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Background and Aims: Non‐alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children and adolescents today. In comparison with adult disease, paediatric NAFLD may show a periportal localization, which is associated with advanced fibrosis. This study aimed to assess the role of genetic risk variants for histological disease pattern and severity in childhood NAFLD. Methods: We studied 14 single nucleotide polymorphisms (SNP) in a cohort of 70 adolescents with biopsy‐proven NAFLD. Genotype was compared to an adult control cohort (n = 200) and analysed in relation to histological disease severity and liver tissue proteomics. Results: Three of the 14 SNPs were significantly associated with paediatric NAFLD after FDR adjustment, rs738409 (PNPLA3, P = 2.80 × 10−06), rs1044498 (ENPP1, P = 0.0091) and rs780094 (GCKR, P = 0.0281). The severity of steatosis was critically associated with rs738409 (OR=3.25; 95% CI: 1.72‐6.52, FDR‐adjusted P = 0.0070). The strongest variants associated with severity of fibrosis were rs1260326, rs780094 (both GCKR) and rs659366 (UCP2). PNPLA3 was associated with a portal pattern of steatosis, inflammation and fibrosis. Proteome profiling revealed decreasing levels of GCKR protein with increasing carriage of the rs1260326/rs780094 minor alleles and downregulation of the retinol pathway in rs738409 G/G carriers. Computational metabolic modelling highlighted functional relevance of PNPLA3, GCKR and UCP2 for NAFLD development. Conclusions: This study provides evidence for the role of PNPLA3 as a determinant of portal NAFLD localization and severity of portal fibrosis in children and adolescents, the risk variant being associated with an impaired hepatic retinol metabolism. See Editorial on Page 440 [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) represents the most common and most malignant type of primary brain tumor and significantly contributes to cancer morbidity and mortality. Invasion into the healthy brain parenchyma is a major feature of glioblastoma aggressiveness. Reelin (RELN) is a large secreted extracellular matrix glycoprotein that regulates neuronal migration and positioning in the developing brain and sustains functionality in the adult brain. We here show that both RELN and its main downstream effector DAB1 are silenced in glioblastoma as compared to non‐neoplastic tissue and mRNA expression is inversely correlated with malignancy grade. Furthermore, RELN expression is positively correlated with patient survival in two large, independent clinically annotated datasets. RELN silencing occurs via promoter hypermethylation as shown by both database mining and bisulfite sequencing of the RELN promoter. Consequently, treatment with 5′‐Azacytidine and trichostatin A induced RELN expression in vitro. On the functional level, we found RELN to regulate glioblastoma cell migration both in a DAB1 (tyrosine phosphorylation)‐dependent and ‐independent fashion, depending on the substrate provided. Moreover, stimulation of RELN signaling strongly reduced proliferation in glioblastoma cells. This phenotype depends on DAB1 stimulation by RELN, as a mutant that lacks all RELN induced tyrosine phosphorylation sites (DAB1‐5F) failed to induce a growth arrest. Proteomic analyzes revealed that these effects are mediated by a reduction in E2F targets and dephosphorylation of ERK1/2. Taken together, our data establish a relevance of RELN signaling in glioblastoma pathology and thereby might unearth novel, yet unrecognized treatment options. [ABSTRACT FROM AUTHOR]

Abstract: Aims: The EMPA‐REG OUTCOME study showed reduced mortality and hospitalization due to heart failure (HF) in diabetic patients treated with empagliflozin. Overexpression and Ca2+‐dependent activation of Ca2+/calmodulin‐dependent kinase II (CaMKII) are hallmarks of HF, leading to contractile dysfunction and arrhythmias. We tested whether empagliflozin reduces CaMKII‐ activity and improves Ca2+‐handling in human and murine ventricular myocytes. Methods and results: Myocytes from wild‐type mice, mice with transverse aortic constriction (TAC) as a model of HF, and human failing ventricular myocytes were exposed to empagliflozin (1 μmol/L) or vehicle. CaMKII activity was assessed by CaMKII–histone deacetylase pulldown assay. Ca2+ spark frequency (CaSpF) as a measure of sarcoplasmic reticulum (SR) Ca2+ leak was investigated by confocal microscopy. [Na+]i was measured using Na+/Ca2+‐exchanger (NCX) currents (whole‐cell patch clamp). Compared with vehicle, 24 h empagliflozin exposure of murine myocytes reduced CaMKII activity (1.6 ± 0.7 vs. 4.2 ± 0.9, P < 0.05, n = 10 mice), and also CaMKII‐dependent ryanodine receptor phosphorylation (0.8 ± 0.1 vs. 1.0 ± 0.1, P < 0.05, n = 11 mice), with similar results upon TAC. In murine myocytes, empagliflozin reduced CaSpF (TAC: 1.7 ± 0.3 vs. 2.5 ± 0.4 1/100 μm−1 s−1, P < 0.05, n = 4 mice) but increased SR Ca2+ load and Ca2+ transient amplitude. Importantly, empagliflozin also significantly reduced CaSpF in human failing ventricular myocytes (1 ± 0.2 vs. 3.3 ± 0.9, P < 0.05, n = 4 patients), while Ca2+ transient amplitude was increased (F/F0: 0.53 ± 0.05 vs. 0.36 ± 0.02, P < 0.05, n = 3 patients). In contrast, 30 min exposure with empagliflozin did not affect CaMKII activity nor Ca2+‐handling but significantly reduced [Na+]i. Conclusions: We show for the first time that empagliflozin reduces CaMKII activity and CaMKII‐dependent SR Ca2+ leak. Reduced Ca2+ leak and improved Ca2+ transients may contribute to the beneficial effects of empagliflozin in HF. [ABSTRACT FROM AUTHOR]

Summary Vacuolar solute accumulation is an important process during plant development, growth and stress responses. Although several vacuolar carriers have been identified recently, knowledge regarding the regulation of transport is still limited. Solute accumulation may be controlled by various factors, such as alterations in carrier abundance or activity. Phosphorylation via kinases is a well-known principle for activation or deactivation of proteins. Several phosphorylated proteins have been identified in the tonoplast proteome; however, kinases that catalyse the phosphorylation of tonoplast proteins are currently unknown. The tonoplast monosaccaride transporter from Arabidopsis ( AtTMT1) and its homologue from barley have multiple phosphorylation sites in their extremely large loops. Here we demonstrate that the loop of AtTMT1 interacts with a mitogen-activated triple kinase-like protein kinase (VIK), that an aspartate-rich loop domain is required for effective interaction, and that the presence of VIK stimulates glucose import into isolated vacuoles. Furthermore, the phenotype of VIK loss-of-function plants strikingly resembles that of plants lacking AtTMT1/2. These data suggest that VIK-mediated phosphorylation of the AtTMT1 loop enhances carrier activity and consequently vacuolar sugar accumulation. As many phosphorylated proteins have been identified in the tonoplast, differential phosphorylation may be a general mechanism regulating vacuolar solute import. [ABSTRACT FROM AUTHOR]

R3452 --> Introduction: Brief episodes of non‐lethal myocardial ischemia/reperfusion preceding more sustained ischemia reduce lethal ischemia/reperfusion injury in all species tested so far, including humans. Although this phenomenon is known for more than 3 decades, the underlying signaling mechanisms are unclear in their details. Among various cardioprotective mediators, neuronal acetylcholine (ACh) and its signaling through cholinergic receptors is certainly involved. However, a non‐neuronal cardiac cholinergic system (NNCCS) has been recently identified, suggesting a nervous system‐independent, cardiomyocyte‐derived ACh signaling with signal amplification by autocrine cholinergic receptor activation. Aim: We aimed to examine the role of NNCCS‐derived ACh as a mediator of cardioprotection. We therefore in a reductionist approach used isolated adult cardiomyocytes with hypoxic preconditioning (HPC). Methods: Hearts from male Lewis rats were harvested, and adult ventricular cardiomyocytes were isolated. In suspended cardiomyocytes HPC was induced by one cycle of 10 min brief hypoxia and 20 min reoxygenation. Thirty min normoxia served as control. Afterwards, cardiomyocytes were exposed to 30 min sustained hypoxia and 5 min reoxygenation (H/R) or to 35 min normoxia as time control (TC). Cardiomyocyte viability was quantified before and after H/R or TC, respectively, as the percent fraction of rod‐shaped, unstained (0.5% trypan blue) cells over all cells. In parallel experiments, physostigmine (0.2 mmol/L) was added to cardiomyocyte suspensions to block the ubiquitous ACh esterase. Intracellular ACh was quantified with and without HPC via liquid chromatography‐coupled tandem mass spectrometry. In subsets, the nicotinic cholinergic receptor antagonists hexamethonium (1.0 µmol/L) and α‐bungarotoxine (0.1 µmol/L) or the muscarinic cholinergic receptor antagonist atropine (0.1 µmol/L) were added to cardiomyocyte suspensions with and without HPC, respectively, and exposure to H/R or TC, respectively, afterwards. Again, cardiomyocyte viability was quantified. Results: With HPC, the viability of cardiomyocytes subjected to H/R was better preserved than without (24±1% vs. 7±1%, figure A). Also, intracellular ACh was slightly (n.s.) increased after sustained hypoxia (6±2 pmol/mg protein), but significantly with HPC (13±3 pmol/mg protein) in comparison to baseline (3±1 pmol/mg protein) or TC (3±1 pmol/mg protein, figure B). However, this increase was only detectable in the presence of physostigmine (figure B). Addition of nicotinic and muscarinic cholinergic receptor antagonists attenuated the protection by HPC to the same extent (hexamethonium: 12±2%, α‐bungarotoxine: 11±1%, atropine: 10±1%, figure A). Conclusion: The NNCCS is activated by HPC and causal for HPC´s protection. HPC increases intracellular ACh, and cardiomyocyte protection is mediated via both nicotinic and muscarinic cholinergic receptors, suggesting an autocrine signal amplification on the cardiomyocyte level. [ABSTRACT FROM AUTHOR]