[Show abstract][Hide abstract] ABSTRACT: Monoglyceride lipase (MGL) degrades 2-arachidonoyl glycerol (2-AG), an endogenous agonist of cannabinoid receptors (CB1/2 ). Since CB1 receptor is involved in the control of gut function, we investigated the effects of pharmacological inhibition and genetic deletion of MGL on intestinal motility. Furthermore, we were interested whether defective 2-AG degradation affects μ-opioid receptor (μ-receptor) signaling, a parallel pathway regulating gut motility.
Gut motility was investigated by monitoring Evan's blue transit and colonic bead propulsion in response to MGL inhibition and CB1 receptor or μ-receptor stimulation. Ileal contractility was investigated by electrical field stimulation. CB1 receptor expression in ileum and colon was studied by immunohistochemical analyses.
Pharmacological inhibition of MGL slows down whole gut transit in a CB1 receptor-dependent manner. Conversely, genetic deletion of MGL does not affect gut transit despite increased 2-AG levels. Notably, MGL-deficiency causes complete insensitivity to CB1 receptor agonist-mediated inhibition of whole gut transit and ileal contractility suggesting local desensitization of CB1 receptors. Accordingly, immunohistochemical analyses of myenteric ganglia of MGL-deficient mice revealed that CB1 receptors are trapped in endocytic vesicles. Finally, MGL-deficient mice display accelerated colonic propulsion and respond hypersensitive to μ-opioid receptor agonist-mediated inhibition of colonic motility. This phenotype can be reproduced by chronic pharmalogical inhibition of MGL.
Constantly elevated 2-AG levels induce severe desensitization of intestinal CB1 receptors and increased sensitivity to μ-receptor-mediated inhibition of colonic motility. These changes should be considered for the use of cannabinoid-based drugs in the therapy of gastrointestinal diseases.
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British Journal of Pharmacology 06/2015; 172(17). DOI:10.1111/bph.13224 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In spite of some progress in understanding the molecular basis of lipid-associated disorders, major questions about the regulation of synthesis and degradation of lipids and the interaction of these processes with other aspects of cellular physiology are still unresolved. Studies in reference organisms such as various yeast species, the fruit fly Drosophila melanogaster, or the nematode Caenorhabditis elegans complement efforts in mouse models as well as clinical studies in humans to address these questions. Imaging techniques play a pivotal role in understanding lipid droplet biology, and the implementation of imaging-based high-content screens of mutant collections has led to the identification of novel molecular players. This study focuses on novel fluorescent probes as well as spectroscopic imaging techniques to investigate lipid droplet formation and turnover in yeast. The application and limitations of such techniques in understanding lipid storage and turnover are discussed.
[Show abstract][Hide abstract] ABSTRACT: To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1+/− mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.
The EMBO Journal 01/2015; 34(8). DOI:10.15252/embj.201489363 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microscopic imaging techniques play a pivotal role in the life sciences. Here we describe labeling and imaging methods for live yeast cell imaging. Yeast is an excellent reference organism for biomedical research to investigate fundamental cellular processes, and has gained great popularity also for large-scale imaging-based screens. Methods are described to label live yeast cells with organelle-specific fluorescent dyes or GFP-tagged proteins, and how cells are maintained viable over extended periods of time during microscopy. We point out common pitfalls and potential microscopy artifacts arising from inhomogeneous labeling and depending on cellular physiology. Application and limitation of bleaching techniques to address dynamic processes in the yeast cell are described.
[Show abstract][Hide abstract] ABSTRACT: In the multidisciplinary field of heart research it is of utmost importance, for the description of phenomena like mechanoelectric feedback or heart wall thickening, to identify accurate myocardium material properties. Therefore this study aims to determine biaxial tensile and triaxial shear properties of the passive human ventricular myocardium. This novel combination of biaxial and shear testing, together with the investigation of the myocardial microstructure, will yield new innovative and essential information about material properties to fulfil the short term goals of constructing realistic myocardial models capable of capturing the mechanics of the heart, as well as aiding the long term goals of improving methods of medical treatment and quality of life for people suffering from heart diseases.
For the biaxial tests, squared specimens (25x25x2mm) were prepared with their sides aligned with the fiber and cross-fiber axis. During the experiments, the specimens were submerged in a cardioplegic solution at physiological conditions and different stretch ratios were applied consecutively. For the triaxial shear testing, three adjoining cubic specimens (4x4x4mm) were prepared with their sides aligned according to the fiber axis, sheet axis and sheet-normal axis. Three cycles of sinusoidal simple shear (0.1-0.5 in 0.1 steps of specimen thickness) were applied to each cubic specimens in two orthogonal directions. A novel combination of optical clearing and multiphoton microscopy was utilized to explore the 3D microstructure of the tissue emphasizing the 3-D orientation and dispersion of the muscle fibers and adjacent collagen fabrics.
The tissue showed pronounced nonlinear and highly orientation dependent behavior. The donor's age was greatly influencing the mechanical behavior of the myocardial tissue. Microstructural investigations affirmed an orthotropic composition of the investigated tissue and showed highly aligned myofibers with small dispersion in the healthy human myocardium. An invariant-based constitutive model showed the ability to give a good representation of both the biaxial tensile and the triaxial shear responses.
The material data from this study is intended to be used in numerical (Finite Element) simulations for better understanding of fundamental underlying ventricular mechanics, a step needed in the improvement of medical treatment of heart diseases.
Cardiovascular Research 07/2014; 103(suppl 1):S95. DOI:10.1093/cvr/cvu091.190 · 5.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription.
[Show abstract][Hide abstract] ABSTRACT: Cytosolic lipid droplets (LD) are ubiquitous organelles in pro- and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAG) and steryl esters which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy was identified, which includes the core autophagic machinery, but not Atg11 or Atg20. Thus, LD autophagy is distinct from ER-phagy, pexophagy or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles, and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, are required for LD formation, in yeast.
Molecular biology of the cell 11/2013; 25(2). DOI:10.1091/mbc.E13-08-0448 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nanostructured substrates have been recognized to initiate transcriptional programs promoting cell proliferation. Specifically β-catenin has been identified as transcriptional regulator, activated by adhesion to nanostructures. We set out to identify processes responsible for nanostructure-induced endothelial β-catenin signaling. Transmission electron microscopy (TEM) of cell contacts to differently sized polyethylene terephthalate (PET) surface structures (ripples with 250 to 300 nm and walls with 1.5 μm periodicity) revealed different patterns of cell-substrate interactions. Cell adhesion to ripples occurred exclusively on ripple peaks, while cells were attached to walls continuously. The Src kinase inhibitor PP2 was active only in cells grown on ripples, while the Abl inhibitors dasatinib and imatinib suppressed β-catenin translocation on both structures. Moreover, Gd(3+) sensitive Ca(2+) entry was observed in response to mechanical stimulation or Ca(2+) store depletion exclusively in cells grown on ripples. Both PP2 and Gd(3+) suppressed β-catenin nuclear translocation along with proliferation in cells grown on ripples but not on walls. Our results suggest that adhesion of endothelial cells to ripple structured PET induces highly specific, interface topology-dependent changes in cellular signalling, characterized by promotion of Gd(3+) -sensitive Ca(2+) entry and Src/Abl activation. We propose that these signaling events are crucially involved in nanostructure-induced promotion of cell proliferation.
Journal of Nanomaterials 10/2013; 2013. DOI:10.1155/2013/251063 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 'discovery' of lipid droplets as a metabolically highly active subcellular organelle has sparked great scientific interest in its research in recent years. The previous view of a rather inert storage pool of neutral lipids-triacylglycerol and sterols or steryl esters-has markedly changed. Driven by the endemic dimensions of lipid-associated disorders on the one hand, and the promising biotechnological application to generate oils ('biodiesel') from single-celled organisms on the other, multiple model organisms are exploited in basic and applied research to develop a better understanding of biogenesis and metabolism of this organelle. This article summarizes the current status of LD research in yeast and experimental approaches to obtain insight into the regulatory and structural components driving lipid droplet formation and their physiological and pathophysiological roles in lipid homeostasis.
Current Genetics 09/2013; 59(4). DOI:10.1007/s00294-013-0407-9 · 2.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We showed earlier that nutritional stress like starvation or high fat diet resulted in phenotypic changes in the lipidomes of hepatocyte lipid droplets (LDs), representative for the pathophysiological status of the mouse model. Here we extend our former study by adding genetic stress due to knock-out (KO) of adipocyte triglyceride lipase (ATGL), the rate limiting enzyme in LD lipolysis. An intervention trial for 6 weeks with male wild-type (WT) and ATGL-KO mice was carried out; both genotypes were fed lab chow or were exposed to short-time starvation. Isolated LDs were analyzed by LC-MS/MS. Triacylglycerol, diacylglycerol and phosphatidylcholine lipidomes, in that order, provided best phenotypic signatures characteristic for respective stresses applied to the animals. This was evidenced at lipid species level by principal component analysis, calculation of average values for chain-lengths and numbers of double bonds, and by visualization in heat maps. Structural backgrounds for analyses and metabolic relationships were elaborated at lipid molecular species level. Relating our lipidomic data to non-alcoholic fatty liver diseases of nutritional and genetic etiologies with or without accompanying insulin resistance, phenotypic distinction in hepatocyte LDs dependent on insulin status emerged. Taken together, lipidomes of hepatocyte lipid droplets are sensitive responders to nutritional and genetic stress.
The Journal of Lipid Research 06/2013; 54(8). DOI:10.1194/jlr.M037952 · 4.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We present a novel approach allowing for a simple, fast and automated morphological analysis of three-dimensional image stacks (z-stacks) featuring fibrillar structures from optically cleared soft biological tissues. Five non-atherosclerotic tissue samples from human abdominal aortas were used to outline the multi-purpose methodology, applicable to various tissue types. It yields a three-dimensional orientational distribution of relative amplitudes, representing the original collagen fibre morphology, identifies regions of isotropy where no preferred fibre orientations are observed and determines structural parameters throughout anisotropic regions for the analysis and numerical modelling of biomechanical quantities such as stress and strain. Our method combines optical tissue clearing with second-harmonic generation imaging, Fourier-based image analysis and maximum-likelihood estimation for distribution fitting. With a new sample preparation method for arteries, we present, for the first time to our knowledge, a continuous three-dimensional distribution of collagen fibres throughout the entire thickness of the aortic wall, revealing novel structural and organizational insights into the three arterial layers.
Journal of The Royal Society Interface 03/2013; 10(80):20120760. DOI:10.1098/rsif.2012.0760 · 3.92 Impact Factor