20.500.12556/RUL-189328 Vpliv inzulina in proinzulina na aktivnost signalne poti protein kinaze Akt v kulturi človeških in podganjih skeletnomišičnih celic The effect of insulin and proinsulin on the activity of the protein kinase Akt signaling pathway in culture of human and rat skeletal muscle cells Uvod: Inzulin je polipeptidni hormon, sestavljen iz 51 aminokislin. Z aktivacijo signalne poti PI3K/Akt uravnava presnovo glukoze v tarčnih tkivih. Eno izmed ključnih tkiv so skeletne mišice, v katerih inzulin spodbuja privzem glukoze in tvorbo glikogena. Prekurzor inzulina je proinzulin, ki se prav tako lahko veže na inzulinski receptor, vendar ima zaradi nižje afinitete bistveno manjšo biološko aktivnost kot zreli inzulin. Za preučevanje inzulinske signalne poti se pogosto uporabljajo celični modeli skeletnih mišic, med katerimi sta uveljavljeni podganja celična linija L6 ter človeške skeletnomišične celice HSMC. Zaradi možnih medvrstnih razlik je pomembno ovrednotiti primerljivost njunega odziva na inzulin. Namen: Raziskati vpliv humanega inzulina in podganjega proinzulina na aktivnost signalne poti protein kinaze Akt v kulturi človeških in podganjih skeletnomišičnih celic ter primerjati odzivnost obeh celičnih modelov. Hipoteze: I) Humani inzulin je pri aktivaciji signalne poti Akt enako učinkovit v kulturah človeških in podganjih skeletnomišičnih celic. II) Podganji proinzulin spodbuja aktivnost signalne poti Akt v kulturi podganjih in človeških skeletnomišičnih celic. III) Podganji proinzulin zavira s humanim inzulinom sproženo aktivacijo signalne poti Akt v kulturi podganjih in človeških skeletnomišičnih celic. Metode: Poskuse smo opravili na diferenciranih kulturah podganjih in človeških skeletnomišičnih celic. Celice smo tretirali z različnimi koncentracijami humanega inzulina in podganjega proinzulina. Aktivacijo signalne poti Akt smo spremljali s pomočjo prenosa Western, z določanjem stopnje fosforilacije signalnih molekul Akt (Ser473), AS160 (Thr642) in S6RP (Ser235/236). Rezultati: Odziv kultur L6 in HSMC na humani inzulin je bil kvalitativno primerljiv, pri molekulah Akt in AS160 ni bilo statistično značilnih razlik, te pa so se pojavile pri nekaterih koncentracijah pri molekuli S6RP. Kvantitativna sprememba v fosforilaciji Akt in AS160 pod vplivom inzulina je bila večja v celicah L6 kot HSMC. Podganji proinzulin sam po sebi ni povzročil statistično značilnega povečanja aktivnosti signalne poti Akt v nobeni od celičnih kultur. V kulturi L6 je dodatek podganjega proinzulina značilno zmanjšal stopnjo fosforilacije, ki jo je sprožil humani inzulin, kar nakazuje na zaviralni učinek. V kulturi HSMC dodatek proinzulina humanemu inzulinu ni imel takšnega učinka. Zaključki: Enake učinkovitosti delovanja humanega inzulina v obeh celičnih kulturah na podlagi analiz nismo mogli v celoti potrditi. Podganji proinzulin sam po sebi ni povečal aktivnosti signalne poti Akt, v kombinaciji s humanim inzulinom pa je le v kulturi L6 povzročil zaviranje njegovega učinka. Background: Insulin is a polypeptide hormone composed of 51 amino acids. By activating the PI3K/Akt signaling pathway, it regulates glucose metabolism in target tissues. One of the key target tissues is skeletal muscle, where insulin promotes glucose uptake and glycogen synthesis. The precursor of insulin is proinsulin, which can also bind to the insulin receptor but has substantially lower biological activity than mature insulin due to its lower affinity. Skeletal muscle cell models are commonly used to study insulin signaling, among which the rat L6 cell line and human skeletal muscle cells (HSMC) are well-established models. Due to potential interspecies differences, it is important to evaluate the comparability of their responses to insulin. Aim: The aim of this study was to investigate the effects of human insulin and rat proinsulin on the activity of the protein kinase Akt signaling pathway in cultures of human and rat skeletal muscle cells, and to compare the responsiveness of the two cell models. Hypotheses: I) Human insulin is equally effective in activating the Akt signaling pathway in human and rat skeletal muscle cell cultures.II) Rat proinsulin stimulates the activity of the Akt signaling pathway in human and rat skeletal muscle cell cultures.III) Rat proinsulin inhibits human insulin-induced activation of the Akt signaling pathway in human and rat skeletal muscle cell cultures. Methods: Experiments were performed on differentiated cultures of rat and human skeletal muscle cells. Cells were treated with different concentrations of human insulin and rat proinsulin. Activation of the Akt signaling pathway was monitored by Western blot analysis, assessing the phosphorylation levels of the signaling molecules Akt (Ser473), AS160 (Thr642) and S6RP (Ser235/236). Results: The responses of L6 and HSMC cultures to human insulin were qualitatively comparable. No statistically significant differences were observed for Akt and AS160, whereas statistically significant differences occurred at some concentrations for S6RP. The quantitative increase in Akt and AS160 phosphorylation induced by insulin was greater in L6 cells than in HSMC. Rat proinsulin alone did not cause a statistically significant increase in Akt signaling pathway activity in either cell culture. In L6 cultures, the addition of rat proinsulin significantly reduced the level of phosphorylation induced by human insulin, suggesting an inhibitory effect. In HSMC cultures, the addition of proinsulin to human insulin did not have such an effect. Conclusions: Equivalent efficacy of human insulin in the two cell cultures could not be fully confirmed based on the analyses performed. Rat proinsulin alone did not increase Akt signaling pathway activity, whereas in combination with human insulin, it inhibited the insulin-induced effect only in the L6 culture. inzulin proinzulin celične kulture skeletnomišične celice medvrstna primerjava signalna pot Akt. insulin proinsulin cell cultures skeletal muscle cells interspecies comparison Akt signaling pathway true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-10-03 08:45:09 2026-10-03 08:45:20 2026-10-04 04:15:34 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 133323 35027.pdf 35027.pdf 1 3B01F02B251329806E11096478D350EE a4d763a5b8b39aa57e1e9aa42e3889e318e884c7443a332b3d496704b1ae3dd4 e169e328-bef5-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=249800 Fakulteta za farmacijo 0 0 0