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Utrecht, Netherlands

Kranendonk M.E.G.,University Utrecht | Kranendonk M.E.G.,Molecular Cancer Research | Visseren F.L.J.,University Utrecht | Van Balkom B.W.M.,UMC Utrecht | And 10 more authors.
Obesity | Year: 2014

Objective Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)-explants play a role in the paracrine interaction between adipocytes and macrophages, a key mechanism in AT inflammation, leading to metabolic complications like insulin resistance (IR) were determined. Methods EVs released from in vitro differentiated adipocytes and AT-explants ex vivo were characterized by electron microscopy, Western blot, multiplex adipokine-profiling, and quantified by flow cytometry. Primary monocytes were stimulated with EVs from adipocytes, subcutaneous (SCAT) or omental-derived AT (OAT), and phenotyped. Macrophage supernatant was subsequently used to assess the effect on insulin signaling in adipocytes. Results Adipocyte and AT-derived EVs differentiated monocytes into macrophages characteristic of human adipose tissue macrophages (ATM), defined by release of both pro- and anti-inflammatory cytokines. The adiponectin-positive subset of AT-derived EVs, presumably representing adipocyte-derived EVs, induced a more pronounced ATM-phenotype than the adiponectin-negative AT-EVs. This effect was more evident for OAT-EVs versus SCAT-EVs. Furthermore, supernatant of macrophages pre-stimulated with AT-EVs interfered with insulin signaling in human adipocytes. Finally, the number of OAT-derived EVs correlated positively with patients HOMA-IR. Conclusions A possible role for human AT-EVs in a reciprocal pro-inflammatory loop between adipocytes and macrophages, with the potential to aggravate local and systemic IR was demonstrated. Copyright © 2013 The Obesity Society.

Pereboom T.C.,University Utrecht | Bondt A.,University Utrecht | Pallaki P.,University Utrecht | Klasson T.D.,University Utrecht | And 10 more authors.
Experimental Hematology | Year: 2014

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome linked to mutations in ribosomal protein (RP) genes that result in the impaired proliferation of hematopoietic progenitor cells. The etiology of DBA is not completely understood; however, the ribosomal nature of the genes involved has led to speculation that these mutations may alter the landscape of messenger RNA (mRNA) translation. Here, we performed comparative microarray analysis of polysomal mRNA transcripts isolated from lymphoblastoid cell lines derived from DBA patients carrying various haploinsufficient mutations in either RPS19 or RPL11. Different spectrums of changes were observed depending on the mutant gene, with large differences found in RPS19 cells and very few in RPL11 cells. However, we find that the small number of altered transcripts in RPL11 overlap for the most part with those altered inRPS19 cells. We show specifically that levels of branched-chain aminotransferase-1 (BCAT1) transcripts are significantly decreased on the polysomes of both RPS19 and RPL11 cells and that translation of BCAT1 protein is especially impaired in cells with small RP gene mutations, and we provide evidence that this effect may be due in part to the unusually long 5'UTR of the BCAT1 transcript. The BCAT1 enzyme carries out the final step in the biosynthesis and the first step of degradation of the branched-chain amino acids leucine, isoleucine, and valine. Interestingly, several animal models of DBA have reported that leucine ameliorates the anemia phenotypes generated by RPS19 loss. Our study suggests thatRP mutations affect the synthesis of specific proteins involved in regulating amino acid levelsthat are important for maintaining the normal proliferative capacity of hematopoietic cells. © 2014 ISEH - Society for Hematology and Stem Cells.

Overlack K.,Max Planck Institute of Molecular Physiology | Primorac I.,Max Planck Institute of Molecular Physiology | Vleugel M.,Molecular Cancer Research | Krenn V.,Max Planck Institute of Molecular Physiology | And 7 more authors.
eLife | Year: 2015

The spindle assembly checkpoint (SAC) monitors and promotes kinetochoremicrotubule attachment during mitosis. Bubl and BubRl, SAC components, originated from duplication of an ancestor gene. Subsequent subfunctionalization established subordination: Bubl, recruited first to kinetochores, promotes successive BubRl recruitment. Because both Bubl and BubRl hetero- dimerize with Bub3, a targeting adaptor for phosphorylated kinetochores, the molecular basis for such sub-functionalization is unclear. We demonstrate that Bubl, but not BubRl, enhances binding of Bub3 to phosphorylated kinetochores. Grafting a short motif of Bubl onto BubRl promotes Bubl-independent kinetochore recruitment of BubRl. Such gain-of-function BubRl mutant cannot sustain a functional checkpoint. We demonstrate that kinetochore localization of BubRl relies on direct hetero-dimerization with Bubl at a pseudo-symmetric interface. Such pseudo-symmetric interaction underpins a template-copy relationship crucial for kinetochore-microtubule attachment and SAC signaling. Our results illustrate how gene duplication and sub-functionalization shape the workings of an essential molecular network. © 2015, eLife Sciences Publications Ltd. All rights reserved.

Chakrabarty K.,University Utrecht | Von Oerthel L.,University Utrecht | Von Oerthel L.,University of Amsterdam | Hellemons A.,University Utrecht | And 7 more authors.
Biology Open | Year: 2012

Meso-diencephalic dopaminergic (mdDA) neurons are critical for motor control and cognitive functioning and their loss or dysfunction is associated with disorders such as Parkinson's disease (PD), schizophrenia and addiction. However, relatively little is known about the molecular mechanisms underlying mdDA neuron development and maintenance. Here, we determined the spatiotemporal map of genes involved in the development of mdDA neurons to gain further insight into their molecular programming. Genome-wide gene expression profiles of the developing ventral mesencephalon (VM) were compared at different developmental stages leading to the identification of novel regulatory roles of neuronal signaling through nicotinic acthylcholine receptors (Chrna6 and Chrnb3 subunits) and the identification of novel transcription factors (Oc2 and 3) involved in the generation of the mdDA neuronal field. We show here that Pitx3, in cooperation with Nurr1, is the critical component in the activation of the Chrna6 and Chrnb3 subunits in mdDA neurons. Furthermore, we provide evidence of two divergent regulatory pathways resulting in the expression of Chrna6 and Chrnb3 respectively. © 2012 Published by The Company of Biologists Ltd.

Kranendonk M.E.G.,University Utrecht | Kranendonk M.E.G.,Molecular Cancer Research | Visseren F.L.J.,University Utrecht | Van Herwaarden J.A.,UMC Utrecht | And 4 more authors.
Obesity | Year: 2014

Objective: Insulin resistance (IR) is a key mechanism in obesity-induced cardiovascular disease. To unravel mechanisms whereby human adipose tissue (AT) contributes to systemic IR, the effect of human AT-extracellular vesicles (EVs) on insulin signaling in liver and muscle cells was determined. Methods: EVs released from human subcutaneous (SAT) and omental AT (OAT)-explants ex vivo were used for stimulation of hepatocytes and myotubes in vitro. Subsequently, insulin-induced Akt phosphorylation and expression of gluconeogenic genes (G6P, PEPCK) was determined. AT-EV adipokine levels were measured by multiplex immunoassay, and AT-EVs were quantified by high-resolution flow cytometry. Results: In hepatocytes, AT-EVs from the majority of patients inhibited insulin-induced Akt phosphorylation, while EVs from some patients stimulated insulin-induced Akt phosphorylation. In myotubes AT-EVs exerted an ambiguous effect on insulin signaling. Hepatic Akt phosphorylation related negatively to G6P-expression by both SAT-EVs (r= -0.60, P=0.01) and OAT-EVs (r= -0.74, P=0.001). MCP-1, IL-6, and MIF concentrations were higher in OAT-EVs compared to SAT-EVs and differently related to lower Akt phosphorylation in hepatocytes. Finally, the number of OAT-EVs correlated positively with liver enzymes indicative for liver dysfunction. Conclusions: Human AT-EVs can stimulate or inhibit insulin signaling in hepatocytes- possibly depending on their adipokine content- and may thereby contribute to systemic IR. © 2014 The Obesity Society.

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