| Abstract | Cilj: Gangliozidi su važni za stabilizaciju i organizaciju membranskih mikrodomena koji služe kao komunikacijska središta za prenošenje inzulinske signalizacije u stanicu. Ometanje sinteze gangliozida trebalo bi poremetiti inzulinsku signalizaciju pojačavajući je ili snižavajući, ovisno o tome koji je enzim poremećen. To bi pak trebalo uzrokovati promjene u staničnoj neuroplastičnosti, morfologiji i toleranciji na inzulin. Ustroj studije: Stanična linija humanog neuroblastoma SH-SY5Y diferencirana je 10 dana retinoičnom kiselinom i tretirana 48 sati s dvije izoforme P4 inhibitora sinteze gangliozida, miglustat inhibitorom sinteze gangliozida i inhibitorom razgradnje glikolipida - conduritol B epoksidom. Materijali i metode: Za MTT stanice su uzgajane, diferencirane i tretirane u pločicama s 96 jažica presvučenih kolagenom. Nakon tretmana utvrđena je održivost stanica. Stanice su uzgajane i diferencirane na pokrovnim staklima presvučenim kolagenom za imunocitokemiju i morfologiju i obojane specifičnim antitijelima za epitope od interesa te FITC bojom za morfologiju. Za Aneksin V, western blot i MALDI-TOF analize, stanice su uzgajane u šest bioloških replika za svaku koncentraciju u pločicama sa 6 jažica, nakon što su tretirane stanice sastrugane, homogenirane i pripremljene za daljnju analizu protočnom citometrijom, western blotom ili MALDI- TOF MS. Rezultati: ometanje biosinteze glikolipida uzrokuje o dozi ovisnu staničnu smrt i promjenu morfologije. Miglustat je uzrokovao očekivane morfološke promjene u obliku produljenja neurita, dok je CBE u sufektivnim koncentracijama imao reverzni učinak na staničnu morfologiju. Put signalizacije inzulina bio je pojačan u svim tretmanima. Stanice su pokazale promjene u neuroplastičnosti i različit odgovor na inducirani metabolički stres, ovisno o tome koji je inhibitor primijenjen. Zaključak: Ometanje sastava glikolipida ometat će inzulinsku signaliziraciju, uzrokovati morfološke promjene i poremetiti neuroplastičnost stanica. |
| Abstract (english) | Objectives: Gangliosides are essential for stabilizing and organization of the membrane microdomains, which serve as communication hubs for relaying insulin signaling into the cell. Interfering with ganglioside synthesis should disrupt insulin signaling by downregulation or upregulation, depending on which enzyme is disrupted. This, in turn, should cause changes in cell neuroplasticity, morphology, and tolerance to insulin challenge. Study design: SH-SY5Y human neuroblastoma cell line was differentiated for 10 days with retinoic acid and treated for 48 hours with two isoforms of P4 inhibitor of ganglioside synthesis, miglustat inhibitor of ganglioside synthesis, and inhibitor of glycolipid degradation – conduritol B epoxide. Material and methods: For MTT, cells were grown, differentiated, and treated in collagen-coated 96 healthy plates. After treatment, cell viability was determined. Cells were grown and differentiated on collagen-coated glass slides for immunocytochemistry and morphology and stained with specific antibodies for epitopes of interest, and FITC dye for morphology. For Annexin V, Western blots, and MALDI-TOF, cells were grown in six biological replicas for each treatment concentration and protocol in 6-well plates. After treatment cells were scraped, homogenated, and prepared for further analysis by flow cytometry, Western blot, or MALDI-TOF MS. Results: Interfering with glycolipid biosynthesis caused dose-dependent cell death and change in morphology. Miglustat caused expected morphology changes in the form of neurite elongation, whilst CBE in sub-effective concentrations had the reverse effect on cell morphology. The insulin signaling pathway was upregulated with all treatments. Cells demonstrated changes in neuroplasticity and different responses to induced metabolic stress depending on which inhibitor was applied. Conclusion: Disrupting glycolipid composition will interfere with insulting signaling, cause morphological changes, and disrupt the neuroplasticity of the cells. |
