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Aims/hypothesis Recently, hedgehog (Hh) was identified as a crucial player in adipose tissue development and energy expenditure. Therefore, we tested whether Hh ligands are regulated in obesity. Further, we aimed at identifying potential target cells of Hh signalling and studied the functional impact of Hh signalling on adipose tissue inflammation and glucose metabolism.
Methods Hh ligands and receptors were analysed in adipose tissue or serum from lean and obese mice as well as in humans. To study the impact on adipose tissue inflammation and glucose metabolism, Hh signalling was specifically blocked in myeloid cells using a conditional knockout approach (Lys-Smo−/−).
Results Desert Hh (DHH) and Indian Hh (IHH) are local Hh ligands, whereas Sonic Hh is not expressed in adipose tissue from mice or humans. In mice, obesity leads to a preferential upregulation of Hh ligands (Dhh) and signalling components (Ptch1, Smo and Gli1) in subcutaneous adipose tissue. Further, adipose tissue macrophages are Hh target cells owing to the expression of Hh receptors, such as Patched1 and 2. Conditional knockout of Smo (which encodes Smoothened, a mandatory Hh signalling component) in myeloid cells increases body weight and adipose tissue inflammation and attenuates glucose tolerance, suggesting an anti-inflammatory effect of Hh signalling. In humans, adipose tissue expression of DHH and serum IHH decrease with obesity and type 2 diabetes, which might be explained by the intake of metformin. Interestingly, metformin reduced Dhh and Ihh expression in mouse adipose tissue explants.
Conclusions/interpretation Hh signalling in myeloid cells affects adipose tissue inflammation and glucose metabolism and may be a potential target to treat type 2 diabetes.
Keywords Adipose tissue . Diabetes . Glucose tolerance . Hedgehog . Inflammation . Macrophages . Obesity . Smo

Validation of antibody reagents for mucin analysis in chronic inflammatory airway diseases

Posted by T Krause, K Ramaker, H Sinnecker, et al. on 2017-03-20 18:37:05

In chronic inflammatory airway diseases, mucins display disease-related alterations in quantity,composition and glycosylation. This opens the possibility to diagnose and monitor inflammatory airway disorders and their exacerbation based on mucin properties. For such an approach to be reasonably versatile and diagnostically meaningful, the mucin of interest must be captured in a reliable, patientindependent way. To identify appropriate mucin-specific reagents, we tested anti-mucin antibodies on mucin-content-standardized, human bronchoalveolar lavage fluid samples in immunoblot assays. All commercially available monoclonal antibodies against the major airway mucin MUC5AC were screened, except for those with known specificity for carbohydrates, as glycosylation patterns are not mucin-specific. Our results indicated considerable inter-patient and inter-antibody variability in mucin recognition for all antibodies and samples tested. The best results in terms of signal strength and reproducibility were
obtained with antibodies Mg-31, O.N.457 and 45M1. Additional epitope mapping experiments revealed that only one of the antibodies with superior binding to MUC5AC recognized linear peptide epitopes on the protein backbone.

Effects of cranberry (Vaccinum macrocarpon) supplementation on iron status and inflammatory markers in rowers

Posted by A Skarpańska - Stejnborn, P Basta, J Trzeciak, et al. on 2017-03-17 01:33:15

Background: The aim of this study was to analyze the effect of supplementation with cranberry (Vaccinum macrocarpon) on the levels of pro-inflammatory cytokines, hepcidin and selected markers of iron metabolism in rowers subjected to exhaustive exercise.
Methods: This double-blind study included 16 members of the Polish Rowing Team. The subjects were randomly assigned to the supplemented group (n = 9), receiving 1200 mg of cranberry extract for 6 weeks, or to the placebo group (n = 7). The participants performed a 2000-m test on a rowing ergometer at the beginning and at the end of the preparatory camp. Blood samples were obtained from the antecubital vein prior to each exercise test, one minute after completing the test, and after a 24-h recovery period. The levels of hepcidin, interleukin 6 (IL-6), tumor necrosis
factor alpha (TNF-alpha), ferritin, iron, soluble transferrin receptor (sTfR) and myoglobin were determined, along with total iron-binding capacity (TIBC), unbound iron-binding capacity (UIBC) and total antioxidant capacity (TAC).
Results: Both prior and after the supplementation, a significant post-exercise increase in the concentration of IL-6 was observed in both groups. At the end of the study period, cranberry-supplemented athletes presented with significantly higher resting, post-exercise and post-recovery levels of TAC than the controls. However, a significant exercise-induced increase in the concentrations of TNF-alpha, myoglobin and hepcidin was observed solely in the control group.
Conclusion: Supplementation with cranberry extract contributed to a significant strengthening of antioxidant potential in individuals exposed to strenuous physical exercise. However, supplementation did not exert direct effects on other analyzed parameters: inflammatory markers and indices of iron metabolism (TNF-alpha, hepcidin and myoglobin).
Keywords: Cranberry, Supplementation, Strenuous exercise, Inflammation

Abstract Adenosine impacts cerebral ischemia reperfusion (IR) through the inhibitory A1 and the excitatory A2 receptors. The present study aimed at investigating the contrasting role of pERK1/2 in mediating adenosine A1R (protective) versus A2AR (deleterious) effects in IR. Male Wistar rats subjected to bilateral carotid occlusion (45 min) followed by reperfusion (24 h) exhibited increased pERK1/2 activity, downstream from DAG pathway, along with increases in hippocampal glutamate, c-Fos, NF-κB, TNF-α, iNOS, TBARS, cytochrome c, caspase-3, BDNF, Nrf2, and IL-10 contents. Further, hippocampal microglial reactivity, glial TNF-α, and BDNF expression were observed. Although unilateral intrahippocampal injection of either the A1R agonist CHA or the A2AR agonist CGS21680 increased pERK1/2, only CHA mitigated histopathological and behavioral deficits along with reducing glutamate, microglial activation, c-Fos, TNF-α, iNOS, TBARS, cytochrome c and caspase-3 and elevating Nrf2 and IL-10 levels in IR rats. These results yield insight into the double-faceted nature of pERK1/2 in mediating protective and deleterious effects of A1R and A2AR signaling, respectively, against IR injury.

Differential decrease in soluble and DNA-bound telomerase in senescent human fibroblasts

Posted by S Yehuda, H Yanai, E Priel, et al. on 2017-03-15 20:20:02

Abstract The role of telomere shortening in the induction of replicative cellular senescence (CS) is well known and as a result, the involvement of telomerase and in particular its catalytic subunit, the telomerase reverse transcriptase (TERT) in CS has also been investigated. However, the majority of studies were conducted on cells that generally express high levels of TERT (cancer and immortalized cells) while the role of telomerase in CS in normal cells has been investigated to a much lesser extent. In particular, it was reported that active TERT is expressed in early passages of cultured human keratinocytes but rapidly diminished towards entry to CS, without telomere shortening. With the putative importance of TERT/telomerase in CS and the aging process in mind, we investigated the expression of TERT and telomerase activity in primary cultures of adult human dermal fibroblasts (HDFs) in the in vitro model of replicative CS. We found that (i) HDFs expressed active TERT; (ii) TERT protein levels and telomerase activity were markedly decreased in senescent HDFs; and (iii) the reduction of TERT in the soluble fraction was more pronounced than in the DNA-bound one. The results suggest the importance of the non-canonical (telomere-unrelated) functions of TERT in cellular senescence.

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