Classical and alternative NF-κB signaling cooperate in regulating adipocyte differentiation and function.

BACKGROUND AND OBJECTIVE: Inflammation of adipose tissue (AT) is a central mediator of insulin resistance. However, the molecular mechanisms triggered by inflammatory cells are not fully understood. The aim of this study was to analyze the metabolic functions of lymphotoxin-ß-receptor (LTßR)-mediated alternative NF-κB signaling in adipocytes and to reveal its effects on body weight and insulin sensitivity in vivo. METHODS: RelB(FatKO) mice and littermate controls were treated with LTßR agonistic antibody (α-LTßR) or a LTßR antagonist (LTßR:Ig fusion protein) after feeding a high-fat diet or standard diet. Mice were analyzed by insulin tolerance and glucose tolerance tests prior to analysis by necropsy and qRT-PCR of abdominal white adipose tissue. 3T3-L1 preadipocytes and mouse embryonic fibroblasts were used for differentiation and expression analysis after treatment with α-LTßR and differentiation to adipocytes. The molecular mechanism was elucidated by chromatin immunoprecipitation and combinatorial treatment with α-LTßR and tumor necrosis factor (TNF). RESULTS: RelB(FatKO) mice showed improved insulin sensitivity despite increased adiposity and adipocyte hypertrophy. LTßR-induced activation of p52-RelB in 3T3-L1 cells attenuated adipogenesis and modulated adipocyte functions via transcriptional downregulation of peroxisome proliferator-activated receptor γ (PPARγ). This LTßR-mediated pathway was synergistically regulated via a TNF-induced increase in p100 and RelB expression and nuclear translocation. CONCLUSIONS: Our data describe an anti-adipogenic action of LTßR signaling and a novel synergism of alternative and classical NF-κB signaling in the regulation of adipocytes. In conclusion, this strong synergism between the two NF-κB pathways shows a method to inhibit adipocyte differentiation and to improve insulin sensitivity and can be a potential target to treat metabolic disorders more efficiently than with other known drugs.

Surgical stress influences cytokine content in autologous conditioned serum.

REASONS FOR PERFORMING STUDY: No recommendations have been made regarding the relative timing of blood collection for autologous conditioned serum (ACS) preparation and surgical procedures. OBJECTIVES: 1) To identify effects of surgical stress on cytokine levels in ACS, 2) identify haematological markers for prediction of cytokine production in ACS and 3) investigate the necessity for specialised ACS containers when preparing a cytokine-rich serum. STUDY DESIGN: Experimental in vitro study. METHODS: Blood was drawn from 15 stallions admitted for elective castration preoperatively and 22-24 h post operatively and incubated in ACS containers and plastic vacutainer tubes containing Z Serum Clot Activator. Concentrations of interleukin (IL)-1 receptor agonist (IL-1Ra), IL-10, IL-1ß, tumour necrosis factor (TNF)-α, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-ß were determined in all serum samples and compared between preparation methods and sampling time by ANOVA. Changes in cytokine levels induced by incubation, defined as delta cytokine, were calculated by subtracting the baseline levels from the levels in incubated samples. Based on post operative serum amyloid A (SAA), horses were grouped into 'mild', moderate' and 'marked' surgical stress; delta cytokine levels in post operative samples were compared between these groups by ANOVA. RESULTS: Delta IGF-1 was significantly lower in post operative samples compared with preoperative. Horses in the 'marked' surgical stress group had significantly lower delta IL-1Ra and delta TGF-ß than the 'moderate' group and significantly lower delta IGF-1 than the 'mild' group. No association between cytokine levels and haematology variables were identified. Cytokine levels were comparable between serum prepared in blood tubes and in specialised ACS containers. CONCLUSIONS: Surgical stress influences the cytokine content in ACS. Useful predictors of cytokine production in ACS were not identified. Specialised ACS containers may not be necessary for preparation of a cytokine-rich serum.

T-piece improves arterial and central venous oxygenation in trachestomized patients as compared to continuous positive airway pressure/pressure support ventilation.

BACKGROUND: T-piece has been widely used as T-piece trial to identify patients who are ready for extubation but it is seldom used as a weaning tool. Our objective was to investigate the effects of breathing via T-piece on gas exchange as compared to continuous positive airway pressure with pressure support (CPAP+PS) and CPAP with automatic tube compensation (CPAP+ATC) as it has not been evaluated yet. METHODS: Tracheostomized, "ready to be weaned" critically ill patients were enrolled in this prospective, auto-control clinical trial. Arterial oxygen tension (PaO2) was determined on CPAP+PS (t0), 15 minutes later on CPAP+ATC (t1), then on T-piece at 15, 30 and 60 minutes (t2-4). ScvO2 was measured at t0 and t4. Settings of fraction of inspired oxygen (FiO2) and positive end-expiratory pressure (PEEP) were kept constant throughout the investigation. RESULTS: Twenty-five patients were enrolled. T-piece trial was interrupted in 4 cases after t2, due to pulmonary oedema, hypertension or fatigue. PaO2/FiO2 was significantly higher on T-piece (t3,4) then on CPAP (t0,1), P<0.05, PaO2/FiO2 did not change significantly on CPAP+PS (t0) vs. CPAP+ATC (t1) modes: median=208 (interquartile range: 175-266) vs. 223 (186-290) mmHg, P=0.102, but significantly increased from t0-t4: 208 (175-266) vs. 249 (215-325) mmHg, P=0.003, respectively. ScvO2 was significantly higher on T-piece at t4: 80% (75-82%) than on CPAP+PS at t0: 73% (71-78%), P<0.001. CONCLUSION: On the same FiO2 and PEEP setting, breathing via T-piece improved oxygenation and resulted in increased ScvO2 as compared to breathing on CPAP with PS. Our observations suggest a potential role of T-piece during weaning from mechanical ventilation.

Cross talk between stimulated NF-kappaB and the tumor suppressor p53.

Nuclear factor-kappaB (NF-kappaB) and p53 critically determine cancer development and progression. Defining the cross talk between these transcription factors can expand our knowledge on molecular mechanisms of tumorigenesis. Here, we show that induction of replicational stress activates NF-kappaB p65 and triggers its interaction with p53 in the nucleus. Experiments with knockout cells show that p65 and p53 are both required for enhanced NF-kappaB activity during S-phase checkpoint activation involving ataxia-telangiectasia mutated and checkpoint kinase-1. Accordingly, the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) also triggers formation of a transcriptionally active complex containing nuclear p65 and p53 on kappaB response elements. Gene expression analyses revealed that, independent of NF-kappaB activation in the cytosol, TNF-induced NF-kappaB-directed gene expression relies on p53. Hence, p53 is unexpectedly necessary for NF-kappaB-mediated gene expression induced by atypical and classical stimuli. Remarkably, data from gain- and loss-of function approaches argue that anti-apoptotic NF-kappaB p65 activity is constitutively evoked by a p53 hot-spot mutant frequently found in tumors. Our observations suggest explanations for the outstanding question why p53 mutations rather than p53 deletions arise in tumors of various origins.

Tissue-specific signal(s) activate the promoter of a metallocarboxypeptidase inhibitor gene family in potato tuber and berry.

The molecular basis of the differential expression of the GM7-type metallocarboxypeptidase inhibitor (MCPI) genes in tuberizing (StMCPI) and non-tuberizing Solanum species (SbMCPI) was investigated. It was shown that the StMCPI is encoded by a gene family in Solanum tuberosum (potato), but SbMCPI might be a single-copy gene in the non-tuberizing species Solanum brevidens. The StMCPI promoter shows evolutionary relatedness to the S. brevidens-derived SbMCPI and to the fruit-specific tomato promoter 2A11. Both StMCPI and SbMCPI promoter regions were able to confer tuber- and berry-specific expression for the beta-glucuronidase reporter gene in potato suggesting that the difference in MCPI gene expression is in trans regulatory factors between the tuberizing and the non-tuberizing Solanum species. The MCPI promoters did not respond to metabolic, environmental or hormonal signals in leaves. Thus, the MCPI genes are regulated in a different way than the other known tuber-specific genes and potentially are suitable for biotechnological application in potato to provide specific transgene expression in tuber and berry.