Randomized, double-blind trial of F14512, a polyamine-vectorized anticancer drug, compared with etoposide phosphate, in dogs with naturally occurring lymphoma.

Purpose: F14512 is an epipodophyllotoxin derivative from etoposide, combined with a spermine moiety introduced as a cell delivery vector. The objective of this study was to compare the safety and antitumor activity of F14512 and etoposide phosphate in dogs with spontaneous non-Hodgkin lymphoma (NHL) and to investigate the potential benefit of F14512 in P-glycoprotein (Pgp) overexpressing lymphomas. Experimental Design: Forty-eight client-owned dogs with intermediate to high-grade NHL were enrolled into a randomized, double-blind trial of F14512 versus etoposide phosphate. Endpoints included safety and therapeutic efficacy. Results: Twenty-five dogs were randomized to receive F14512 and 23 dogs to receive etoposide phosphate. All adverse events (AEs) were reversible, and no treatment-related death was reported. Hematologic AEs were more severe with F14512 and gastrointestinal AEs were more frequent with etoposide phosphate. F14512 exhibited similar response rate and progression-free survival (PFS) as etoposide phosphate in the global treated population. Subgroup analysis of dogs with Pgp-overexpressing NHL showed a significant improvement in PFS in dogs treated with F14512 compared with etoposide phosphate. Conclusion: F14512 showed strong therapeutic efficacy against spontaneous NHL and exhibited a clinical benefice in Pgp-overexpressing lymphoma superior to etoposide phosphate. The results clearly justify the evaluation of F14512 in human clinical trials.

The nuclear bile acid receptor FXR is a PKA- and FOXA2-sensitive activator of fasting hepatic gluconeogenesis.

BACKGROUND & AIMS: Embedded into a complex signaling network that coordinates glucose uptake, usage and production, the nuclear bile acid receptor FXR is expressed in several glucose-processing organs including the liver. Hepatic gluconeogenesis is controlled through allosteric regulation of gluconeogenic enzymes and by glucagon/cAMP-dependent transcriptional regulatory pathways. We aimed to elucidate the role of FXR in the regulation of fasting hepatic gluconeogenesis. METHODS: The role of FXR in hepatic gluconeogenesis was assessed in vivo and in mouse primary hepatocytes. Gene expression patterns in response to glucagon and FXR agonists were characterized by quantitative reverse transcription PCR and microarray analysis. FXR phosphorylation by protein kinase A was determined by mass spectrometry. The interaction of FOXA2 with FXR was identified by cistromic approaches and in vitro protein-protein interaction assays. The functional impact of the crosstalk between FXR, the PKA and FOXA2 signaling pathways was assessed by site-directed mutagenesis, transactivation assays and restoration of FXR expression in FXR-deficient hepatocytes in which gene expression and glucose production were assessed. RESULTS: FXR positively regulates hepatic glucose production through two regulatory arms, the first one involving protein kinase A-mediated phosphorylation of FXR, which allowed for the synergistic activation of gluconeogenic genes by glucagon, agonist-activated FXR and CREB. The second arm involves the inhibition of FXR's ability to induce the anti-gluconeogenic nuclear receptor SHP by the glucagon-activated FOXA2 transcription factor, which physically interacts with FXR. Additionally, knockdown of Foxa2 did not alter glucagon-induced and FXR agonist enhanced expression of gluconeogenic genes, suggesting that the PKA and FOXA2 pathways regulate distinct subsets of FXR responsive genes. CONCLUSIONS: Thus, hepatic glucose production is regulated during physiological fasting by FXR, which integrates the glucagon/cAMP signal and the FOXA2 signal, by being post-translationally modified, and by engaging in protein-protein interactions, respectively. LAY SUMMARY: Activation of the nuclear bile acid receptor FXR regulates gene expression networks, controlling lipid, cholesterol and glucose metabolism, which are mostly effective after eating. Whether FXR exerts critical functions during fasting is unknown. The results of this study show that FXR transcriptional activity is regulated by the glucagon/protein kinase A and the FOXA2 signaling pathways, which act on FXR through phosphorylation and protein-protein interactions, respectively, to increase hepatic glucose synthesis.

The RBM14/CoAA-interacting, long intergenic non-coding RNA Paral1 regulates adipogenesis and coactivates the nuclear receptor PPARγ.

Adipocyte differentiation and function relies on a network of transcription factors, which is disrupted in obesity-associated low grade, chronic inflammation leading to adipose tissue dysfunction. In this context, there is a need for a thorough understanding of the transcriptional regulatory network involved in adipose tissue pathophysiology. Recent advances in the functional annotation of the genome has highlighted the role of non-coding RNAs in cellular differentiation processes in coordination with transcription factors. Using an unbiased genome-wide approach, we identified and characterized a novel long intergenic non-coding RNA (lincRNA) strongly induced during adipocyte differentiation. This lincRNA favors adipocyte differentiation and coactivates the master adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARγ) through interaction with the paraspeckle component and hnRNP-like RNA binding protein 14 (RBM14/NCoAA), and was therefore called PPARγ-activator RBM14-associated lncRNA (Paral1). Paral1 expression is restricted to adipocytes and decreased in humans with increasing body mass index. A decreased expression was also observed in diet-induced or genetic mouse models of obesity and this down-regulation was mimicked in vitro by TNF treatment. In conclusion, we have identified a novel component of the adipogenic transcriptional regulatory network defining the lincRNA Paral1 as an obesity-sensitive regulator of adipocyte differentiation and function.

Interspecies NASH disease activity whole-genome profiling identifies a fibrogenic role of PPARα-regulated dermatopontin.

Nonalcoholic fatty liver disease prevalence is soaring with the obesity pandemic, but the pathogenic mechanisms leading to the progression toward active nonalcoholic steatohepatitis (NASH) and fibrosis, major causes of liver-related death, are poorly defined. To identify key components during the progression toward NASH and fibrosis, we investigated the liver transcriptome in a human cohort of NASH patients. The transition from histologically proven fatty liver to NASH and fibrosis was characterized by gene expression patterns that successively reflected altered functions in metabolism, inflammation, and epithelial-mesenchymal transition. A meta-analysis combining our and public human transcriptomic datasets with murine models of NASH and fibrosis defined a molecular signature characterizing NASH and fibrosis and evidencing abnormal inflammation and extracellular matrix (ECM) homeostasis. Dermatopontin expression was found increased in fibrosis, and reversal of fibrosis after gastric bypass correlated with decreased dermatopontin expression. Functional studies in mice identified an active role for dermatopontin in collagen deposition and fibrosis. PPARα activation lowered dermatopontin expression through a transrepressive mechanism affecting the Klf6/TGFß1 pathway. Liver fibrotic histological damages are thus characterized by the deregulated expression of a restricted set of inflammation- and ECM-related genes. Among them, dermatopontin may be a valuable target to reverse the hepatic fibrotic process.

The logic of transcriptional regulator recruitment architecture at cis-regulatory modules controlling liver functions.

Control of gene transcription relies on concomitant regulation by multiple transcriptional regulators (TRs). However, how recruitment of a myriad of TRs is orchestrated at cis-regulatory modules (CRMs) to account for coregulation of specific biological pathways is only partially understood. Here, we have used mouse liver CRMs involved in regulatory activities of the hepatic TR, NR1H4 (FXR; farnesoid X receptor), as our model system to tackle this question. Using integrative cistromic, epigenomic, transcriptomic, and interactomic analyses, we reveal a logical organization where trans-regulatory modules (TRMs), which consist of subsets of preferentially and coordinately corecruited TRs, assemble into hierarchical combinations at hepatic CRMs. Different combinations of TRMs add to a core TRM, broadly found across the whole landscape of CRMs, to discriminate promoters from enhancers. These combinations also specify distinct sets of CRM differentially organized along the genome and involved in regulation of either housekeeping/cellular maintenance genes or liver-specific functions. In addition to these TRMs which we define as obligatory, we show that facultative TRMs, such as one comprising core circadian TRs, are further recruited to selective subsets of CRMs to modulate their activities. TRMs transcend TR classification into ubiquitous versus liver-identity factors, as well as TR grouping into functional families. Hence, hierarchical superimpositions of obligatory and facultative TRMs bring about independent transcriptional regulatory inputs defining different sets of CRMs with logical connection to regulation of specific gene sets and biological pathways. Altogether, our study reveals novel principles of concerted transcriptional regulation by multiple TRs at CRMs.

Nuclear bile acid signaling through the farnesoid X receptor.

Bile acids (BAs) are amphipathic molecules produced from cholesterol by the liver. Expelled from the gallbladder upon meal ingestion, BAs serve as fat solubilizers in the intestine. BAs are reabsorbed in the ileum and return via the portal vein to the liver where, together with nutrients, they provide signals to coordinate metabolic responses. BAs act on energy and metabolic homeostasis through the activation of membrane and nuclear receptors, among which the nuclear receptor farnesoid X receptor (FXR) is an important regulator of several metabolic pathways. Highly expressed in the liver and the small intestine, FXR contributes to BA effects on metabolism, inflammation and cell cycle control. The pharmacological modulation of its activity has emerged as a potential therapeutic strategy for liver and metabolic diseases. This review highlights recent advances regarding the mechanisms by which the BA sensor FXR contributes to global signaling effects of BAs, and how FXR activity may be regulated by nutrient-sensitive signaling pathways.

Glucose sensing O-GlcNAcylation pathway regulates the nuclear bile acid receptor farnesoid X receptor (FXR).

UNLABELLED: Bile acid metabolism is intimately linked to the control of energy homeostasis and glucose and lipid metabolism. The nuclear receptor farnesoid X receptor (FXR) plays a major role in the enterohepatic cycling of bile acids, but the impact of nutrients on bile acid homeostasis is poorly characterized. Metabolically active hepatocytes cope with increases in intracellular glucose concentrations by directing glucose into storage (glycogen) or oxidation (glycolysis) pathways, as well as to the pentose phosphate shunt and the hexosamine biosynthetic pathway. Here we studied whether the glucose nonoxidative hexosamine biosynthetic pathway modulates FXR activity. Our results show that FXR interacts with and is O-GlcNAcylated by O-GlcNAc transferase in its N-terminal AF1 domain. Increased FXR O-GlcNAcylation enhances FXR gene expression and protein stability in a cell type-specific manner. High glucose concentrations increased FXR O-GlcNAcylation, hence its protein stability and transcriptional activity by inactivating corepressor complexes, which associate in a ligand-dependent manner with FXR, and increased FXR binding to chromatin. Finally, in vivo fasting-refeeding experiments show that FXR undergoes O-GlcNAcylation in fed conditions associated with increased direct FXR target gene expression and decreased liver bile acid content. CONCLUSION: FXR activity is regulated by glucose fluxes in hepatocytes through a direct posttranslational modification catalyzed by the glucose-sensing hexosamine biosynthetic pathway.

Sleep architecture when sleeping at an unusual circadian time and associations with insulin sensitivity.

UNLABELLED: Circadian misalignment affects total sleep time, but it may also affect sleep architecture. The objectives of this study were to examine intra-individual effects of circadian misalignment on sleep architecture and inter-individual relationships between sleep stages, cortisol levels and insulin sensitivity. Thirteen subjects (7 men, 6 women, age: 24.3±2.5 y; BMI: 23.6±1.7 kg/m²) stayed in a time blinded respiration chamber during three light-entrained circadian cycles (3x21h and 3x27h) resulting in a phase advance and a phase delay. Sleep was polysomnographically recorded. Blood and salivary samples were collected to determine glucose, insulin and cortisol concentrations. Intra-individually, a phase advance decreased rapid eye movement (REM) sleep and slow-wave sleep (SWS), increased time awake, decreased sleep and REM sleep latency compared to the 24h cycle. A phase delay increased REM sleep, decreased stage 2 sleep, increased time awake, decreased sleep and REM sleep latency compared to the 24h cycle. Moreover, circadian misalignment changed REM sleep distribution with a relatively shorter REM sleep during the second part of the night. Inter-individually, REM sleep was inversely associated with cortisol levels and HOMA-IR index. Circadian misalignment, both a phase advance and a phase delay, significantly changed sleep architecture and resulted in a shift in rem sleep. Inter-individually, shorter REM sleep during the second part of the night was associated with dysregulation of the HPA-axis and reduced insulin sensitivity. TRIAL REGISTRATION: International Clinical Trials Registry Platform NTR2926 http://apps.who.int/trialsearch/

Palmitate increases Nur77 expression by modulating ZBP89 and Sp1 binding to the Nur77 proximal promoter in pancreatic ß-cells.

Nur77 is a stress sensor in pancreatic ß-cells, which negatively regulates glucose-stimulated insulin secretion. We recently showed that a lipotoxic shock caused by exposure of ß-cells to the saturated fatty acid palmitate strongly increases Nur77 expression. Here, using dual luciferase reporter assays and Nur77 promoter deletion constructs, we identified a regulatory cassette between -1534 and -1512 bp upstream from the translational start site mediating Nur77 promoter activation in response to palmitate exposure. Chromatin immunoprecipitation, transient transfection and siRNA-mediated knockdown assays revealed that palmitate induced Nur77 promoter activation involves Sp1 recruitment and ZBP89 release from the gene promoter.

Palmitate increases Nur77 expression by modulating ZBP89 and Sp1 binding to the Nur77 proximal promoter in pancreatic ß-cells.

Nur77 is a stress sensor in pancreatic ß-cells, which negatively regulates glucose-stimulated insulin secretion. We recently showed that a lipotoxic shock caused by exposure of ß-cells to the saturated fatty acid palmitate strongly increases Nur77 expression. Here, using dual luciferase reporter assays and Nur77 promoter deletion constructs, we identified a regulatory cassette between -1534 and -1512 bp upstream from the translational start site mediating Nur77 promoter activation in response to palmitate exposure. Chromatin immunoprecipitation, transient transfection and siRNA-mediated knockdown assays revealed that palmitate induced Nur77 promoter activation involves Sp1 recruitment and ZBP89 release from the gene promoter.

Effect of a phase advance and phase delay of the 24-h cycle on energy metabolism, appetite, and related hormones.

BACKGROUND: The disruption of the circadian system has been associated with the development of obesity. OBJECTIVE: We examined the effects of circadian misalignment on sleep, energy expenditure, substrate oxidation, appetite, and related hormones. DESIGN: Thirteen subjects [aged 24.3 ± 2.5 (mean ± SD) y; BMI (in kg/m²): 23.6 ± 1.7 (mean ± SD)] completed a randomized crossover study. For each condition, subjects stayed time blinded in the respiration chamber during 3 light-entrained circadian cycles that resulted in a phase advance (3 × 21 h) and a phase delay (3 × 27 h) compared with during a 24-h cycle. Sleep, energy expenditure, substrate oxidation, and appetite were quantified. Blood and saliva samples were taken to determine melatonin, glucose, insulin, ghrelin, leptin, glucagon-like peptide 1 (GLP-1), and cortisol concentrations. RESULTS: Circadian misalignment, either phase advanced or phase delayed, did not result in any changes in appetite or energy expenditure, whereas meal-related blood variables (glucose, insulin, ghrelin, leptin, and GLP-1) followed the new meal patterns. However, phase-advanced misalignment caused flattening of the cortisol-secretion pattern (P < 0.001), increased insulin concentrations (P = 0.04), and increased carbohydrate oxidation (P = 0.03) and decreased protein oxidation (P = 0.001). Phase-delayed misalignment increased rapid eye movement sleep (P < 0.001) and the sleeping metabolic rate (P = 0.02), increased glucose (P = 0.02) and decreased GLP-1 (P = 0.02) concentrations, and increased carbohydrate oxidation (P = 0.01) and decreased protein oxidation (P = 0.003). CONCLUSIONS: The main effect of circadian misalignment, either phase advanced or phase delayed, is a concomitant disturbance of the glucose-insulin metabolism and substrate oxidation, whereas the energy balance or sleep is not largely affected. Chronically eating and sleeping at unusual circadian times may create a health risk through a metabolic disturbance. This trial was registered at the International Clinical Trials Registry Platform (http://apps.who.int/trialsearch/) as NTR2926.

DNA alteration and programmed cell death during ageing of sunflower seed.

Sunflower (Helianthus annuus L.) seed viability is affected by moisture content (MC) during ageing and is related to accumulation of hydrogen peroxide and changes in energy metabolism. The aim of the present work was to investigate the effect of ageing on DNA alteration events by RAPD (random amplification of polymorphic DNA) analysis and to determine whether loss of seed viability might correspond to a controlled programmed cell death (PCD). Ageing of sunflower seeds was carried out at 35 °C for 7 d at different MCs. The higher the MC, the lower was the seed viability. RAPD analysis showed that DNA alterations occurred during ageing especially in seeds containing a high MC. In addition, PCD, as revealed by DNA fragmentation and TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick-end labelling) assay, was detected in aged seeds at MCs which resulted in ∼50% seed viability. At the cellular level, TUNEL assay and propidium iodide staining showed that cell death concerns all the cells of the embryonic axis. The quantification of the adenylate pool highlights mitochondrial dysfunction in aged seeds containing a high MC. The involvement of oxidative burst, mitochondria dysfunction, and PCD in seed loss of viability is proposed.