N6-trimetillizīna dioksigenāzes nozīme muskuļu enerģijas metabolismā un funkcijā.
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Date
2020
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Rīgas Stradiņa universitāte
Rīga Stradiņš University
Rīga Stradiņš University
Abstract
Karnitīns ir būtiska molekula, kas regulē šūnu enerģijas metabolismu muskuļu šūnās. Ir pierādīts, ka karnitīna deficīts, ko izraisa galvenokārt karnitīna transporta traucējumi, veicina muskuļu vājuma un miopātiju attīstību. Pētījuma mērķis bija noskaidrot karnitīna biosintēzes ceļa enzīma N6-trimetillizīna dioksiogenāzes (TMLD) ietekmi uz muskuļu darbību un mitohondriju funkciju, izmantojot TMLD-izgriezta gēna peles.
TMLD -/- peļu modelis tika izstrādāts sadarbība ar Tartu Universitāti. Karnitīna daudzumu muskuļos noteica, izmantojot UPLC/MS/MS metodi. No peles muskuļu audiem tika sagatavotas permeabilizētās šķiedras, un mitohondriju funkciju noteikšanu veica, izmantojot augstas izšķirtspējas respirometriju (Oxygraph-O2k, Oroboros Instruments). Lai noteiktu no substrātiem (LEAK stāvoklis) un no oksidatīvās fosforilēšanas atkarīgo (OXPHOS stāvoklis) elpošanu, tika izmantoti dažādi substrāti un inhibitori. Muskuļu veiktspēju novērtēja, izmantojot saķeres spēka pārbaudi un skrejceliņa izsīkuma testu.
Karnitīna daudzums TMLD -/- peļu muskuļu audos bija 5 reizes mazāks, salīdzinot ar kontroles grupas dzīvnieku audiem. Saķeres spēka un skrejceļš izsīkuma testi parādīja, ka muskuļu veiktspēja neatšķiras starp kontroles un TMLD -/- pelēm. TMLD gēna izgriešana izraisīja LEAK elpošanas palielināšanos ar piruvātu un malātu par 27% un ar kompleksu I un I&II saistīto elpošanu OXPHOS stāvoklī par aptuveni 30%. Turklāt, TMLD -/- pelēm elpošanas ātrums, izmantojot palmitoilkarnitīnu kā taukskābju oksidācijas substrātu, bija palielināts apmēram 2 reizes LEAK stāvoklī un par 35% OXPHOS stāvoklī, salīdzinot ar kontroles grupu.
Pētījumu rezultāti parāda, ka karnitīna trūkums, ko izraisa TMLD gēna izgriešana, neietekmē muskuļu darbību. Turklāt karnitīna biosintēzes ceļa pārtraukšana uzlabo mitohondriju bioenerģētiku muskuļu audos.
Carnitine is an essential molecule for regulating cellular energy metabolism in muscle cells. It has been shown that carnitine deficiency, mainly caused by disruption in carnitine transport, leads to muscle weakness and myopathy. The aim of the study was to investigate the effect of the deletion of the N6-trimethyllysine dioxygenase (TMLD), the enzyme of carnitine biosynthesis pathway, on muscle performance and mitochondrial function, using TMLD knock-out mice. TMLD-/- mice model was developed in collaboration with Tartu University. Carnitine content in muscle was measured using UPLC/MS/MS. Permeabilized fibers were prepared from mice muscle tissues and mitochondrial respiration measurements were performed using high- resolution respirometry (Oxygraph-O2k, Oroboros Instruments). Different substrates and inhibitors were used to determine substrate-dependent (LEAK state) and oxidative phosphorylation-dependent (OXPHOS state) respiration. Muscle performance was evaluated using grip-strength test and treadmill-exhaustion test. The carnitine content in muscle tissue of TMLD-/- mice was decreased 5 times compared to wild type group. Grip-strength and treadmill exhaustion tests showed that there is no difference in muscle performance between wild type and TMLD-/- animals. Deletion of the TMLD gene caused an increase in LEAK respiration with pyruvate and malate by 27% and in complex I- and complex I&II-linked respiration at OXPHOS state by about 30%. Moreover, in TMLD-/- mice, the respiration rate using palmitoylcarnitine as fatty acid oxidation substrate was increased by about 2 times at LEAK state and by 35% at OXPHOS state compared to wild type control. Our results demonstrate that carnitine deficiency induced by deletion of TMLD gene does not affect muscle performance. Moreover, the disruption of carnitine biosynthesis pathway improves mitochondrial bioenergetics in muscle tissue.
Carnitine is an essential molecule for regulating cellular energy metabolism in muscle cells. It has been shown that carnitine deficiency, mainly caused by disruption in carnitine transport, leads to muscle weakness and myopathy. The aim of the study was to investigate the effect of the deletion of the N6-trimethyllysine dioxygenase (TMLD), the enzyme of carnitine biosynthesis pathway, on muscle performance and mitochondrial function, using TMLD knock-out mice. TMLD-/- mice model was developed in collaboration with Tartu University. Carnitine content in muscle was measured using UPLC/MS/MS. Permeabilized fibers were prepared from mice muscle tissues and mitochondrial respiration measurements were performed using high- resolution respirometry (Oxygraph-O2k, Oroboros Instruments). Different substrates and inhibitors were used to determine substrate-dependent (LEAK state) and oxidative phosphorylation-dependent (OXPHOS state) respiration. Muscle performance was evaluated using grip-strength test and treadmill-exhaustion test. The carnitine content in muscle tissue of TMLD-/- mice was decreased 5 times compared to wild type group. Grip-strength and treadmill exhaustion tests showed that there is no difference in muscle performance between wild type and TMLD-/- animals. Deletion of the TMLD gene caused an increase in LEAK respiration with pyruvate and malate by 27% and in complex I- and complex I&II-linked respiration at OXPHOS state by about 30%. Moreover, in TMLD-/- mice, the respiration rate using palmitoylcarnitine as fatty acid oxidation substrate was increased by about 2 times at LEAK state and by 35% at OXPHOS state compared to wild type control. Our results demonstrate that carnitine deficiency induced by deletion of TMLD gene does not affect muscle performance. Moreover, the disruption of carnitine biosynthesis pathway improves mitochondrial bioenergetics in muscle tissue.
Description
Farmācija
Pharmacy
Veselības aprūpe
Health Care
Pharmacy
Veselības aprūpe
Health Care
Keywords
N6-trimetillizīna dioksigenāze, enerģijas metabolisms, mitochondriji, muskuļu funkcija, karnitīna trūkums., N6-trimethyllysine dioxygenase, energy metabolism, mitochondria, muscle performance, carnitine deficiency.