20.500.12556/RUL-184016 Vpliv lizosomskih zaviralcev na izražanje piruvat dehidrogenaza kinaze 1 v kulturi podganjih skeletnomišičnih celic L6 The effect of lysosomal inhibitors on pyruvate dehydrogenase kinase 1 expression in cultured L6 rat skeletal muscle cells Piruvat dehidrogenaza kinaza 1 (PDK1) uravnava piruvat dehidrogenazni kompleks in s tem presnovo glukoze, motnje v uravnavanju pa so povezane z različnimi boleznimi. Dikloroacetat (DCA) zavira aktivnost PDK1 in hkrati zmanjša njegovo izražanje. Lizosom je odgovoren za razgradnjo znotrajceličnega materiala, ki do lizosoma pride z avtofagijo. Lizosomsko razgradnjo prek avtofagije lahko preprečimo z zaviranjem zlitja avtofagosoma in lizosoma, z zvišanjem pH v svetlini lizosoma ali z zaviranjem lizosomskih encimov. V nalogi smo preučili vpliv lizosomskih zaviralcev na razgradnjo piruvat dehidrogenaza kinaze 1 v kulturi podganjih skeletnomišičnih celic L6. Namen naloge smo strnili v tri hipoteze: 1) klorokin, amonijev klorid in leupeptin povečajo raven PDK1 v skeletnomišičnih celicah L6; 2) klorokin, amonijev klorid in leupeptin v kombinaciji bolj učinkovito povečajo raven PDK1 v skeletnomišičnih celicah L6 kot posamezni zaviralci; 3) amonijev klorid in leupeptin zmanjšata zaviralni učinek DCA na izražanje in delovanje PDK1 v skeletnomišičnih celicah L6. Poskuse smo izvedli na kulturi podganjih skeletnomišičnih celic L6. Izražanje izbranih proteinov smo določili z metodo prenos western in rezultate statistično ovrednotili. Amonijev klorid in leupeptin sta povišala raven PDK1, medtem ko klorokin ni povzročil spremembe. Kombinaciji lizosomskih zaviralcev nista pokazali večjega učinka na izražanje PDK1 kot posamezni zaviralci. DCA je zavrl aktivnost PDK1, njegova souporaba z amonijevim kloridom in leupeptinom pa je zmanjšala raven PDK1. Zaviranje lizosoma smo potrdili z zvišanjem ravni označevalcev avtofagije lahke verige 3 z mikrotubuli povezanega proteina (LC3B II) in sekvestosoma-1 (SQSTM1/p62), ki sta ga povzročila amonijev klorid in leupeptin, delovanje DCA pa z zmanjšanjem fosforilirane α1 podenote piruvat dehidrogenaze. Rezultati delno podpirajo prvo hipotezo o lizosomski razgradnji, medtem ko druge in tretje hipoteze ne podpirajo. Pyruvate dehydrogenase kinase 1 (PDK1) regulates the pyruvate dehydrogenase complex and thus glucose metabolism, its dysregulation is linked to various diseases. Dichloroacetate (DCA) inhibits PDK1 activity and simultaneously lowers its expression. The lysosome is responsible for degradation of intracellular material delivered through autophagy. Lysosomal degradation via autophagy can be prevented by inhibiting autophagosome-lysosome fusion, raising lysosomal pH, or inhibiting lysosomal enzymes. We investigated the effect of lysosomal inhibitors on PDK1 degradation in cultured L6 rat skeletal muscle cells. We summarized the aim in three hypotheses: 1) chloroquine, ammonium chloride and leupeptin increase PDK1 levels in cultured L6 rat skeletal muscle cells; 2) in combination chloroquine, ammonium chloride and leupeptin increase PDK1 levels more effectively than individual inhibitors in cultured L6 rat skeletal muscle cells; 3) ammonium chloride and leupeptin reduce the inhibitory effect of DCA on PDK1 expression and activity in cultured L6 rat skeletal muscle cells. Experiments were performed on cultured L6 rat skeletal muscle cells. The expression of selected proteins was determined by the western blot method, followed by statistical evaluation of results. Ammonium chloride and leupeptin increased PDK1 levels, while chloroquine had no effect. Combinations of lysosomal inhibitors did not show greater effects on PDK1 levels than individual inhibitors. DCA inhibited PDK1 activity, while co-treatment with ammonium chloride or leupeptin reduced PDK1 levels. Lysosomal inhibition was confirmed by elevated levels of autophagy markers microtubule-associated protein light chain 3 (LC3B II) and sequestosome-1 (SQSTM1/p62), induced by ammonium chloride and leupeptin, while DCA activity was confirmed by reduced phosphorylation of the pyruvate dehydrogenase α1 subunit. The results partially support the first hypothesis of lysosomal PDK1 degradation, but not the second or third hypotheses. piruvat dehidrogenaza kinaza 1 avtofagija lizosom skeletnomišične celice dikloroacetat pyruvate dehydrogenase kinase 1 autophagy lysosome skeletal muscle cells dichloroacetate true false false [C. Prevodnik] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-06-24 07:45:07 2026-06-24 07:45:20 2026-07-07 15:21:46 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 616-074(043.2) 125557 282658307 32305.pdf 32305.pdf 1 98B64A7DB0FCC62347862AA2FDCE8C5B 62fe6cbfbb5f175fbfde3005286a562d088708443148e24c6f57906993a20fde cde2b202-6f8f-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=236460 Fakulteta za farmacijo 0 0 0