CONCLUSIONS

We recently found that feeding healthy mice a diet with reduced levels of branched-chain amino acids (BCAAs), which are associated with insulin resistance in both humans and rodents, modestly improves glucose tolerance and slows fat mass gain. In the present study, we show that a reduced BCAA diet promotes rapid fat mass loss without calorie restriction in obese mice. Selective reduction of dietary BCAAs also restores glucose tolerance and insulin sensitivity to obese mice, even as they continue to consume a high-fat, high-sugar diet. A low BCAA diet transiently induces FGF<em>21</em> (<em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>) and increases energy expenditure. We suggest that dietary protein quality (i.e. the precise macronutrient composition of dietary protein) may impact the effectiveness of weight loss diets.

UNASSIGNED

Obesity and diabetes are increasing problems around the world, and although even moderate weight loss can improve metabolic health, reduced calorie diets are notoriously difficult to sustain. Branched-chain amino acids (BCAAs; leucine, isoleucine and valine) are elevated in the blood of obese, insulin-resistant humans and rodents. We recently demonstrated that specifically reducing dietary levels of BCAAs has beneficial effects on the metabolic health of young, growing mice, improving glucose tolerance and modestly slowing fat mass gain. In the present study, we examine the hypothesis that reducing dietary BCAAs will promote weight loss, reduce adiposity, and improve blood glucose control in diet-induced obese mice with pre-existing metabolic syndrome. We find that specifically reducing dietary BCAAs rapidly reverses diet-induced obesity and improves glucoregulatory control in diet-induced obese mice. Most dramatically, mice eating an otherwise unhealthy high-calorie, high-sugar Western diet with reduced levels of BCAAs lost weight and fat mass rapidly until regaining a normal weight. Importantly, this normalization of weight was mediated not by caloric restriction or increased activity, but by increased energy expenditure, and was accompanied by a transient induction of the energy balance regulating hormone FGF<em>21</em> (<em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>). Consumption of a Western diet reduced in BCAAs was also accompanied by a dramatic improvement in glucose tolerance and insulin resistance. Our results link dietary BCAAs with the regulation of metabolic health and energy balance in obese animals, and suggest that specifically reducing dietary BCAAs may represent a highly translatable option for the treatment of obesity and insulin resistance.

The role of ras proteins in signal transduction was assessed by studying inositol phospholipid metabolism and inositol phospholipid-mediated cellular responsiveness to agonists in cells transformed by ras and other oncogenes. Specific alterations were observed in the inositol phospholipid cycle of ras-transformed <em>fibroblasts</em>, but similar changes were also produced by spontaneous transformation or transformation mediated by either membrane-associated oncogenes, such as src, met, or trk, or cytoplasmic oncogenes, mos and raf; the nuclear oncogenes fos and myc did not produce these changes. The alterations included (i) stimulation of phospholipase A2 activity as indicated by elevated levels of glycerophosphoinositol and nonesterified arachidonic acid and (ii) specific uncoupling between surface receptor-mediated stimulation by platelet-derived <em>growth</em> <em>factor</em>, bombesin, or serum and activation of intracellular phospholipase C. These findings suggest the existence of common biochemical pathways for transformation by cytoplasmic and membrane-associated oncogenes and are not consistent with the hypothesis that <em>21</em>-kDa ras proteins (p<em>21</em>) are direct or distinct regulatory elements of phospholipase C or phospholipase A2 in inositol phospholipid signal transduction pathways.

BACKGROUND

Cardiovascular morbidity and mortality are massively increased in patients with chronic kidney disease (CKD). Sevelamer hydrochloride has been shown to attenuate cardiovascular calcifications in CKD and end-stage renal disease (ESRD) patients. We assessed how sevelamer hydrochloride influences the evolution of serum fetuin-A and other serological factors predicting cardiovascular outcome and survival in haemodialysis patients.

METHODS

Fifty-seven prevalent haemodialysis patients were included in a three-phase prospective interventional trial (A-B-A design; 8 weeks per phase). Sevelamer was only administered in the middle phase of the study. Within the other two phases,>>or=90% of the patients received calcium acetate for phosphate binding. Detailed time courses of serum biochemistries were analysed in order to obtain detailed insight into the influence of sevelamer upon CKD-mineral and bone disorder (MBD) parameters as well as serum fetuin-A, fibroblast growth factor 23 (FGF23) and uraemic toxin levels [uric acid, indoxyl sulphate, hippuric acid, indole acetic acid, p-cresol and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF)].

RESULTS

Forty-one patients finished the three prospective study phases (intention-to-treat analysis). After treatment with sevelamer, serum fetuin-A significantly increased (+21%), showing a delayed increase outlasting the third (non-sevelamer) study period. Total and low-density lipoprotein (LDL) cholesterol levels, as well as serum calcium, decreased significantly. The opposite occurred with albumin, C-reactive protein and intact parathyroid hormone (iPTH). FGF23, uric acid, indoxyl sulphate, hippuric acid, indole acetic acid, CMPF and serum phosphate did not change significantly during sevelamer treatment. In contrast, in parallel to sevelamer treatment, there was a significant rise in serum P-cresol.

CONCLUSIONS

In haemodialysis patients, treatment with sevelamer over 8 weeks was associated with a delayed yet long-lasting increase in serum fetuin-A levels. Increasing the serum level of fetuin-A, a negative acute-phase protein and systemic calcification inhibitor, might be one of the potential anti-calcification mechanisms of sevelamer. Since we failed to detect a decrease in systemic inflammation and uraemic toxins, the exact mechanisms by which sevelamer treatment affects serum fetuin-A remain to be determined.

Despite the emerging importance of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) as a metabolic hormone regulating energy balance, its direct effects on renal function remain unexplored. FGF<em>21</em> was injected ip daily for 12 weeks into db/db mice. Compared with control vehicle injection, FGF<em>21</em> treatment significantly improved lipid profiles and insulin resistance and resulted in significantly higher serum adiponectin levels. In contrast, serum insulin and 8-isoprostane levels were significantly decreased. Interestingly, FGF<em>21</em> and its receptor components in the kidneys were found to be significantly up-regulated in db/db mice, which suggests an FGF<em>21</em>-resistant state. FGF<em>21</em> treatment significantly down-regulated FGF<em>21</em> receptor components and activated ERK phosphorylation. FGF<em>21</em> administration also markedly decreased urinary albumin excretion and mesangial expansion and suppressed profibrotic molecule synthesis. Furthermore, FGF<em>21</em> improved renal lipid metabolism and oxidative stress injury. In cultured renal cells, FGF<em>21</em> was mainly expressed in mesangial cells, and knockdown of FGF<em>21</em> expression by stealth small interfering RNA further aggravated high-glucose-induced profibrotic cytokine synthesis in mesangial cells. Our results suggest that FGF<em>21</em> improves insulin resistance and protects against renal injury through both improvement of systemic metabolic alterations and antifibrotic effects in type 2 diabetic nephropathy. Targeting FGF<em>21</em> could therefore provide a potential candidate approach for a therapeutic strategy in type 2 diabetic nephropathy.

OBJECTIVE

The purpose of this study was to evaluate the clinical activity and toxicity of recombinant human Interleukin (IL)-12 in patients with relapsed and refractory non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD).

METHODS

Forty-two previously treated patients (32 patients with NHL and 10 patients with HD) were enrolled on the study. Patients were treated with either intravenous (n = 11) or subcutaneous (n = 31) administration of IL-12. The patients had received a median of three prior treatment regimens, and 16 patients had undergone prior autologous stem cell transplantation.

RESULTS

All patients were assessable for toxicity, and 39 of 42 (93%) patients were assessable for response. Six of 29 (<em>21</em>%) patients with NHL had a partial or complete response, whereas none of the 10 patients with HD responded. Furthermore, 15 patients had stable disease that lasted for up to 54 months. Progression-free survival in patients with indolent NHL, aggressive NHL, and HD was 6, 2, and 2.5 months, respectively. Treatment was well tolerated, and the most common toxicity was flu-like symptoms. Reversible grade 3 hepatic toxicity was observed in three patients requiring dose reduction. IL-12 therapy increased the median number of peripheral blood CD8 T lymphocytes from 423/microl to 576/microl (P = 0.0019). Furthermore, IL-12 therapy decreased serum vascular endothelial <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> concentrations in 37% of the patients.

CONCLUSIONS

The ability of recombinant human IL-12 therapy to increase the number of circulating CD8+ cells and induce clinical remissions in patients with relapsed NHL warrants further investigation of the drug.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-19 and FGF-<em>21</em> are novel metabolic regulators that improve insulin resistance and obesity in rodents. The aim of the study was to assess the effects of laparoscopic sleeve gastrectomy (LSG) on serum concentrations of FGF-19 and FGF-<em>21</em> along with circulating bile acids and other relevant hormonal and biochemical parameters.

METHODS

Seventeen females with obesity undergoing LSG and 15 lean healthy females were included into the study. Anthropometric and biochemical parameters, serum concentrations of FGF-19 and -<em>21</em>, insulin, adiponectin, leptin, C-reactive protein, resistin, amylin (total), ghrelin (active), glucagon-like peptide 1 (GLP-1, active), glucose-dependent insulinotropic peptide (GIP, total), peptide YY (PYY, total), pancreatic polypeptide (PP), and bile acids, and mRNA expression of selected adipokines and inflammatory markers in bioptic samples of subcutaneous fat were assessed at baseline and 6, 12, and 24 months after LSG.

RESULTS

LSG markedly decreased body weight, BMI, waist circumference, and insulin levels and improved systemic inflammation and lipid levels. FGF-19 concentrations increased and FGF-<em>21</em> concentrations decreased after LSG along with increased adiponectin and decreased leptin, amylin, and ghrelin levels. GLP-1, GIP, PP, and circulating bile acids were not affected by LSG. PYY decreased significantly 24 months after surgery only. mRNA expression analysis in subcutaneous fat showed markedly reduced proinflammatory state.

CONCLUSIONS

Our results indicate that increased FGF-19 and decreased ghrelin concentrations could have partially contributed to the improvement of systemic inflammation and some metabolic parameters after LSG, while changes of FGF-<em>21</em> are rather secondary because of weight loss.

BACKGROUND

Thalidomide is a putative antiangiogenesis agent with activity in several hematologic malignancies.

METHODS

Forty-four patients who had myelofibrosis with myeloid metaplasia received treatment with thalidomide in a Phase II clinical trial at a dose of 200 mg daily with escalation by 200 mg weekly until the best tolerated dose (maximum, 800 mg) was reached.

RESULTS

Seventeen of 41 evaluable patients (41%) who received treatment for at least 15 days had a response. A complete response (without reversal of bone marrow fibrosis) was achieved in 4 patients (10%), a partial response was achieved in 4 patients (10%), and hematologic improvements in anemia, thrombopenia, and/or splenomegaly were observed in 9 patients (<em>21</em>%). Improvements in anemia occurred in 7 of 35 patients (20%) with hemoglobin levels <10.0 g/dL, and improvements in thrombopenia occurred in 5 of 24 patients (<em>21</em>%) with platelet counts <100 x 10(9)/L. Five of 24 patients (<em>21</em>%) became transfusion-independent. Major or minor regression of splenomegaly was noted in 9 of 29 evaluable patients (31%), and complete regression was noted in 5 patients. Responders had a lower baseline median vascular endothelial <em>growth</em> <em>factor</em> levels (77.9 pg/mL vs. 97.7 pg/mL; P <.01) and higher median basis <em>fibroblast</em> <em>growth</em> <em>factor</em> levels (60.8 pg/mL vs. 37.4 pg/mL; P <.01) compared with nonresponders. Nine patients (22%) had deterioration that was attributed to thalidomide (resolved after withdrawal) with either progressive cytopenias or excessive proliferation. Two patients developed Grade 3 neutropenia with recovery and resumed therapy with dose reductions, and both later achieved a complete response. Dose-related toxicities included fatigue (50%), constipation (48%), rash or pruritus (37%), sedation (35%), peripheral edema (29%), tremors (23%), peripheral neuropathy (22%), and orthostasis (16%).

CONCLUSIONS

Thalidomide warrants further evaluation in patients with MMM, particularly in combination regimens, along with the investigation of newer analogs.

Acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) belong to a family of mitogenic polypeptides that are involved in cellular proliferation and differentiation. In this study we investigated the potential role of aFGF and bFGF in chronic pancreatitis (CP), a fibrotic condition associated with acinar cell dedifferentiation and atrophy, and <em>fibroblast</em>ic proliferation. By immunohistochemistry, aFGF and bFGF were abundant in pancreatic ductal and acinar cells in pancreatic tissues from CP patients. Immunoblotting with the same highly specific monoclonal antibodies demonstrated a marked increase in aFGF and bFGF in pancreatic homogenates from CP patients by comparison with the normal pancreas. Northern blot analysis indicated that, by comparison with normal controls, 16 of <em>21</em> CP tissues exhibited a 14-fold increase in aFGF mRNA levels, and 19 of <em>21</em> CP tissues exhibited a 15-fold increase in bFGF mRNA levels. In situ hybridization confirmed that this overexpression occurred in ductal and acinar cells, and indicated that both mRNA moieties colocalized with their respective proteins. These findings suggest that aFGF and bFGF may either be involved in the pathobiological mechanisms that occur in CP, or that their overexpression may be the consequence of other perturbations that occur in this disorder.

OBJECTIVE

To simultaneously identify several genes whose expression is altered in dermal fibroblasts from patients with systemic sclerosis (SSc).

METHODS

Total RNA was prepared from fibroblasts derived from clinically affected and unaffected skin of patients with SSc. The RNA samples were analyzed using differential-display reverse transcription-polymerase chain reaction (DDRT-PCR). Complementary DNA (cDNA) fragments corresponding to differentially expressed messenger RNAs (mRNAs) were eluted, cloned, and sequenced. The differential expression of the corresponding mRNAs was confirmed by ribonuclease protection assay.

RESULTS

We identified 21 differentially expressed mRNAs. Their corresponding cDNAs were sequenced and the sequences obtained were compared with those of known genes entered into the EMBL/GenBank database. Three of the sequences corresponded to transcripts of yet-unidentified genes. Some of the mRNAs shared partial homology with extracellular matrix components, cellular receptors, enzymes, and nuclear factors. Others corresponded to known mRNAs such as those of fibronectin, fibronectin receptor, laminin receptor homolog, beta-tubulin, insulin-like growth factor binding protein 5, KIAA0179 protein, and protease nexin 1.

CONCLUSIONS

The application of DDRT-PCR to scleroderma research has identified many mRNAs whose altered expression in scleroderma has not yet been described, thus providing new information for further investigation and potential targets for the development of novel therapies.

Methionine- and choline-deficient diet (MCD) is a model for nonalcoholic steatohepatitis (NASH) in rodents. However, the mechanism of NASH development by dietary methionine/choline deficiency remains undetermined. To elucidate the early metabolic changes associated with MCD-NASH, serum metabolomic analysis was performed using mice treated with MCD and control diet for 3 days and 1 week, revealing significant increases in oleic and linoleic acids after MCD treatment. These increases were correlated with reduced body weight and white adipose tissue (WAT) mass, increased phosphorylation of hormone-sensitive lipase, and up-regulation of genes encoding carboxylesterase 3 and β2-adrenergic receptor in WAT, indicating accelerated lipolysis in adipocytes. The changes in serum fatty acids and WAT by MCD treatment were reversed by methionine supplementation, and similar alterations were detected in mice fed a methionine-deficient diet (MD), thus demonstrating that dietary methionine deficiency enhances lipolysis in WAT. MD treatment decreased glucose and increased <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> in serum, thus exhibiting a similar metabolic phenotype as the fasting response. Comparison between MCD and choline-deficient diet (CD) treatments suggested that the addition of MD-induced metabolic alterations, such as WAT lipolysis, to CD-induced hepatic steatosis promotes liver injury. Collectively, these results demonstrate an important role for dietary methionine deficiency and WAT lipolysis in the development of MCD-NASH.

1. Airway smooth muscle proliferation is a significant component of the airway wall remodelling that occurs in asthma. In this study, the effects of glucocorticoids on mitogenic responses of human airway smooth muscle have been examined. 2. Pretreatment of smooth muscle cells with dexamethasone (100 nM, 60 min) inhibited thrombin-induced increases in [3H]-thymidine incorporation (DNA synthesis) and cell number. 3. Inhibition of thrombin-induced [3H]-thymidine incorporation was also observed with hydrocortisone (0.01-1 microM) and methylprednisolone (0.001-0.1 microM) pretreatment. In contrast, pretreatment with either testosterone (0.001-1 microM) progesterone (0.001-1 microM), 17 beta-oestradiol (0.001-1 microM), or aldosterone (0.001-1 microM) had no effect on the response to thrombin. 4. Responses to a range of mitogens including thrombin (0.01-. 10 u ml-1), epidermal <em>growth</em> <em>factor</em> (EGF, 3-3000 pM), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF, 0.3-300 pM) and foetal calf serum (FCS, 0.1-10% v/v) were inhibited by dexamethasone (100 nM) pretreatment. However, the magnitude of the inhibitory effect was dependent on the mitogen, with EGF being the least, and thrombin being the most sensitive to the inhibitory effect. 5. The potency of hydrocortisone as an inhibitor of [3H]-thymidine incorporation was reduced when FCS (10% v/v, which caused a 40 fold increase in [3H]-thymidine incorporation) was used as the mitogen in place of thrombin (0.3 u ml-1, which caused a 10 fold increase in [3H]-thymidine incorporation). 6. The effect of post-treatment with dexamethasone (100 nM) indicated that addition of the glucocorticoid up to 17-19 h after thrombin (0.3 u ml-1) produced similar degrees of inhibition to those obtained when it was added as a pretreatment. Dexamethasone no longer produced an inhibitory effect if added <em>21</em> h or more after the addition of thrombin. 7. These results suggest that glucocorticoids regulate airway smooth muscle proliferation initiated by a range of stimuli. This effect may be of importance in the therapeutic actions of these compounds in asthma, particularly when they are used for prolonged periods of time.

BACKGROUND

Disordered extracellular matrix production is a feature of bronchopulmonary dysplasia (BPD). The basis of this phenomenon is not understood.

OBJECTIVE

To assess lysyl oxidase expression and activity in the injured developing lungs of newborn mice and of prematurely born infants with BPD or at risk for BPD.

METHODS

Pulmonary lysyl oxidase and elastin gene and protein expression were assessed in newborn mice breathing <em>21</em> or 85% oxygen, in patients who died with BPD or were at risk for BPD, and in control patients. Signaling by transforming <em>growth</em> <em>factor</em> (TGF-beta) was preemptively blocked in mice exposed to hyperoxia using TGF-beta-neutralizing antibodies. Lysyl oxidase promoter activity was assessed using plasmids containing the lox or loxl1 promoters fused upstream of the firefly luciferase gene.

RESULTS

mRNA and protein levels and activity of lysyl oxidases (Lox, LoxL1, LoxL2) were elevated in the oxygen-injured lungs of newborn mice and infants with BPD or at risk for BPD. In oxygen-injured mouse lungs, increased TGF-beta signaling drove aberrant lox, but not loxl1 or loxl2, expression. Lox expression was also increased in oxygen-injured fibroblasts and pulmonary artery smooth muscle cells.

CONCLUSIONS

Lysyl oxidase expression and activity are dysregulated in BPD in injured developing mouse lungs and in prematurely born infants. In developing mouse lungs, aberrant TGF-beta signaling dysregulated lysyl oxidase expression. These data support the postulate that excessive stabilization of the extracellular matrix by excessive lysyl oxidase activity might impede the normal matrix remodeling that is required for pulmonary alveolarization and thereby contribute to the pathological pulmonary features of BPD.

Low-carbohydrate/high-fat diets (LC-HFDs) in rodent models have been implicated with both weight loss and as a therapeutic approach to treat neurological diseases. LC-HFDs are known to induce ketosis; however, systematic studies analyzing the impact of the macronutrient composition on ketosis induction and weight loss success are lacking. Male Wistar rats were pair-fed for 4 wk either a standard chow diet or one of three different LC-HFDs, which only differed in the relative abundance of fat and protein (percentages of fat/protein in dry matter: LC-75/10; LC-65/20; LC-55/30). We subsequently measured body composition by nuclear magnetic resonance (NMR), analyzed blood chemistry and urine acetone content, evaluated gene expression changes of key ketogenic and gluconeogenic genes, and measured energy expenditure (EE) and locomotor activity (LA) during the first 4 days and after 3 wk on the respective diets. Compared with chow, rats fed with LC-75/10, LC-65/20, and LC-55/30 gained significantly less body weight. Reductions in body weight were mainly due to lower lean body mass and paralleled by significantly increased fat mass. Levels of β-hydroxybutyate were significantly elevated feeding LC-75/10 and LC-65/20 but decreased in parallel to reductions in dietary fat. Acetone was about 16-fold higher with LC-75/10 only (P < 0.001). In contrast, rats fed with LC-55/30 were not ketotic. Serum <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em>, hepatic mRNA expression of hydroxymethylglutaryl-CoA-lyase, peroxisome proliferator-activated receptor-γ coactivator-1α, and peroxisome proliferator-activated receptor-γ coactivator-1β were increased with LC-75/10 only. Expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase was downregulated by 50-70% in LC-HF groups. Furthermore, EE and LA were significantly decreased in all groups fed with LC-HFDs after 3 wk on the diets. In rats, the absence of dietary carbohydrates per se does not induce ketosis. LC-HFDs must be high in fat, but also low in protein contents to be clearly ketogenic. Independent of the macronutrient composition, LC-HFD-induced weight loss is not due to increased EE and LA.

Four members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) family of tyrosine kinases transduce signals of a diverse group of more than 23 <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) ligands. Each prototypic receptor is composed of three immunoglobulin-like extracellular domains, two of which are involved in ligand binding. Alternative RNA splicing of one of two exons results in two different forms of the second half of the third immunoglobulin-like domain, the IIIb or IIIc isoforms. The contribution of each receptor and their isoforms in tumorigenesis remains unknown. In the pituitary, FGFR2 is expressed primarily as the IIIb isoform in normal adenohypophysial cells. In contrast, FGFR2 is significantly down-regulated in mouse corticotroph AtT20 tumor cells where the 5' promoter is methylated. Treatment of AtT20 cells with 5'-azacytidine resulted in FGFR2 re-expression, mainly as the FGFR2-IIIb isoform. Chromatin immunoprecipitation revealed evidence of histone methylation, but not of deacetylation, in the silencing of FGFR2 in AtT20 cells. Exposure of these cells to the cognate FGFR2-IIIb ligand FGF-7 resulted in diminished Rb phosphorylation and accumulation of p<em>21</em> and p27, indicating diminished cell cycle progression. Examination of primary human pituitary adenomas revealed FGFR2 down-regulation in 52% (11 of <em>21</em>) of samples and FGFR2 promoter DNA methylation in 45% (10 of 22) of samples. These data highlight the contribution from DNA and histone methylation as epigenetic mechanisms responsible for FGFR2 silencing in pituitary neoplasia.

Adipose triglyceride lipase (ATGL) catalyzes the first step of lipolysis of cytoplasmic triacylglycerols in white adipose tissue (WAT) and several other organs. We created adipose-specific ATGL-deficient (ATGLAKO) mice. In these mice, in vivo lipolysis, measured as the increase of plasma nonesterified fatty acid and glycerol levels after injection of a β3-adrenergic agonist, was undetectable. In isolated ATGLAKO adipocytes, β3-adrenergic-stimulated glycerol release was 10-fold less than in controls. Under fed conditions, ATGLAKO mice had normal viability, mild obesity, low plasma nonesterified fatty acid levels, increased insulin sensitivity, and increased daytime food intake. After 5 h of fasting, ATGLAKO WAT showed phosphorylation of the major protein kinase A-mediated targets hormone-sensitive lipase and perilipin A and ATGLAKO liver showed low glycogen and triacylglycerol contents. During a 48-h fast, ATGLAKO mice developed striking and complex differences from controls: progressive reduction of oxygen consumption, high respiratory exchange ratio, consistent with reduced fatty acid availability for energy production, lethargy, hypothermia, and undiminished fat mass, but greater loss of lean mass than controls. Plasma of 48 h-fasted ATGLAKO mice had a unique pattern: low 3-hydroxybutyrate, insulin, adiponectin, and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> with elevated leptin and corticosterone. ATGLAKO WAT, liver, skeletal muscle, and heart showed increased levels of mRNA related to autophagy and proteolysis. In murine ATGL deficiency, adipose lipolysis is critical for fasting energy homeostasis, and fasting imposes proteolytic stress on many organs, including heart and skeletal muscle.

BACKGROUND

Myocardial fibrosis is a hallmark of inflammation-triggered end-stage heart disease, a common cause of heart failure in young patients.

OBJECTIVE

We used CD4(+) T-cell-mediated experimental autoimmune myocarditis model to determine the parameters regulating cardiac fibrosis in inflammatory heart disease.

RESULTS

alpha-Myosin heavy chain peptide/complete Freund's adjuvant immunization was used to induce experimental autoimmune myocarditis in BALB/c mice. Chimeric mice, reconstituted with enhanced green fluorescence protein (EGFP)(+) bone marrow, were used to track the fate of inflammatory cells. Prominin-1(+) cells were isolated from the inflamed hearts, cultured in vitro and injected intracardially at different stages of experimental autoimmune myocarditis. Transforming <em>growth</em> <em>factor</em> (TGF)-beta-mediated fibrosis was addressed using anti-TGF-beta antibody treatment. Myocarditis peaked <em>21</em> days after immunization and numbers of cardiac <em>fibroblasts</em> progressively increased on follow-up. In chimeric mice, >60% of cardiac <em>fibroblasts</em> were EGFP(+) 46 days after immunization. At day <em>21</em>, cardiac infiltrates contained approximately 30% of prominin-1(+) progenitors. In vitro and in vivo experiments confirmed that prominin-1(+) but not prominin-1(-) cells isolated from acutely inflamed hearts represented the cellular source of cardiac <em>fibroblasts</em> at late stages of disease, characterized by increased TGF-beta levels within the myocardium. Mechanistically, the in vitro differentiation of heart-infiltrating prominin-1(+) cells into <em>fibroblasts</em> depended on TGF-beta-mediated phosphorylation of Smad proteins. Accordingly, anti-TGF-beta antibody treatment prevented myocardial fibrosis in immunized mice.

CONCLUSIONS

Taken together, heart-infiltrating prominin-1(+) progenitors are the major source of subsequent TGF-beta-triggered cardiac fibrosis in experimental autoimmune myocarditis. Recognizing the critical, cytokine-dependent role of bone marrow-derived progenitors in cardiac remodeling might result in novel treatment concepts against inflammatory heart failure.

This study aimed to assess the effects of carbohydrate (CHO) and fat intake on the expression of key genes related with nutrient partitioning and metabolism in main tissues involved in energy metabolism (white adipose tissue, liver, and skeletal muscle). Rats were studied under different conditions: feeding state, 24 h fasting, and 12 h refeeding after 24 h fasting with isocaloric amounts of CHO or fat. Fat, but not CHO, refeeding was associated with an increase in serum and liver triglyceride content. Main changes in gene expression elicited by CHO compared with fat refeeding were: 1) higher expression levels of genes related with lipogenesis (PPARgamma2, ChREBP, FAS), glucose uptake and metabolism (GLUT4, HKII), fatty acid uptake (LPL, CD36), and lipolysis (ATGL, HSL) in white adipose tissue; 2) higher expression levels of genes related with lipogenesis (FAS, SCD1) but lower ones related with fatty acid uptake (CD36) and oxidation (PPARalpha, CPT1, PDK4) in liver; and 3) higher expression levels of GLUT4 but lower ones related with fatty acid oxidation (PDK4 and UCP3) in muscle. It is worth mentioning that both CHO and fat refeeding resulted in a robust increase in both hepatic mRNA and circulating levels of <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em>, compared with fasted levels. In summary, these results, showing marked differences in gene expression after CHO and fat refeeding, can explain diet-associated differences in fuel handling and partitioning between tissues; in addition, a role of <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> in metabolic adaptations, not only in the ketotic state but also to face an unbalanced nutritional situation, is suggested.

Accumulation of DNA is essential for muscle <em>growth</em>, yet mechanisms of androgen-induced DNA accretion in skeletal muscle are unclear. The purpose of this study was to determine whether androgen receptors (AR) are present in cultured skeletal muscle satellite cells and myotubes and examine the effects of testosterone on satellite cell proliferation and differentiation. Immunoblot analysis using polyclonal AR antibodies (PG-<em>21</em>) revealed an immunoreactive AR protein of approximately 107 kDa in porcine satellite cells and myotubes. Immunocytochemical AR staining was confined to the nuclei of satellite cells, myotubes, and muscle-derived <em>fibroblasts</em>. Administration of 10(-7) M testosterone to satellite cells, myotubes, and muscle-derived <em>fibroblasts</em> increased immunoreactive AR. In satellite cells and myotubes, AR increased incrementally after 6, 12, and 24 h of exposure to testosterone. Testosterone (10(-10) - 10(-6) M), alone or in combination with insulin-like <em>growth</em> <em>factor</em> I, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, or platelet-derived <em>growth</em> <em>factor</em>-BB, had no effect (P>> 0.01) on porcine satellite cell proliferation, and testosterone pretreatment for 24 h did not alter the subsequent responsiveness of cells to these <em>growth</em> <em>factors</em>. Satellite cell differentiation was depressed (20-30%) on days 2-4 of treatment with 10(-7) M testosterone. This effect was not reversible within 48 h after treatment withdrawal and replacement with control medium. These data indicate that satellite cells are direct targets for androgen action, and testosterone administration increases immunoreactive AR protein and reduces differentiation of porcine satellite cells in vitro.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> is an emerging metabolic regulator that was recently proposed to be a fed-state inducible <em>factor</em> in adipose tissue. As mice lacking FGF<em>21</em> were refractory to treatment with rosiglitazone, FGF<em>21</em> was suggested to underlie PPARγ-driven pharmacology and side effect profile (Dutchak et al., 2012 [12]). To evaluate FGF<em>21</em>/PPARγ cross-talk we conducted experiments in control and FGF<em>21</em> null animals and found that rosiglitazone was equally efficacious in both strains. Specifically, diverse endpoints ranging from enhanced glycemic control, improved lipid homeostasis and side effects such as adipose accumulation were evident in both genotypes. Furthermore, the transcriptional signature and cytokine secretion profile of rosiglitazone action were maintained in our FGF<em>21</em>KO animals. Finally, we found that FGF<em>21</em> in adipose was expressed at comparable levels in fasted and fed states. Thus, our data present a new viewpoint on the FGF<em>21</em>/PPARγ interplay whereby FGF<em>21</em> is not necessary for the metabolic events downstream of PPARγ.

A number of clinical studies have demonstrated the prognostic significance of angiogenesis and angiogenic <em>growth</em> <em>factors</em> in solid tumours; however, very little is known about the relevance of these parameters in haematological malignancies. We evaluated circulating levels of angiogenic <em>growth</em> <em>factors</em> and endostatin in 36 non-Hodgkin's lymphoma (NHL) patients. Baseline vascular endothelial <em>growth</em> <em>factor</em> (VEGF) levels of patients in complete remission (CR) after a median follow-up of <em>21</em> months were significantly lower than those of patients with progressive disease (P = 0.016). Event-free survival (EFS) rate was significantly higher in patients who had baseline VEGF and basic-<em>fibroblast</em> <em>growth</em> <em>factor</em> (b.FGF) levels below the median values of 147 and 19.5 pg/ml (P = 0.018 and 0.039 by log-rank test, respectively). Conversely, the levels of endostatin, angiogenin and leptin were not different in CR patients compared to relapsed patients and did not correlate with EFS. Our data suggest that b-FGF and, particularly, VEGF might be considered prognostic <em>factors</em> in NHL staging and management.

BACKGROUND

Paediatric reference values for novel markers of phosphate homeostasis, bone formation and resorption and their putative relationship to growth are lacking.

METHODS

A total of 424 healthy children, adolescents and young adults (221 males) aged 0.1-21 y, were enrolled in this cross-sectional study. Height, weight and height velocity were assessed. Plasma/serum samples for determination of C-terminal fragment of fibroblast growth factor-23 (cFGF-23), sclerostin, bone alkaline phosphatase (BAP) and tartrate-resistant acid phosphatase 5b (TRAP5b) were available from 222, 264, 352 and 338 individuals, respectively. Calculation of cross-sectional centiles and z-scores was based on median (M), standard coefficient of variation (S) and the Box-Cox power (L) of transformation (LMS method) per age cohort. Correlations between variables as well as with growth were assessed.

RESULTS

cFGF-23, BAP and TRAP5b were significantly correlated with age (each P < 0.01), with highest values during infancy and adolescence. Serum levels of BAP and TRAP5b were significantly higher in adolescent boys compared with girls (each P < 0.01). In contrast, sclerostin levels were independent of age and gender. BAP and TRAP5b were strongly correlated and both were significantly associated with cFGF-23 and sclerostin as well (each P < 0.01). cFGF-23 was positively correlated with serum phosphate and renal phosphate threshold concentration (each P < 0.01). Height, weight, body mass index and height velocity were weakly correlated with BAP and TRAP5b (each P < 0.05).

CONCLUSIONS

This study provides age- and gender-related centile charts and z-scores for cFGF-23, BAP, TRAP5b and sclerostin and highlights the link between phosphate homeostasis and markers of bone metabolism during growth.

Successful hematopoietic stem cell (HSC) transplantation is often limited by the numbers of HSCs, and robust methods to expand HSCs ex vivo are needed. We previously showed that angiopoietin-like proteins (Angptls), a group of <em>growth</em> <em>factors</em> isolated from a fetal liver HSC-supportive cell population, improved ex vivo expansion of HSCs. Here, we demonstrate that insulin-like <em>growth</em> <em>factor</em>-binding protein 2 (IGFBP2), secreted by a tumorigenic cell line, also enhanced ex vivo expansion of mouse HSCs. On the basis of these findings, we established a completely defined, serum-free culture system for mouse HSCs, containing SCF, thrombopoietin, <em>fibroblast</em> <em>growth</em> <em>factor</em> 1, Angptl3, and IGFBP2. As measured by competitive repopulation analyses, there was a 48-fold increase in numbers of long-term repopulating mouse HSCs after <em>21</em> days of culture. This is the first demonstration that IGFBP2 stimulates expansion or proliferation of murine stem cells. Our finding also suggests that certain cancer cells synthesize proteins that can stimulate HSC expansion. Disclosure of potential conflicts of interest is found at the end of this article.

BACKGROUND

Fibroblast growth factor-23 (FGF-23) is a phosphorus-regulating substance. Circulating FGF-23 levels increase markedly in dialysis patients and are independently associated with increased risk of mortality. Given the fact that cardiovascular disease is the leading cause of death in dialysis patients, the aim of this study was to test if elevated FGF-23 levels might be associated with left ventricular mass index (LVMI) and left ventricular index of myocardial performance (MPI) in maintenance haemodialysis patients.

METHODS

In this cross-sectional study, plasma FGF-23 concentrations were measured using a C-terminal human enzyme-linked immunosorbent assay kit, and echocardiography was performed in 128 maintenance haemodialysis patients (65 women and 63 men, mean age: 55.5 ± 13 years, mean haemodialysis vintage: 52 ± 10 months, all patients are on haemodialysis thrice a week) and 40 control subjects (21 women and 19 men; mean age: 54 ± 11 years) with normal kidney function (eGFR>> 90 mL/min/1.73 m(2)).

RESULTS

Serum FGF-23 levels were elevated when compared with age- and gender-matched controls with preserved kidney function [(median 958 RU/mL; interquartile range 106-1894 RU/mL) vs (median 27 RU/mL; interquartile range 11-35), P < 0.0001]. Patients with a history of coronary artery disease and aortic valve calcifications had higher levels of log FGF-23 than those without (3.00 ± 0.22 vs 2.82 ± 0.26, P = 0.002; and 3.06 ± 0.19 vs 2.83 ± 0.26, P = 0.0001, respectively). Patients with MPI>> 0.47 had higher serum FGF-23 levels than those with MPI < 0.47 [(median 1156 RU/mL; interquartile range 396-1894 RU/mL) vs (median 657 RU/mL; interquartile range 106-1102 RU/mL), P = 0.0001]. Significant correlations were recorded between log FGF-23 levels and LVMI (r = 0.281, P = 0,007) and MPI (r = 0.555, P = 0.0001). Multivariable-adjusted regression analyses revealed that increased log FGF-23 concentrations were independently associated with increased left ventricular mass index (30% increase per 1-SD increase in log FGF-23 concentration, P = 0.002) and increased MPI (28.5% increase per 1-SD increase in log FGF-23 concentration, P = 0.001).

CONCLUSIONS

Plasma FGF-23 concentration is independently associated with LVMI and MPI in maintenance haemodialysis patients. Further prospective studies are needed to clarify whether increased serum FGF-23 level is a marker or a potential mechanism for left ventricular involvement in patients with end-stage renal disease.

OBJECTIVE

This phase I study was designed to evaluate the safety, tolerability and pharmacokinetics of intra-arterial basic fibroblast growth factor (bFGF) in patients with atherosclerotic peripheral arterial disease (PVD) and intermittent claudication. We also assessed the effects of basic fibroblast growth factor (bFGF) on calf blood flow as a measure of biologic activity.

BACKGROUND

Preclinical studies have shown that bFGF, an angiogenic peptide, promotes collateral development in animal models of myocardial and hind limb ischemia. The safety and efficacy of bFGF in patients is unknown, and early clinical trials are underway in coronary and peripheral arterial disease.

METHODS

A double-blind, placebo-controlled, dose-escalation trial was conducted in patients with claudication demonstrating ankle/brachial index <0.8. Patients were randomly assigned to placebo (n = 6), 10 microg/kg of bFGF (n = 4), 30 microg/kg of bFGF once (n = 5) and 30 microg/kg of bFGF on two consecutive days (n = 4). Study drug was infused into the femoral artery of the ischemic leg. Detailed safety information including retinal photography for neovascularization were obtained through one year. Calf blood flow was measured with strain gauge plethysmography in the two higher dose treatment groups and in four placebo patients at baseline, one month and three to seven months after treatment.

RESULTS

Intra-arterial bFGF was safe and well-tolerated. The half-life was 46 +/- 21 min. Calf blood flow increased at one month by 66 +/- 26% (mean +/- SEM) and at six months by 153 +/- 51% in bFGF-treated patients (n = 9, p = 0.002). Flow did not change significantly in the placebo group.

CONCLUSIONS

In this initial randomized, double-blind, placebo-controlled trial in patients with atherosclerotic PVD and claudication, bFGF was well-tolerated. The data suggest a salutary biologic effect, and initiation of phase 2 trials is warranted.