title: 2-Deoxy-D-glucose treatment changes the Golgi apparatus architecture without blocking synthesis of complex lipids.
abstract: The classic Golgi apparatus organization, an arrangement of highly ordered cisternal stacks with tubular-vesicular membrane specializations on both sides, is the functional image of a continuous flow of contents and membranes with input, metabolization, and output in a dynamic steady state. In response to treatment with 2-deoxy-D-glucose (2-DG), which lowers the cellular ATP level by about 70% within minutes, this organization is rapidly replaced by tubular-glomerular membrane convolutes described as Golgi networks and bodies. 2-DG is a non-metabolizable glucose analogue and competitive inhibitor of glycolysis, which has become attractive in the context of therapeutic approaches for several kinds of tumors specifically targeting glycolysis in cancer. With the question of whether the functions of the Golgi apparatus in lipid synthesis would be influenced by the 2-DG-induced Golgi apparatus reorganization, we focused on lipid metabolism within the Golgi bodies. For this, we applied a fluorophore-labeled short-chain ceramide (BODIPY-Cer) in various combinations with 2-DG treatment to HepG2 cell cultures and followed uptake, enrichment and metabolization to higher ordered lipids. The cellular ATP status in each experiment was controlled with a bioluminescence assay, and the response of the Golgi apparatus was tracked by immunostaining of the trans-Golgi network protein TGN46. For electron microscopy, the fluorescent BODIPY-Cer signals were converted into electron-dense precipitates by photooxidation of diaminobenzidine (DAB); DAB precipitates labeled trans-Golgi areas in control cultures but also compartments at the periphery of the Golgi bodies formed in response to 2-DG treatment, thus indicating that concentration of ceramide takes place in spite of the Golgi apparatus reorganization. Lipid analyses by thin-layer chromatography (TLC) performed in parallel showed that BODIPY-Cer is not only concentrated in compartments of the 2-DG-induced Golgi bodies but is partly metabolized to BODIPY-sphingomyelin. Both, uptake and condensation of BODIPY-Cer and its conversion to complex lipids indicate that functions of the Golgi apparatus in the cellular lipid metabolism persist although the classic Golgi apparatus organization is abolished.
authors.affiliation: Department of Cell Biology and Ultrastructure Research, Center for Anatomy and Cell Biology, Medical University of Vienna, Schwarzspanierstr. 17, 1090, Vienna, Austria.
authors.forename: Carmen
authors.initials: C
authors.lastname: Ranftler
authors.forename: Claudia
authors.initials: C
authors.lastname: Meisslitzer-Ruppitsch
authors.forename: Herbert
authors.initials: H
authors.lastname: Stangl
authors.forename: Clemens
authors.initials: C
authors.lastname: Röhrl
authors.forename: Stefanie
authors.initials: S
authors.lastname: Fruhwürth
authors.forename: Josef
authors.initials: J
authors.lastname: Neumüller
authors.forename: Margit
authors.initials: M
authors.lastname: Pavelka
authors.forename: Adolf
authors.initials: A
authors.lastname: Ellinger
mesh_terms: D000255:Adenosine Triphosphate / Q000172:deficiency; D002855:Chromatography, Thin Layer; D003847:Deoxyglucose / Q000494:pharmacology*; D004734:Energy Metabolism / Q000187:drug effects; D006056:Golgi Apparatus / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D056945:Hep G2 Cells; D006801:Humans; D050155:Lipogenesis / Q000187:drug effects*; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D013997:Time Factors; D021601:trans-Golgi Network / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure
chemical_list: C527864:N-(5-(5,7-dimethyl Bodipy)-1-pentanoyl)-erythrosphingosine; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D013110:Sphingosine