The potential cytotoxic and anti-proliferative activities of ellagic acid (a naturally occurring bioactive compound in berries, grapes, and nuts) was evaluated using human umbilical vein endothelial cells (HUVEC), normal human lung <em>fibroblast</em> cells HEL 299, Caco-2 colon, MCF-7 breast, Hs 578T breast, and DU 145 human prostatic cancer cells. Ellagic acid at concentration in the range 10-100 micromol/L did not affect the viability of normal <em>fibroblast</em> cells during a 24-hour incubation. An increase in adenosine triphosphate (ATP) bioluminescence of approximately 18-<em>21</em>% was observed in normal cells incubated with ellagic acid. In contrast, ellagic acid at 1-100 micromol/L dose-dependently inhibited HUVEC tube formation and proliferation on a reconstituted extracellular matrix and showed strong anti-proliferative activity against the colon, breast, and prostatic cancer cell lines investigated. The most sensitive cells were the Caco-2, and the most resistant were the breast cancer cells. Ellagic acid induced cancer cell death by apoptosis as shown by the microscopic examination of cell gross morphology. Ellagic acid induced reduced cancer cell viability as shown by decreased ATP levels of the cancer cells. After 24 hours incubation of 100 micromol/L of ellagic acid with Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells, ellagic acid suppressed fetal bovine serum (FBS) stimulation of cell migration. The apoptosis induction was accompanied by a decreased in the levels of pro-matrix metalloproteinase-2 (pro-MMP-2 or gelatinase A), pro-matrix metalloproteinase-9 (pro-MMP-9 or gelatinase B), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF(165)) in conditioned media. The results suggest that ellagic acid expressed a selective cytotoxicity and anti-proliferative activity, and induced apoptosis in Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells without any toxic effect on the viability of normal human lung <em>fibroblast</em> cells. It was also observed that the mechanism of apoptosis induction in ellagic acid-treated cancer cells was associated with decreased ATP production, which is crucial for the viability of cancer cells.

The presence of the intermediate filament protein nestin has been the predominant marker used to describe stem and progenitor cells in the mammalian CNS. In this study, a 998-bp fragment in the 3' region of the nestin mRNA was cloned from human fetal brain cells (HFBC). The nucleotide sequence of the cloned cDNA revealed <em>21</em> differences with the previously published human nestin sequence, resulting in 17 amino acid changes. A 150-amino-acid fragment derived from the cloned nestin cDNA was coupled to glutathione S-transferase and used as an immunogen to generate a rabbit polyclonal antiserum that selectively detects human nestin. HFBC that proliferated in response to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> incorporated 5-bromo-2'-deoxyuridine into their nuclei and immunostained for nestin, indicating nestin expression in proliferating CNS progenitor cells. In all cell cultures, nestin costained with the neuroepithelial cell marker vimentin. A small subset of nestin-stained cells (1-2%) immunostained with neuronal marker MAP-2 during the first week and after 4 weeks in culture. However, during the first week in culture, approximately 10-30% of the total cell population of HFBC stained for the glial cell marker GFAP, and nearly all coimmunostained for nestin. After 4 weeks in culture, a subset of GFAP-positive cells emerged that no longer costained with nestin. These results describe nestin expression not only in CNS progenitor cells but also in the cells which were in transition from a progenitor stage to glial differentiation. Collectively, these data suggest a differential temporal regulation of nestin expression during glial and neuronal cell differentiation.

OBJECTIVE

The primary objective of this study was to determine whether intracoronary administration of the adenoviral gene for fibroblast growth factor (Ad5FGF-4) can improve myocardial perfusion compared with placebo.

BACKGROUND

Animal studies and observational clinical studies have shown improvement in perfusion of the ischemic myocardium using genes encoding angiogenic growth factors; however, randomized, double-blind data in humans are lacking.

METHODS

We performed a randomized, double-blind, placebo-controlled trial of intracoronary injection of 10(10) adenoviral particles containing a gene encoding fibroblast growth factor (Ad5FGF-4) to determine the effect on myocardial perfusion. Fifty-two patients with stable angina and reversible ischemia comprising >9% of the left ventricle on adenosine single-photon emission computed tomography (SPECT) imaging were randomized to gene therapy (n = 35) or placebo (n = 17). Clinical follow-up was performed, and 51 (98%) patients underwent a second adenosine SPECT scan after 8 weeks.

RESULTS

Overall (n = 52), the mean total perfusion defect size at baseline was 32.4% of the left ventricle, with 20% reversible ischemia and 12.5% scar. At eight weeks, Ad5FGF-4 injection resulted in a significant reduction of ischemic defect size (4.2% absolute, 21% relative; p < 0.001) and placebo-treated patients had no improvement (p = 0.32). Although the change in reversible perfusion defect size between Ad5FGF-4 and placebo was not significant (4.2% vs. 1.6%, p = 0.14), when a single outlier was excluded a significant difference was observed (4.2% vs. 0.8%, p < 0.05). Ad5FGF-4 was well tolerated and did not result in any permanent adverse sequelae.

CONCLUSIONS

Intracoronary injection of Ad5FGF-4 showed an encouraging trend for improved myocardial perfusion; however, further studies of therapeutic angiogenesis with Ad5FGF-4 will be necessary.

OBJECTIVE

To investigate human umbilical cord-derived mesenchymal stem cells (hUCMSCs) for use in the reversal of mouse hepatic injury.

METHODS

Human umbilical cord-derived mesenchymal stem cells, characterized by flow cytometry, were transplanted into carbon tetrachloride (CCl(4))-injured mice, and then followed for determination of localization and differentiation. Reverse transcriptase-polymerase chain reaction for the human 17alpha gene and fluorescence in situ hybridization analysis for the human X chromosome were used to locate exogenous hUCMSCs in mouse livers. Peripheral blood and liver specimens were collected at 7, 14 and <em>21</em> days after transplantation. For evaluating the recovery of injured liver tissues, serum aminotransferase was measured, and the pathological state of the hepatocytes was assessed.

RESULTS

The hUCMSCs were positive for the human MSC-specific markers CD13, CD29, CD44, CD105 and nerve growth factor receptor, but negative for the haematopoietic lineage markers CD31, CD34, CD38, CD45 and HLA-DR. Under conditions favouring differentiation in vivo, the expression of tryptophan 2,3-dioxygenase, human alpha-fetoprotein, cytokeratin 18, fibroblast secretory protein 1 and alpha-smooth-muscle-actin was detectable after hUCMSCs administration to mice subjected to liver injury. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labelling and proliferating cell nuclear antigen staining showed that transplanted hUCMSCs could inhibit hepatocyte apoptosis and facilitate proliferation. Serum aminotransferases were decreased after transplantation of hUCMSCs into the injured mice, and hepatocyte denaturation was reduced.

CONCLUSIONS

Human umbilical cord-derived mesenchymal stem cells can enhance recovery of CCl(4)-injured mouse liver, providing evidence that such therapy could be useful for liver disorders or injury.

Cardiac fibrosis is characterized by aberrant proliferation of cardiac <em>fibroblasts</em> and exaggerated deposition of extracellular matrix (ECM) in the myocardial interstitial, and ultimately impairs cardiac function. It is still controversial whether microRNA-<em>21</em> (miR-<em>21</em>) participates in the process of cardiac fibrosis. Our previous study confirmed that transforming <em>growth</em> <em>factor</em> beta receptor III (TGFβRIII) is a negative regulator of TGF-β pathway. Here, we aimed to decipher the relationship between miR-<em>21</em> and TGFβRIII in the pathogenic process of myocardial fibrosis. We found that TGF-β1 and miR-<em>21</em> were up-regulated, whereas TGFβRIII was down-regulated in the border zone of mouse hearts in response to myocardial infarction. After transfection of miR-<em>21</em> into cardiac <em>fibroblasts</em>, TGFβRIII expression was markedly reduced and collagen content was increased. And, luciferase results confirmed that TGFβRIII was a target of miR-<em>21</em>. It suggests that up-regulation of miR-<em>21</em> could increase the collagen content and at least in part through inhibiting TGFβRIII. Conversely, we also confirmed that overexpression of TGFβRIII could inhibit the expression of miR-<em>21</em> and reduce collagen production in <em>fibroblasts</em>. Further studies showed that overexpression of TGFβRIII could also deactivate TGF-β1 pathway by decreasing the expression of TGF-β1 and phosphorylated-Smad3 (p-Smad3). TGF-β1 has been proven as a positive regulator of miR-<em>21</em>. Taken together, we found a novel reciprocal loop between miR-<em>21</em> and TGFβRIII in cardiac fibrosis caused by myocardial infarction in mice, and targeting this pathway could be a new strategy for the prevention and treatment of myocardial remodeling.

The liver is a key metabolic organ that controls whole-body physiology in response to nutrient availability. Mammalian target of rapamycin (mTOR) is a nutrient-activated kinase and central controller of <em>growth</em> and metabolism that is negatively regulated by the tumor suppressor tuberous sclerosis complex 1 (TSC1). To investigate the role of hepatic mTOR complex 1 (mTORC1) in whole-body physiology, we generated liver-specific Tsc1 (L-Tsc1 KO) knockout mice. L-Tsc1 KO mice displayed reduced locomotor activity, body temperature, and hepatic triglyceride content in a rapamycin-sensitive manner. Ectopic activation of mTORC1 also caused depletion of hepatic and plasma glutamine, leading to peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)-dependent <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) expression in the liver. Injection of glutamine or knockdown of PGC-1α or FGF<em>21</em> in the liver suppressed the behavioral and metabolic defects due to mTORC1 activation. Thus, mTORC1 in the liver controls whole-body physiology through PGC-1α and FGF<em>21</em>. Finally, mTORC1 signaling correlated with FGF<em>21</em> expression in human liver tumors, suggesting that treatment of glutamine-addicted cancers with mTOR inhibitors might have beneficial effects at both the tumor and whole-body level.

The liver is an important integrator of nutrient metabolism, yet no liver-derived <em>factors</em> regulating nutrient preference or carbohydrate appetite have been identified. Here we show that the liver regulates carbohydrate intake through production of the hepatokine <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), which markedly suppresses consumption of simple sugars, but not complex carbohydrates, proteins, or lipids. Genetic loss of FGF<em>21</em> in mice increases sucrose consumption, whereas acute administration or overexpression of FGF<em>21</em> suppresses the intake of both sugar and non-caloric sweeteners. FGF<em>21</em> does not affect chorda tympani nerve responses to sweet tastants, instead reducing sweet-seeking behavior and meal size via neurons in the hypothalamus. This liver-to-brain hormonal axis likely represents a negative feedback loop as hepatic FGF<em>21</em> production is elevated by sucrose ingestion. We conclude that the liver functions to regulate macronutrient-specific intake by producing an endocrine satiety signal that acts centrally to suppress the intake of "sweets."

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a potent regulator of glucose and lipid metabolism and is currently being pursued as a therapeutic agent for insulin resistance and type 2 diabetes. However, the cellular mechanisms by which FGF<em>21</em> modifies insulin action in vivo are unclear. To address this question, we assessed insulin action in regular chow- and high-fat diet (HFD)-fed wild-type mice chronically infused with FGF<em>21</em> or vehicle. Here, we show that FGF<em>21</em> administration results in improvements in both hepatic and peripheral insulin sensitivity in both regular chow- and HFD-fed mice. This improvement in insulin responsiveness in FGF<em>21</em>-treated HFD-fed mice was associated with decreased hepatocellular and myocellular diacylglycerol content and reduced protein kinase Cε activation in liver and protein kinase Cθ in skeletal muscle. In contrast, there were no effects of FGF<em>21</em> on liver or muscle ceramide content. These effects may be attributed, in part, to increased energy expenditure in the liver and white adipose tissue. Taken together, these data provide a mechanism by which FGF<em>21</em> protects mice from lipid-induced liver and muscle insulin resistance and support its development as a novel therapy for the treatment of nonalcoholic fatty liver disease, insulin resistance, and type 2 diabetes.

BACKGROUND

Bronchial asthma is characterised by airway structural changes, including mucosal inflammatory infiltration and subepithelial collagen deposition, that may represent the morphological basis for the chronicity of the disease. The relationship between airway wall thickness and growth factors in asthma has not been elucidated.

METHODS

Bronchial biopsy specimens were obtained from 21 asthmatic patients and eight healthy subjects and the basement membrane thickness was measured by light microscopy and electron microscopy. At the same time the numbers of eosinophils and fibroblasts were assessed and the expression of transforming growth factor beta 1 (TGF-beta 1), platelet derived growth factor (PDGF), and insulin like growth factor (IGF) I in the bronchial mucosa was examined by immunostaining. The relationship between the degree of thickening of the subepithelial layer and both the clinical data and pulmonary function were also investigated.

RESULTS

The basement membrane of the asthmatic patients was thicker than that of the healthy controls (median 8.09 versus 4.02 microns). Electron microscopic examination of the basement membrane revealed thickening of the subepithelial lamina reticularis; this thickening significantly correlated with the number of fibroblasts in the submucosa in the asthmatic subjects (rs = 0.88) but not in the controls (rs = 0.70). There was a significantly higher number of eosinophils in the airways of the asthmatic subjects than in the healthy subjects (EG1 + cells: 52.0 versus 2.0/mm2, EG2 + cells: 56.0 versus 1.5/mm2). The expression of each growth factor in the bronchial mucosa was similar in asthmatic and healthy subjects (TGF-beta 1: 18.0% versus 16.0%, PDGF: 37.0% versus 32.5%, IGF-I: 15.0% versus 8.0%). A weak but statistically significant correlation was found between the number of fibroblasts and the expression of TGF-beta 1 in asthmatic subjects (rs = 0.50). There was a significant correlation between the thickness of the subepithelial layer in asthmatic subjects and the attack score (rs = 0.58) and a significant inverse correlation between the subepithelial collagen thickness in asthmatic subjects and airway hypersensitivity (rs = -0.65).

CONCLUSIONS

These findings indicate that the thickening of the subepithelial layer in bronchial asthma is due to an increase in fibroblasts, and that the thickness of the subepithelial collagen appears to be linked to an increase in bronchial responsiveness and exacerbation of clinical manifestations.

BACKGROUND

Rheumatoid arthritis (RA) is associated with an increased production of a range of cytokines including tumour necrosis factor (TNF)-alpha and interleukin (IL)-1, which display potent proinflammatory actions that are thought to contribute to the pathogenesis of the disease. Although TNF-alpha seems to be the major cytokine in the inflammatory process, IL-1 is the key mediator with regard to cartilage and bone destruction. Apart from direct blockage of IL-1/TNF, regulation can be exerted at the level of modulatory cytokines such as IL-1 and IL-10. IL-4 is a pleiotropic T-cell derived cytokine that can exert either suppressive or stimulatory effects on different cell types, and was originally identified as a B-cell growth factor and regulator of humoral immune pathways. IL-4 is produced by activated CD4+T cells and it promotes the maturation of TH2 cells. IL-4 stimulates proliferation, differentiation and activation of several cell types, including fibroblasts, endothelial cells and epithelial cells. IL-4 is also known to be a potent anti-inflammatory cytokine that acts by inhibiting the synthesis of proinflammatory cytokines such as IL-1, TNF-alpha, IL-6, IL-8 and IL-12 by macrophages and monocytes. Moreover, IL-4 stimulates the synthesis of several cytokine inhibitors such as interleukin-1 receptor antagonist (IL-1Ra), soluble IL-1-receptor type II and TNF receptors IL-4 suppresses metalloproteinase production and stimulates tissue inhibitor of metalloproteinase-1 production in human mononuclear phagocytes and cartilage explants, indicating a protective effect of IL-4 towards extracellular matrix degradation. Furthermore, IL-4 inhibits both osteoclast activity and survival, and thereby blocks bone resorption in vitro. Of great importance is that IL-4 could not be detected in synovial fluid or in tissues. This absence of IL-4 in the joint probably contributes to the disturbance in the Th1/Th2 balance in chronic RA. Collagen-induced arthritis (CIA) is a widely used model of arthritis that displays several features of human RA. Recently it was demonstrated that the onset of CIA is under stringent control of IL-4 and IL-10. Furthermore, it was demonstrated that exposure to IL-4 during the immunization stage reduced onset and severity of CIA. However, after cessation of IL-4 treatment disease expression increased to control values.

OBJECTIVE

Because it was reported that IL-4 suppresses several proinflammatory cytokines and matrix degrading enzymes and upregulates inhibitors of both cytokines and catabolic enzymes, we investigated the tissue protective effect of systemic IL-4 treatment using established murine CIA as a model. Potential synergy of low dosages of anti-inflammatory glucocorticosteroids and IL-4 was also evaluated.

METHODS

DBA-1J/Bom mice were immunized with bovine type II collagen and boosted at day 21. Mice with established CIA were selected at day 28 after immunization and treated for days with IL-4, prednisolone, or combinations of prednisolone and IL-4. Arthritis score was monitored visually. Joint pathology was evaluated by histology, radiology and serum cartilage oligomeric matrix protein (COMP). In addition, serum levels of IL-1Ra and anticollagen antibodies were determined.

RESULTS

Treatment of established CIA with IL-4 (1microgram/day) resulted in suppression of disease activity as depicted in Figure 1. Of great interest is that, although 1 microgram/day IL-4 had only a moderate effect on the inflammatory component of the disease activity, it strongly reduced cartilage pathology, as determined by histological examination (Fig. 1). Moreover, serum COMP levels were significantly reduced, confirming decreased cartilage involvement. In addition, both histological and radiological analysis showed that bone destruction was prevented (Fig. 1). Systemic IL-4 administration increased serum IL-1Ra levels and reduced anticollagen type II antibody levels. Treatment with low-dose IL-4 (0.1 microgram/day) was ineffective in suppressing disease score, serum COMP or joint destruction. Synergistic suppression of both arthritis severity and COMP levels was noted when low-dose IL-4 was combined with prednisolone (0.05 mg/kg/day), however, which in itself was not effective.

CONCLUSIONS

In the present study, we demonstrate that systemic IL-4 treatment ameliorates disease progression of established CIA. Although clinical disease progression of established CIA. Although clinical disease progression was only arrested and not reversed, clear protection against cartilage and bone destruction was noted. This is in accord with findings in both human RA and animal models of RA that show that inflammation and tissue destruction sometimes are uncoupled processes. Of great importance is that, although inflammation was still present, strong reduction in serum COMP was found after exposure to IL-4. This indicated that serum COMP levels reflected cartilage damage, although a limited contribution of the inflamed synovium cannot be excluded. Increased serum IL-1Ra level (twofold) was found after systemic treatment with IL-4, but it is not likely that this could explain the suppression of CIA. We and others have reported that high dosages of IL-1Ra are needed for marked suppression of CIA. As reported previously, lower dosages of IL-4 did not reduce clinical disease severity of established CIA. Of importance is that combined treatment of low dosages of IL-4 and IL-10 appeared to have more potent anti-inflammatory effects, and markedly protected against cartilage destruction. Improved anti-inflammatory effect was achieved with IL-4/prednisolone treatment. In addition, synergistic effects were found for the reduction of cartilage and bone destruction. This indicates that systemic IL-4/prednisolone treatment may provide a cartilage and bone protective therapy for human RA.

OBJECTIVE

To review the rationale for targeting the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) pathways for anti-angiogenic therapy in patients with ovarian cancer and to summarize the currently available data with agents that block these pathways.

METHODS

Relevant papers and studies were identified by searches conducted on Medline using the terms angiogenesis, ovarian cancer, VEGF, PDGF, FGF, receptor, kinase, and inhibitor alone or in combination as well as by searches by drug name and by review of abstracts presented at recent oncology meetings.

RESULTS

The VEGF pathway is considered to be the key driver of angiogenesis, but the PDGF and FGF pathways also play important roles and may contribute to resistance to VEGF-specific blockade. Each pathway may also promote tumorigenesis; tumor cell overexpression of these growth factors and their receptors have been detected in ovarian tumor specimens, suggesting that autocrine loops may lead to tumor growth and progression. Selective inhibitors of the VEGF pathway (e.g., bevacizumab and VEGF Trap) as well as VEGF/PDGF pathway inhibitors (e.g., sorafenib and sunitinib) and VEGF/PDGF/FGF pathway inhibitors (e.g., cediranib, pazopanib, and BIBF 1120) have shown single-agent activity in women with ovarian cancer in phase II trials. Response rates of up to 21% have been reported with several agents in patients with recurrent ovarian cancer. Phase III trials with many anti-angiogenic agents in the treatment of ovarian cancer are currently ongoing.

CONCLUSIONS

Anti-angiogenic agents may provide an improvement in the treatment of patients with recurrent ovarian cancer and may be useful when incorporated into first-line platinum/taxane therapy. It remains to be determined whether multitargeted agents will offer greater clinical benefit than specific VEGF pathway inhibitors.

Glucagon, an essential regulator of glucose homeostasis, also modulates lipid metabolism and promotes weight loss, as reflected by the wasting observed in glucagonoma patients. Recently, coagonist peptides that include glucagon agonism have emerged as promising therapeutic candidates for the treatment of obesity and diabetes. We developed a novel stable and soluble glucagon receptor (GcgR) agonist, which allowed for in vivo dissection of glucagon action. As expected, chronic GcgR agonism in mice resulted in hyperglycemia and lower body fat and plasma cholesterol. Notably, GcgR activation also raised hepatic expression and circulating levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>). This effect was retained in isolated primary hepatocytes from wild-type (WT) mice, but not GcgR knockout mice. We confirmed this link in healthy human volunteers, where injection of natural glucagon increased plasma FGF<em>21</em> within hours. Functional relevance was evidenced in mice with genetic deletion of FGF<em>21</em>, where GcgR activation failed to induce the body weight loss and lipid metabolism changes observed in WT mice. Taken together, these data reveal for the first time that glucagon controls glucose, energy, and lipid metabolism at least in part via FGF<em>21</em>-dependent pathways.

<em>Growth</em> <em>factors</em> are known to appear during wound healing. We hypothesized that <em>growth</em> <em>factors</em> would also appear during the healing process of a rotator cuff tear. We determined the expression of various <em>growth</em> <em>factors</em> during healing of acute rotator cuff tears in the rabbit. We made a full-thickness defect in the supraspinatus tendon of 27 Japanese white rabbits. The shoulders were harvested on days 1, 3, 5, 7, 9, 11, 14, <em>21</em>, and 28 postoperatively (n = 3 at each time point). We assessed the expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em> 1, platelet-derived <em>growth</em> <em>factor</em>, and transforming <em>growth</em> <em>factor</em> beta. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> appeared with its peak on days 7 and 9, insulin-like <em>growth</em> <em>factor</em> 1 appeared with its peak on day 5, platelet-derived <em>growth</em> <em>factor</em> appeared with a mild expression between days 7 and 14, and transforming <em>growth</em> <em>factor</em> beta appeared with constant mild expression throughout the observation period. It is likely that each of these <em>growth</em> <em>factors</em> plays a role in the early phase of healing of the supraspinatus tendon in rabbits.

Normophosphatemia and normocalcemia are maintained in chronic kidney disease (CKD) by increased levels of <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF-23) and parathyroid hormone (PTH), but the stimuli for secretion of these hormones in early CKD are incompletely understood. Most human physiologic studies have focused on random or fasting measurements of phosphorus, calcium, FGF-23, and PTH, but in this study, the hypothesis was that measurements in the postprandial state may reveal intermittent stimuli that lead to increased FGF-23 and PTH levels. The 4-h postprandial response in 13 patients with CKD and fasting normophosphatemia and normocalcemia (mean GFR 41 +/- 8 ml/min per m(2)) was compared with <em>21</em> healthy volunteers. Compared with healthy subjects, fasting patients with CKD had significantly higher levels of FGF-23 and fractional excretion of phosphorus; lower fractional excretion of calcium; and no difference in serum calcium, phosphorus, and PTH levels. After standardized meals, urinary phosphorus excretion in both groups increased despite unchanged serum phosphorus and FGF-23 levels. Postprandial urinary calcium excretion also increased in both groups, and this was accompanied by significantly reduced serum calcium and increased PTH levels in patients with CKD only; therefore, FGF-23 does not seem to be an acute postprandial regulator of phosphaturia in CKD or in health, but inappropriate postprandial calciuria with episodic, relative hypocalcemia may represent a previously unreported mechanism of secondary hyperparathyroidism in CKD.

OBJECTIVE

Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues. In this study, the interactions between CTGF and transforming growth factor (TGF)-beta were assessed in human corneal fibroblasts, and the levels and location of CTGF protein and mRNA were measured during healing of excimer laser ablation wounds in rat corneas.

METHODS

Human corneal fibroblasts were incubated with TGF-beta1, -beta2, and -beta3 isoforms, and CTGF mRNA and protein were measured. CTGF was immunolocalized in the cultured fibroblasts by using a specific antibody. Regulation of collagen synthesis by TGF-beta and CTGF was assessed in human corneal fibroblasts with a neutralizing antibody and an antisense oligonucleotide to CTGF. CTGF mRNA and protein were measured in rat corneas up to day 21 after excimer ablation of the cornea. CTGF protein was immunolocalized in rat corneas after photorefractive keratectomy (PRK), and the presence of CTGF mRNA and protein in ex vivo rat corneal scrapings was established.

RESULTS

All three TGF-beta isoforms stimulated expression of CTGF in human corneal fibroblasts, and CTGF was immunolocalized in the cells. Both TGF-beta and CTGF increased collagen synthesis in corneal fibroblasts. Furthermore, CTGF antibody or antisense oligonucleotide blocked TGF-beta-stimulated collagen synthesis. CTGF protein and mRNA increased in rat corneas through day 21 after PRK. CTGF expression was also detected in ex vivo scrapings of rat corneas.

CONCLUSIONS

These data demonstrate that CTGF is expressed by corneal cells after stimulation by TGF-beta, that CTGF expression increases significantly during corneal wound healing, and that CTGF mediates the effects of TGF-beta induction of collagen synthesis by corneal fibroblasts. These data support the hypothesis that CTGF promotes corneal scar formation and imply that regulating CTGF synthesis and action may be an important goal for reducing corneal scarring.

Bile acids activate farnesoid X receptor (FXR) and G protein-coupled bile acid receptor-1 (aka Takeda G protein-coupled receptor-5 [TGR5]) to regulate bile acid metabolism and glucose and insulin sensitivity. FXR and TGR5 are coexpressed in the enteroendocrine L cells, but their roles in integrated regulation of metabolism are not completely understood. We reported recently that activation of FXR induces TGR5 to stimulate glucagon-like peptide-1 (GLP-1) secretion to improve insulin sensitivity and hepatic metabolism. In this study, we used the intestine-restricted FXR agonist fexaramine (FEX) to study the effect of activation of intestinal FXR on the gut microbiome, bile acid metabolism, and FXR and TGR5 signaling. The current study revealed that FEX markedly increased taurolithocholic acid, increased secretion of <em>fibroblast</em> <em>growth</em> <em>factors</em> 15 and <em>21</em> and GLP-1, improved insulin and glucose tolerance, and promoted white adipose tissue browning in mice. Analysis of 16S ribosomal RNA sequences of the gut microbiome identified the FEX-induced and lithocholic acid-producing bacteria Acetatifactor and Bacteroides. Antibiotic treatment completely reversed the FEX-induced metabolic phenotypes and inhibited taurolithocholic acid synthesis, adipose tissue browning, and liver bile acid synthesis gene expression but further increased intestinal FXR target gene expression. FEX treatment effectively improved lipid profiles, increased GLP-1 secretion, improved glucose and insulin tolerance, and promoted adipose tissue browning, while antibiotic treatment reversed the beneficial metabolic effects of FEX in obese and diabetic mice.

CONCLUSIONS

This study uncovered a mechanism in which activation of intestinal FXR shaped the gut microbiota to activate TGR5/GLP-1 signaling to improve hepatic glucose and insulin sensitivity and increase adipose tissue browning; the gut microbiota plays a critical role in bile acid metabolism and signaling to regulate metabolic homeostasis in health and disease. (Hepatology 2018).

Brown fat generates heat through uncoupled respiration, protecting against hypothermia and obesity. Adult humans have brown fat, but the amounts and activities are substantially decreased in obesity, by unknown mechanisms. Here we show that elevated microRNA 34a (miR-34a) in obesity inhibits fat browning in part by suppressing the browning activators <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) and SIRT1. Lentivirus-mediated downregulation of miR-34a in mice with diet-induced obesity reduced adiposity, improved serum profiles, increased the mitochondrial DNA copy number, and increased oxidative function in adipose tissue in both BALB/c and C57BL/6 mice. Remarkably, downregulation of miR-34a increased coexpression of the beige fat-specific marker CD137 and the browning marker UCP1 in all types of white fat, including visceral fat, and promoted additional browning in brown fat. Mechanistically, downregulation of miR-34a increased expression of the FGF<em>21</em> receptor components, FGFR1 and βKL, and also that of SIRT1, resulting in FGF<em>21</em>/SIRT1-dependent deacetylation of PGC-1α and induction of the browning genes Ucp1, Pgc-1α, and Prdm16. Importantly, anti-miR-34a-mediated beneficial effects, including decreased adiposity, are likely from multiple tissues, since downregulation of miR-34a also improves hepatic FGF<em>21</em> signaling and lipid oxidation. This study identifies miR-34a as an inhibitor of beige and brown fat formation, providing a potential target for treating obesity-related diseases.

BACKGROUND

Non-alcoholic fatty liver disease is a major health problem worldwide. Resveratrol is a natural polyphenol found in edible plants that has a variety of biochemical and physiological effects.

OBJECTIVE

To evaluate the effect of resveratrol on insulin resistance, glucose and lipid metabolism in non-alcoholic fatty liver disease.

METHODS

Double-blind, randomized, placebo-controlled trial: 60 subjects with non-alcoholic fatty liver disease were given 2 placebo capsules (placebo group) or 2 150mg resveratrol capsules (resveratrol group) twice daily for three months. Liver ultrasound imaging, anthropometric profile, serum liver enzymes, insulin, glucose, C-peptide, lipid profile, and inflammation-related cytokines were compared pre and post-treatment.

RESULTS

Compared with the placebo group, resveratrol significantly decreased aspartate aminotransferase, glucose and low-density lipoprotein cholesterol [-6.00 (-9.00, -3.00) IU/L, -0.64±0.31mmol/L, and -0.41±0.35mmol/L, respectively, P≤0.001] alanine aminotransferase, total cholesterol [-7.00 (-11.0, -2.50) IU/L and -0.67±0.50mmol/L, respectively, P=0.002], and homeostasis model assessment insulin resistance index (-0.60±1.15, P=0.016). In the resveratrol group significant reductions of the levels of tumour necrosis <em>factor</em>-alpha, cytokeratin 18 fragment, and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> [-0.53±1.30pg/mL, -26.9 (-70.3, 5.12) IU/L and -23.3 (-43.0, 0.31) pg/mL, respectively, P<0.05] and elevation of adiponectin level [1.22 (-0.37, 1.60) ng/mL, P=0.025] were observed.

CONCLUSIONS

Resveratrol supplementation may benefit patients with non-alcoholic fatty liver disease.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em> is an endocrine <em>factor</em> actively involved in glucose and lipid metabolism in rodents. However, little is known about its physiological function and regulation in humans. This study investigated the diurnal changes in circulating FGF<em>21</em> concentrations and their association with other metabolic markers in both obese and lean individuals.

METHODS

A total of 36 volunteers were assigned to 2 groups. One group received 3 standardized meals and another group was fasted for 24 h. Blood samples were drawn every 30 min throughout a 24-h period. Circulating FGF<em>21</em> concentrations were measured with an in-house chemiluminescence immunoassay. The effects of fatty acids on hepatic production of FGF<em>21</em> were determined by using real-time PCR.

RESULTS

In both the fasting and standardized meals groups, circulating FGF<em>21</em> began to rise at midnight, reaching a peak in the early morning and then declining to basal concentrations early in the afternoon. Baseline concentrations of circulating FGF<em>21</em> were much higher in obese individuals than in lean individuals (P < 0.05). However, the magnitude of the nocturnal rise in circulating FGF<em>21</em> was significantly blunted in obese individuals. The 24-h oscillatory pattern of circulating FGF<em>21</em> resembled that of free fatty acids and cortisol, but was opposite to the patterns of insulin and glucose. Unsaturated fatty acids induced time-dependent expression of FGF<em>21</em> mRNA in human hepatocytes.

CONCLUSIONS

These findings support the role of FGF<em>21</em> as an important metabolic regulator that integrates the circadian rhythm with energy homeostasis in humans. Diurnal rhythms of circulating FGF<em>21</em> could be partly caused by the oscillation of free fatty acids.

Sodium-glucose cotransporter (SGLT) 2 inhibitors increase urinary glucose excretion (UGE), leading to blood glucose reductions and weight loss. However, the impacts of SGLT2 inhibition on energy homeostasis and obesity-induced insulin resistance are less well known. Here, we show that empagliflozin, a SGLT2 inhibitor, enhanced energy expenditure and attenuated inflammation and insulin resistance in high-fat-diet-induced obese (DIO) mice. C57BL/6J mice were pair-fed a high-fat diet (HFD) or a HFD with empagliflozin for 16weeks. Empagliflozin administration increased UGE in the DIO mice, whereas it suppressed HFD-induced weight gain, insulin resistance, and hepatic steatosis. Moreover, empagliflozin shifted energy metabolism towards fat utilization, elevated AMP-activated protein kinase and acetyl-CoA carbolxylase phosphorylation in skeletal muscle, and increased hepatic and plasma <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> levels. Importantly, empagliflozin increased energy expenditure, heat production, and the expression of uncoupling protein 1 in brown fat and in inguinal and epididymal white adipose tissue (WAT). Furthermore, empagliflozin reduced M1-polarized macrophage accumulation while inducing the anti-inflammatory M2 phenotype of macrophages within WAT and liver, lowering plasma TNFα levels and attenuating obesity-related chronic inflammation. Thus, empagliflozin suppressed weight gain by enhancing fat utilization and browning and attenuated obesity-induced inflammation and insulin resistance by polarizing M2 macrophages in WAT and liver.

OBJECTIVE

The aim of this study was to examine the effect of the p38 mitogen-activated protein kinase (MAPK) inhibitor, RWJ-67657 (RWJ), on left ventricular (LV) dysfunction and remodeling post-myocardial infarction (MI) in rats.

BACKGROUND

p38 MAPK signaling has been implicated in the progression of chronic heart failure.

METHODS

From day 7 post-MI (coronary artery ligation), rats received either RWJ (50 mg/day, by gavage, n = 8, MI+RWJ) or vehicle (by gavage, n = 8, MI+V) for <em>21</em> days. Echocardiography was performed on day 6, before the commencement of treatment, and on day 27. In vivo hemodynamic measurements were made on day 28. Sham-operated rats served as controls.

RESULTS

The LV end-diastolic pressure and lung/body weight ratio were reduced, whereas the maximum rate of rise of LV pressure was increased towards sham levels in MI+RWJ compared with MI+V. Baseline echocardiographic studies demonstrated uniform LV remodeling and dysfunction in MI rats. Fractional shortening (FS) further deteriorated in MI+V, whereas FS was preserved in MI+RWJ. Progressive LV dilation and infarct expansion observed in MI+V were inhibited in MI+RWJ. MI+RWJ also demonstrated increased myocyte hypertrophy in the peri-infarct and non-infarct zones, and reduced myocardial collagen and alpha-smooth muscle actin (SMA) immunoreactivity compared with MI+V. The antifibrotic effects of RWJ in vivo may reflect direct effects on cardiac fibroblasts, because RWJ attenuated transforming growth factor beta-1-stimulated collagen synthesis and alpha-SMA expression in isolated cardiac fibroblasts. RWJ also protected cultured myocytes from hydrogen peroxide-induced apoptosis.

CONCLUSIONS

RWJ-67657 treatment post-MI had beneficial effects on LV remodeling and dysfunction, supporting a key role for p38 MAPK in pathologic cell signaling in these processes and its inhibition as a novel therapy.

Graves' disease (GD), an autoimmune process involving thyroid and orbital tissue, is associated with lymphocyte abnormalities including expansion of memory T cells. Insulin-like <em>growth</em> <em>factor</em> receptor-1 (IGF-1R)-bearing <em>fibroblasts</em> overpopulate connective tissues in GD. IGF-1R on <em>fibroblasts</em>, when ligated with IgGs from these patients, results in the expression of the T cell chemoattractants, IL-16 and RANTES. We now report that a disproportionately large fraction of peripheral blood T cells express IGF-1R (CD3+IGF-R+). CD3+IGF-1R+ T cells comprise 48 +/- 4% (mean +/- SE; n = 33) in patients with GD compared with 15 +/- 3% (n = <em>21</em>; p < 10(-8)) in controls. This increased population of IGF-1R+ T cells results, at least in part, from an expansion of CD45RO+ T cells expressing the receptor. In contrast, the fraction of CD45RA+IGF-1R+ T cells is similar in GD and controls. T cells harvested from affected orbital tissues in GD reflect similar differences in the proportion of IGF-1R+CD3+ and IGF-1R+CD4+CD3+ cells as those found in the peripheral circulation. GD-derived peripheral T cells express durable, constitutive IGF-1R expression in culture and receptor levels are further up-regulated following CD3 complex activation. IGF-1 enhanced GD-derived T cell incorporation of BrdU (p < 0.02) and inhibited Fas-mediated apoptosis (p < 0.02). These findings suggest a potential role for IGF-1R displayed by lymphocytes in supporting the expansion of memory T cells in GD.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> 19 (FGF19) and <em>21</em> (FGF<em>21</em>) have emerged as key regulators of energy metabolism. Several studies have been conducted to understand the mechanism of FGF19 and FGF<em>21</em> action, however, the data presented has often been inconsistent and at times contradictory. Here in a single study we compare the mechanisms mediating FGF19/FGF<em>21</em> actions, and how similarities/differences in actions at the cellular level between these two <em>factors</em> translate to common/divergent physiological outputs. Firstly, we show that in cell culture FGF19/FGF<em>21</em> are very similar, however, key differences are still observed differentiating the two. In vitro we found that both FGF's activate FGFRs in the context of βKlotho (KLB) expression. Furthermore, both <em>factors</em> alter ERK phosphorylation and glucose uptake with comparable potency. Combination treatment of cells with both <em>factors</em> did not have additive effects and treatment with a competitive inhibitor, the FGF<em>21</em> delta N17 mutant, also blocked FGF19's effects, suggestive of a shared receptor activation mechanism. The key differences between FGF<em>21</em>/FGF19 were noted at the receptor interaction level, specifically the unique ability of FGF19 to bind/signal directly via FGFR4. To determine if differential effects on energy homeostasis and hepatic mitogenicity exist we treated DIO and ob/ob mice with FGF19/FGF<em>21</em>. We find comparable efficacy of the two proteins to correct body weight and serum glucose in both DIO and ob/ob mice. Nevertheless, FGF<em>21</em> and FGF19 had distinctly different effects on proliferation in the liver. Interestingly, in vivo blockade of FGF<em>21</em> signaling in mice using ΔN17 caused profound changes in glycemia indicative of the critical role KLB and FGF<em>21</em> play in the regulation of glucose homeostasis. Overall, our data demonstrate that while subtle differences exist in vitro the metabolic effects in vivo of FGF19/FGF<em>21</em> are indistinguishable, supporting a shared mechanism of action for these two hormones in the regulation of energy balance.

The identification of leptin as a mediator of body weight regulation provided much initial excitement for the treatment of obesity. Unfortunately, leptin monotherapy is insufficient in reversing obesity in rodents or humans. Recent findings suggest that amylin is able to restore leptin sensitivity and when used in combination with leptin enhances body weight loss in obese rodents and humans. However, as the uniqueness of this combination therapy remains unclear, we assessed whether co-administration of leptin with other weight loss-inducing hormones equally restores leptin responsiveness in diet-induced obese (DIO) mice. Accordingly, we report here the design and characterization of a series of site-specifically enhanced leptin analogs of high potency and sustained action that, when administered in combination with exendin-4 or <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), restores leptin responsiveness in DIO mice after an initial body weight loss of 30%. Using either combination, body weight loss was enhanced compared with either exendin-4 or FGF<em>21</em> monotherapy, and leptin alone was sufficient to maintain the reduced body weight. In contrast, leptin monotherapy proved ineffective when identical weight loss was induced by caloric restriction alone over a comparable time. Accordingly, we find that a hypothalamic counter-regulatory response to weight loss, assessed using changes in hypothalamic agouti related peptide (AgRP) levels, is triggered by caloric restriction, but blunted by treatment with exendin-4. We conclude that leptin re-sensitization requires pharmacotherapy but does not appear to be restricted to a unique signaling pathway. Our findings provide preclinical evidence that high activity, long-acting leptin analogs are additively efficacious when used in combination with other weight-lowering agents.