaks associated with acute or chronic inflammation, respectively, and to decrease arthritis severity. At these doses, thiazolidinediones reduced synovitis and synovial expression of TNF-alpha, IL-1beta and basic fibroblast growth factor without affecting neovascularization or the expression of vascular endothelial growth factor. Thiazolidinediones failed to prevent cartilage lesions and arthritis-induced inhibition of proteoglycan synthesis, aggrecan mRNA level or glycosaminoglycan content in patellar cartilage, but reduced bone erosions and inflammatory bone loss. A trend towards lower urinary levels of deoxipyridinolin was also noted in arthritic rats treated with thiazolidinediones. Rosiglitazone 10 mg/kg/day or pioglitazone 30 mg/kg/day increased the expression of PPAR-gamma and adiponectin in adipose tissue, confirming that they were activating PPAR-gamma in inflammatory conditions, although an increase in fat mass percentage was observed for the most anti-arthritic dose.

CONCLUSIONS

These data emphasize that higher dosages of thiazolidinediones are required for the treatment of arthritis than for restoring insulin sensitivity but that thiazolidinediones prevent inflammatory bone loss despite exposing animals to increased fatness possibly resulting from excessive activation of PPAR-gamma.

OBJECTIVE

To assess neurotrophic factor upregulation in the retina after damage to the optic nerve and relate that regulation to changes in photoreceptor stability and function.

METHODS

Retinas of adult pigmented (Long-Evans) rats were examined at successive times (1-60 days) after unilateral optic nerve section. The distribution and expression of ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (FGF-2) and their receptor elements FGFR1 and CNTFRalpha were studied with immunohistochemistry and Western blot analysis. FGF-2 and CNTF mRNA levels were also assessed, with semiquantitative reverse transcription-PCR. Levels and localization of the intracellular signaling molecule ERK and its activated, phosphorylated form pERK, were examined by immunohistochemistry. To assess the correlation between neurotrophic factor levels and their protective effect against light damage, albino (Sprague-Dawley) rats were exposed to bright continuous light (1000 lux) for 24 or 48 hours at successive times after nerve section. The TUNEL technique was used to visualize neuronal cell death in the retina.

RESULTS

CNTF upregulation was detected 1 week after optic nerve section, peaked at 2 weeks, and fell to control levels at 4 weeks. CNTF appeared first in the inner retina in the ganglion cells, then in the Muller cells in which it became prominent at the outer limiting membrane (OLM) and in the outer segment (OS) region of photoreceptors. FGF-2 upregulation became prominent, particularly in photoreceptors, 21 to 28 days after surgery, continued to 2 months, and slowly declined thereafter. Double labeling with antibodies to ligand and the receptor showed colocalization of CNTF to its receptor at the OS region, whereas FGF-2-to-FGFR1 binding was found in the outer nuclear (ONL) and outer plexiform (OPL) layers. Optic nerve section provided a significant protective effect against light-induced damage in the first 2 weeks. There was no protection when animals were exposed to damaging light 1 month after nerve section.

CONCLUSIONS

The upregulation of CNTF 7 to 14 days after nerve section correlates with a reduction in the a-wave described previously. Colocalization of CNTF and CNTFRalpha on the outer segments suggests that CNTF acts at the photoreceptor membrane. The slower upregulation of FGF-2 correlates with a reduction of the b-wave. FGF-2/FGFR1 colocalization in the OPL suggests that this factor acts at the synaptic terminals of photoreceptors, modulating the release of neurotransmitters. The time course of pERK upregulation suggests that the successive upregulation of CNTF and FGF-2 activates the ERK pathway. Based on the time course of protection against bright continuous light, it seems that CNTF plays a major role in this effect, and FGF-2 has a less important role in the protection against light-induced damage.

BACKGROUND

The relationships among cortical volumetric bone mineral density (CortBMD) and comprehensive measures of mineral metabolism have not been addressed in chronic kidney disease (CKD).

OBJECTIVE

The aim of the study was to identify the determinants of CortBMD in childhood CKD. A secondary objective was to assess whether CortBMD was associated with subsequent fracture.

METHODS

This prospective cohort study included 171 children, adolescents, and young adults (aged 5-<em>21</em> years) with CKD stages 2-5D at enrollment and 89 1 year later.

RESULTS

Serum measures included vitamin D [25-hydroxyvitamin D (25[OH]D), 1,25-dihydroxyvitamin D (1,25(OH)₂D), 24,25-dihydroxyvitamin D], vitamin D-binding protein, intact PTH, fibroblast growth factor 23, calcium, and phosphorus. Tibia quantitative computed tomography measures of CortBMD were expressed as sex-, race-, and age-specific Z-scores based on 675 controls. Multivariable linear regression identified the independent correlates of CortBMD Z-scores and the change in CortBMD Z-scores.

RESULTS

Lower calcium (β = .31/1 mg/dL, P = .01) and 25(OH)D (β = .18/10 ng/mL, P = .04) and higher PTH (β = -.02/10%, P = .002) and 1,25(OH)₂D (β = -.07/10%, P < .001) were independently associated with lower CortBMD Z-scores at baseline. The correlations of total, free, and bioavailable 25(OH)D with CortBMD did not differ. Higher baseline 1,25(OH)₂D (P < .05) and greater increases in PTH (P < .001) were associated with greater declines in CortBMD Z-scores. Greater increases in calcium concentrations were associated with greater increases in CortBMD Z-scores in growing children (interaction P = .009). The hazard ratio for fracture was 1.75 (95% confidence interval 1.15-2.67; P = .009) per SD lower baseline CortBMD.

CONCLUSIONS

Greater PTH and 1,25(OH)₂D and lower calcium concentrations were independently associated with baseline and progressive cortical deficits in childhood CKD. Lower CortBMD Z-score was associated with increased fracture risk.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) plays an important role in energy homoeostasis. The unaddressed question of FGF<em>21</em>'s effect on the development and progression of diabetic cardiomyopathy (DCM) is investigated here with FGF<em>21</em> knockout (FGF<em>21</em>KO) diabetic mice. Type 1 diabetes was induced in both FGF<em>21</em>KO and C57BL/6J wild-type (WT) mice via streptozotocin. At 1, 2 and 4 months after diabetes onset, the plasma FGF<em>21</em> levels were significantly decreased in WT diabetic mice compared to controls. There was no significant difference between FGF<em>21</em>KO and WT diabetic mice in blood glucose and triglyceride levels. FGF<em>21</em>KO diabetic mice showed earlier and more severe cardiac dysfunction, remodelling and oxidative stress, as well as greater increase in cardiac lipid accumulation than WT diabetic mice. Western blots showed that increased cardiac lipid accumulation was accompanied by further increases in the expression of nuclear <em>factor</em> (erythroid-derived 2)-like 2 (Nrf2) and its target protein CD36, along with decreases in the phosphorylation of AMP-activated protein kinase and the expression of hexokinase II and peroxisome proliferator-activated receptor gamma co-activator 1α in the heart of FGF<em>21</em>KO diabetic mice compared to WT diabetic mice. Our results demonstrate that FGF<em>21</em> deletion-aggravated cardiac lipid accumulation is likely mediated by cardiac Nrf2-driven CD36 up-regulation, which may contribute to the increased cardiac oxidative stress and remodelling, and the eventual development of DCM. These findings suggest that FGF<em>21</em> may be a therapeutic target for the treatment of DCM.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), a glucose and lipid metabolic regulator, has recently been demonstrated to be associated with cardiovascular diseases (CVD) such as carotid atherosclerosis, coronary heart disease and carotid artery plaques. However, the relationship between circulating FGF<em>21</em> and subclinical atherosclerosis or atherosclerosis of other arteries such as the femoral and iliac artery remains unclear. In this study, we evaluated the association of serum FGF<em>21</em> with intima-media thickness (IMT) and subclinical atherosclerosis in type 2 diabetic patients.

METHODS

Serum FGF<em>21</em> levels were detected by enzyme-linked immunosorbent assay in <em>21</em>2 newly diagnosed type 2 diabetic patients without clinical symptoms of atherosclerosis or cardiovascular diseases. IMT of the carotid, femoral, and iliac arteries were measured by high-resolution B-mode ultrasound to determine the presence of subclinical atherosclerosis, which was defined as having an IMT>> 1.0 mm and/or plaque on one or more of the three arteries without any clinical manifestations. The relationship between serum FGF<em>21</em> levels and subclinical atherosclerosis was analyzed.

RESULTS

Serum FGF<em>21</em> levels were significantly higher in patients with subclinical atherosclerosis compared to those without [261.3 (135.1-396.4) versus 144.9 (95.9-223.0) ng/L, P < 0.001]. These differences were also observed in both men and women with subclinical atherosclerosis compared to their respective groups without [men: 243.2 (107.6-337.0) versus 136.8 (83.6-<em>21</em>2.8) ng/L, P = 0.048; women: 292.4 (174.2-419.9) versus 160.4 (115.3-258.5) ng/L, P = 0.001]. Moreover, serum FGF<em>21</em> levels showed a significantly positive correlation with carotid IMT in women (r = 0.23, P = 0.018) and with iliac IMT in both genders (women: r = 0.27, P = 0.005; men: r = 0.22, P = 0.024). Multiple logistic regression analysis further showed that serum FGF<em>21</em> was an independent impact <em>factor</em> for subclinical atherosclerosis in patients with type 2 diabetes.

CONCLUSIONS

Serum FGF<em>21</em> is elevated in newly diagnosed type 2 diabetes, and positively correlates with carotid and iliac lesions in patients with subclinical atherosclerosis, especially in women. High levels of FGF<em>21</em> may be a compensatory reaction to offset atherosclerosis.

OBJECTIVE

Mitochondria and the endoplasmic reticulum (ER) physically interact by close structural juxtaposition, via the mitochondria-associated ER membrane. Inter-organelle communication between the ER and mitochondria has been shown to regulate energy metabolism and to be central to the modulation of various key processes such as ER stress. We aimed to clarify the role of mitochondrial fission in this communication.

METHODS

We generated mice lacking the mitochondrial fission protein dynamin-related protein 1 (DRP1) in the liver (Drp1LiKO mice).

RESULTS

Drp1LiKO mice showed decreased fat mass and were protected from high-fat diet (HFD)-induced obesity. Analysis of liver gene expression profiles demonstrated marked elevation of ER stress markers. In addition, we observed increased expression of the <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) gene through induction of activating transcription <em>factor</em> 4, master regulator of the integrated stress response.

CONCLUSIONS

Disruption of mitochondrial fission in the liver provoked ER stress, while inducing the expression of FGF<em>21</em> to increase energy expenditure and protect against HFD-induced obesity.

BACKGROUND

Improving the biochemical and structural qualities of the new tissue that fills deep osteochondral defects is critical to enhance articular cartilage repair. We developed a novel molecular therapy to increase articular cartilage repair based on a combined strategy to stimulate chondrogenesis by co-transfection of the human insulin-like growth factor I (IGF-I) and fibroblast growth factor 2 (FGF-2) in a xenogenic transplantation model.

METHODS

NIH 3T3 cells were transfected with expression plasmid vectors containing a cDNA for the E. coli lacZ gene (lacZ implants), the human IGF-I gene (IGF-I implants) or both the human IGF-I and FGF-2 genes (IGF-I/FGF-2 implants). The expression patterns of the transgenes were monitored in vitro for 21 days. LacZ, IGF-I and IGF-I/FGF-2 implants were transplanted into osteochondral defects in the trochlear groove of rabbits. At 3 weeks, the quality of articular cartilage repair was evaluated qualitatively and quantitatively.

RESULTS

Both IGF-I and IGF-I/FGF-2 implants secreted increased levels of the corresponding recombinant proteins in vitro. In vivo, transplantation of the co-transfected IGF-I/FGF-2 implants increased the DNA content of the repair tissue, accelerated the formation of the subchondral bone and improved articular cartilage repair in a magnitude that was larger than with IGF-I alone or when compared to lacZ implants.

CONCLUSIONS

These results suggest that gene delivery of a combination of IGF-I and FGF-2 to cartilage defects may be more beneficial than application of IGF-I alone.

Alcohol consumption leads to adipose tissue lipoatrophy and mobilization of FFAs, which contributes to hepatic fat accumulation in alcoholic liver disease. This study aimed to investigate the role of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>21</em>, a metabolic regulator, in the regulation of chronic-binge alcohol-induced adipose tissue lipolysis. FGF<em>21</em> KO mice were subjected to chronic-binge alcohol exposure, and epididymal white adipose tissue lipolysis and liver steatosis were investigated. Alcohol exposure caused adipose intracellular cAMP elevation and activation of lipolytic enzymes, leading to FFA mobilization in both WT and FGF<em>21</em> KO mice. However, alcohol-induced systemic elevation of catecholamine, which is known to be a major player in adipose lipolysis by binding to the β-adrenergic receptor, was markedly inhibited in KO mice. Supplementation with recombinant human FGF<em>21</em> to alcohol-exposed FGF<em>21</em> KO mice resulted in an increase in fat loss in parallel with an increase of circulating norepinephrine concentration. Furthermore, alcohol consumption-induced fatty liver was blunted in the KO mice, indicating an inhibition of fatty acid reverse transport from adipose to the liver in the KO mice. Taken together, our studies demonstrate that FGF<em>21</em> KO mice are protected from alcohol-induced adipose tissue excess-lipolysis through a mechanism involving systemic catecholamine release.

Our previous studies showed that both exogenous and endogenous FGF21 inhibited cardiac apoptosis at the early stage of type 1 diabetes. Whether FGF21 induces preventive effect on type 2 diabetes-induced cardiomyopathy was investigated in the present study. High-fat-diet/streptozotocin-induced type 2 diabetes was established in both wild-type (WT) and FGF21-knockout (FGF21-KO) mice followed by treating with FGF21 for 4 months. Diabetic cardiomyopathy (DCM) was diagnosed by significant cardiac dysfunction, remodeling, and cardiac lipid accumulation associated with increased apoptosis, inflammation, and oxidative stress, which was aggravated in FGF21-KO mice. However, the cardiac damage above was prevented by administration of FGF21. Further studies demonstrated that the metabolic regulating effect of FGF21 is not enough, contributing to FGF21-induced significant cardiac protection under diabetic conditions. Therefore, other protective mechanisms must exist. The in vivo cardiac damage was mimicked in primary neonatal or adult mouse cardiomyocytes treated with HG/Pal, which was inhibited by FGF21 treatment. Knockdown of AMPKα1/2, AKT2, or NRF2 with their siRNAs revealed that FGF21 protected cardiomyocytes from HG/Pal partially via upregulating AMPK-AKT2-NRF2-mediated antioxidative pathway. Additionally, knockdown of AMPK suppressed fatty acid β-oxidation via inhibition of ACC-CPT-1 pathway. And, inhibition of fatty acid β-oxidation partially blocked FGF21-induced protection in cardiomyocytes. Further, in vitro and in vivo studies indicated that FGF21-induced cardiac protection against type 2 diabetes was mainly attributed to lipotoxicity rather than glucose toxicity. These results demonstrate that FGF21 functions physiologically and pharmacologically to prevent type 2 diabetic lipotoxicity-induced cardiomyopathy through activation of both AMPK-AKT2-NRF2-mediated antioxidative pathway and AMPK-ACC-CPT-1-mediated lipid-lowering effect in the heart.

Platelet-derived <em>growth</em> <em>factor</em> (PDGF), a polypeptide mitogen, is a dimer composed of PDGF-AA and -BB chains. In rats, PDGF-BB is the prevalent circulating form, whereas in bone, PDGF-AA is the isoform secreted by unstimulated normal bone cells. Although PDGF-BB increased DNA synthesis in fetal rat calvariae, the effects on collagen synthesis were small and inconsistent. To localize the cells in the cranial periosteum that were responding to PDGF isoforms AA and BB, we cultured <em>21</em>-day-old fetal rat calvariae to assess the effects of human recombinant PDGF-AA and -BB on bone cell replication and matrix formation. Changes were assessed using histomorphometry and autoradiography and correlated with effects on collagen synthesis and [3H]thymidine incorporation, using biochemical assays. PDGF-AA and -BB at 0.03-3.3 nM (1-100 ng/ml) for 24-72 h increased DNA synthesis by 1.5- to 3-fold; PDGF-BB was more potent than PDGF-AA. Although PDGF increased cell replication in all cell zones, the effects of both PDGF-AA and -BB were preferentially greater in the periosteal <em>fibroblast</em> zone, in which, at 3.3 nM, the labeling index (LI) was increased by 3-fold with AA and by 5-fold with BB. Cell replication of the bone surface cell (osteoblast) layer was increased by 2-fold with AA and by 2.5-fold with BB, whereas replication in the intermediate osteoprogenitor zone increased by 50% with AA and by 2.5-fold with BB. The increase in cell replication was associated with a significant inhibition of bone matrix-forming surfaces, with PDGF-BB being more potent at equivalent doses than -AA after 24-72 h of continuous treatment. Continuous or intermittent exposure to PDGF-AA or PDGF-BB for 24-72 h stimulated neither the rate of collagen synthesis nor organized bone matrix formation in rat calvariae. In addition, PDGF-BB at 0.03-3.3 nM increased the number of osteoclasts and the percent eroded surface by 2- to 3-fold. Our studies show that PDGF-AA and -BB are mitogens affecting multiple bone cells, including those of the osteoblast and osteoclast lineage. Treatment with PDGF severely disrupted and inhibited bone matrix formation, and there was no evidence to show that cells incorporating [3H]thymidine differentiated into mature osteoblasts within the time frame of these experiments. In fetal rat calvaria, the most significant consequence of treatment with PDGF was the selective stimulation of <em>fibroblast</em> replication and function.

Based on prior work showing that human pituitary tumors overexpress epidermal and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors, we hypothesized that downstream components of <em>growth</em> <em>factor</em> signaling pathways may also be dysregulated. Epidermal <em>growth</em> <em>factor</em> pathway substrate number 8 (Eps8) was identified as a transcript overexpressed (5.9-fold) in human pituitary tumors compared with normal pituitary by DNA microarrays. Eps8 mRNA up-regulation was confirmed by semiquantitative RT-PCR. Immunoblot analysis showed that Eps8 protein levels and its downstream target phosphorylated ERK were also up-regulated in human pituitary tumors. Stable overexpression of Eps8 in LbetaT2 gonadotrope pituitary cells augmented colony formation in soft agar at d <em>21</em>. Eps8 cells proliferated more robustly compared with controls in <em>growth</em> <em>factor</em> replete as well as <em>growth</em>-restricted conditions. In addition, the Eps8 overexpressing cells were protected from serum withdrawal-induced apoptosis compared with controls as assessed by caspase-3 cleavage. Epidermal <em>growth</em> <em>factor</em> activated a robust amplification of ERK and modest up-regulation of Akt in Eps8-overexpressing pituitary cells compared with vector controls. MAPK kinase inhibition or silencing of Eps8 blunted the proliferation of the cells in response to <em>growth</em> <em>factor</em> stimulation. Blockade of the phosphatidylinositol 3-kinase pathway or silencing of Eps8 resulted in a loss of the Eps8 protection from <em>growth</em> <em>factor</em> withdrawal-induced apoptosis. Together these data support a role of Eps8 in amplifying <em>growth</em> <em>factor</em> receptor signaling in human pituitary tumors to promote proliferation and cell survival.

Circulating <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) levels are elevated in diabetic subjects and correlate directly with abnormal glucose metabolism, while pharmacologically administered FGF<em>21</em> can ameliorate hyperglycemia. The pancreatic islet is an FGF<em>21</em> target, yet the actions of FGF<em>21</em> in the islet under normal and diabetic conditions are not fully understood. This study investigated the effects of high glucose on islet FGF<em>21</em> actions in a diabetic mouse model by investigating db/db mouse islet responses to exogenous FGF<em>21</em>, the direct effects of glucose on FGF<em>21</em> signaling, and the involvement of peroxisome proliferator-activated receptor γ (PPARγ) in FGF<em>21</em> pathway activation. Results showed that both adult db/db mouse islets and normal islets treated with high glucose ex vivo displayed reduced β-klotho expression, resistance to FGF<em>21</em>, and decreased PPARγ expression. Rosiglitazone, an antidiabetic PPARγ ligand, ameliorated these effects. Our data indicate that hyperglycemia in type 2 diabetes mellitus may lead to FGF<em>21</em> resistance in pancreatic islets, probably through reduction of PPARγ expression, which provides a novel mechanism for glucose-mediated islet dysfunction.

Controlled differentiation of multi-potent mesenchymal stem cells (MSCs) into vocal fold-specific, <em>fibroblast</em>-like cells in vitro is an attractive strategy for vocal fold repair and regeneration. The goal of the current study was to define experimental parameters that can be used to control the initial <em>fibroblast</em>ic differentiation of MSCs in vitro. To this end, connective tissue <em>growth</em> <em>factor</em> (CTGF) and micro-structured, fibrous scaffolds based on poly(glycerol sebacate) (PGS) and poly(ɛ-caprolactone) (PCL) were used to create a three-dimensional, connective tissue-like microenvironment. MSCs readily attached to and elongated along the microfibers, adopting a spindle-shaped morphology during the initial 3 days of preculture in an MSC maintenance medium. The cell-laden scaffolds were subsequently cultivated in a conditioned medium containing CTGF and ascorbic acids for up to <em>21</em> days. Cell morphology, proliferation, and differentiation were analyzed collectively by quantitative PCR analyses, and biochemical and immunocytochemical assays. F-actin staining showed that MSCs maintained their <em>fibroblast</em>ic morphology during the 3 weeks of culture. The addition of CTGF to the constructs resulted in an enhanced cell proliferation, elevated expression of <em>fibroblast</em>-specific protein-1, and decreased expression of mesenchymal surface epitopes without markedly triggering chondrogenesis, osteogenesis, adipogenesis, or apoptosis. At the mRNA level, CTGF supplement resulted in a decreased expression of collagen I and tissue inhibitor of metalloproteinase 1, but an increased expression of decorin and hyaluronic acid synthesase 3. At the protein level, collagen I, collagen III, sulfated glycosaminoglycan, and elastin productivity was higher in the conditioned PGS-PCL culture than in the normal culture. These findings collectively demonstrate that the fibrous mesh, when combined with defined biochemical cues, is capable of fostering MSC <em>fibroblast</em>ic differentiation in vitro.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2), a multifunctional polypeptide that affects cell <em>growth</em> and differentiation and becomes upregulated by stress, is expressed as AUG-initiated 18 kDa FGF-2 or CUG-initiated <em>21</em>-34 kDa (hi-FGF-2) isoforms. Animal models have provided strong evidence that FGF-2 is essential for the manifestation of overload- and angiotensin-induced cardiac hypertrophy. Nevertheless, studies to-date have not discriminated between the activities of 18 kDa FGF-2 and hi-FGF-2. Our recent work has pointed to a potent pro-hypertrophic effect of added hi-FGF-2, and a pro-apoptotic effect of sustained intracrine hi-FGF-2 signaling. In the future, it will be important to differentiate between the activities of the different FGF-2 isoforms in the context of adaptive and maladaptive myocardial hypertrophy and heart failure. Based on all available evidence, we propose that while the 18-kDa FGF-2 is a component of an adaptive trophic response, a switch to hi-FGF-2 accumulation would exacerbate hypertrophy and contribute to cell death, thus driving the myocardium towards a maladaptive phenotype.

Increasing incidence of type 2 diabetes is a major health problem of the modern world and requires new diagnostic tools to assess early metabolic disorders, particularly insulin resistance. The link between obesity, inflammation and insulin resistance indicates the important secretory role of adipose tissue. Proinflammatory <em>factors</em> (cytokines, adipokines) produced by enlarged adipose tissue are related to impaired glucose metabolism. Adipokines act as paracrine <em>factors</em> in adipose tissue and as endocrine hormones in the liver, muscles and central nervous system. Novel adipokines secreted from adipocytes such as retinol binding protein-4 (RBP-4), vaspin, omentin, chemerin, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), adipocyte fatty acid-binding protein (A-FABP) and dipeptidyl peptidase 4 (DPP4) demonstrate pleiotropic activity and their insulin-sensitizing or enhancing insulin resistance properties have not been clearly confirmed yet. In spite of the lack of standardized automated assay methods currently available for these novel biomarkers, promising results from several studies emphasize that they might potentially be useful prognostic <em>factors</em> for diabetes and its complications, especially in individuals without the typical symptoms of metabolic syndrome.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) was originally identified as a member of the FGF family in homology studies and is a member of the endocrine FGF subfamily that lacks heparin binding domains and is released into the circulation. A potential role as a metabolic regulator emerged when FGF<em>21</em> was shown to increase glucose uptake in adipocytes. Subsequently, marked elevations in FGF<em>21</em> expression were observed in mice that ate a ketogenic diet and when fasting, which suggests that FGF<em>21</em> expression plays a role in the adaptation to metabolic states that require increased fatty acid oxidation. Consistent with this evidence, FGF<em>21</em> knockout mice were not able to respond appropriately to consumption of a ketogenic diet. FGF<em>21</em> expression is downstream of peroxisome proliferator-activated receptor (PPAR) α in the liver and PPARγ in adipose tissue. FGF<em>21</em> concentrations are higher in both rodent and human obesity, and recent data suggest that obesity may be an FGF<em>21</em>-resistant state. Recent data increasingly suggest that FGF<em>21</em> is an important metabolic regulator that may have potential clinical implications.

After incubation with human serum or plasma, 125I-basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) (molecular mass 18.5 kDa) exhibits molecular mass forms greater than 200 kDa as determined by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. These high molecular mass forms of bFGF are immunoprecipitable with antiserum raised against alpha 2-macroglobulin (alpha 2M). Purified alpha 2M and 125I-bFGF form a covalent complex in a specific, saturable manner. Excess unlabeled bFGF competes with 125I-bFGF for complex formation. Complex formation is complete after 4 h and is inhibited by pretreating alpha 2M with dithiothreitol, iodoacetamide, iodoacetic acid, and N-ethylmaleimide. The complex is resistant to acidic conditions and denaturants such as urea. Heparin, which binds bFGF, has no effect on complex formation. Methylamine, which blocks protease binding to alpha 2M, increases the amount of 125I-bFGF that can be bound 2-fold. Plasmin and trypsin treatment of alpha 2M has no effect on 125I-bFGF binding. The ability of <em>growth</em> <em>factors</em> to compete for binding is specific, as aFGF and TGF-beta compete for binding to alpha 2M, whereas platelet-derived <em>growth</em> <em>factor</em> does not. 125I-bFGF.alpha 2M complexes do not bind to low affinity bFGF binding sites and bind poorly to high affinity bFGF binding sites on BHK-<em>21</em> cells. In addition, 125I-bFGF bound to alpha 2M has decreased ability to stimulate plasminogen activator production in bovine capillary epithelial cells.

OBJECTIVE

To investigate the mechanism and regulation of differentiation from bone marrow mesenchymal stem cells (MSCs) into hepatocytes and to find a new source of cell types for therapies of hepatic diseases.

METHODS

MSCs were isolated by combining gradient density centrifugation with plastic adherence. The cells were cultured in osteogenic or adipogenic differentiation medium and determined by histochemical staining. MSCs were plated in plastic culture flasks that were not coated with components of extracellular matrix (ECM). When MSCs reached 70% confluence, they were cultured in low glucose Dulbecco's modified Eagle's medium supplemented with 10 mL/L fetal bovine serum, 20 ng/mL hepatocyte growth factor (HGF) and 10 ng/mL fibroblast growth factor-4 (FGF-4). The medium was changed every 3 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Glycogen store of hepatocytes was determined by periodic acid-Schiff staining.

RESULTS

By combining gradient density centrifugation with plastic adherence, we isolated a homogeneous population of cells from rat bone marrow and differentiated them into osteocytes and adipocytes. When MSCs were cultured with FGF-4 and HGF, approximately 56.6% of cells became small round and epithelioid on d 24 by morphology. Compared with the control, levels of AFP increased significantly from d 12 to 15.5+/-1.4 microg/L (t = 2.31, P<0.05) in MSCs cultured with FGF-4 and HGF, and were higher (46.2+/-1.5 microg/L) on d 21 (t = 41.926, P<0.01), then decreased to 24.8+/-2.2 microg/L on d 24 (t = 10.345, P<0.01). Albumin increased significantly on d 21 (t = 3.325, P<0.01) to 1.4+/-0.2 microg/mL, and to 2.1+/-0.7 microg/mL on d 24 (t = 3.646, P<0.01). Urea (2.3+/-0.4 mmol/L) was first detected on d 21 (t = 6.739, P<0.01), and continued to increase to 2.6+/-0.9 mmol/L on d 24 (t = 4.753, P<0.01). Glycogen storage was first seen on d 21.

CONCLUSIONS

The method combining gradient density centrifugation with plastic adherence can isolate MSCs. Rat MSCs may be differentiated into hepatocytes by FGF-4 and HGF. Cytokines may play a more important role in differentiation from rat MSCs into hepatocytes.