Farmakološko uravnavanje fosforilacije z AMP aktivirane protein kinaze v kulturi skeletnomišičnih celic

Konjc, Anja (author), Pirkmajer, Sergej (mentor)

Z AMPK aktivirana protein kinaza (AMPK) je pomemben senzor energijskega stanja celice. V skeletni mišici se aktivira med telesno dejavnostjo in prispeva k uravnavanju presnovnih procesov. Ker aktivacija AMPK izboljša homeostazo glukoze in delovanja inzulina, je AMPK pomembna tarča za razvoj novih oblik zdravljenja metabolnih bolezni, med drugim tudi sladkorne bolezni tipa 2. Pri razvoju novih farmakoloških učinkovin z delovanjem v skeletni mišici so pomembni zanesljivi eksperimentalni modeli in vitro. Namen diplomskega dela je bil preveriti, kakšna je zanesljivost spremljanja farmakološke aktivacije AMPK v skeletnomišičnih celicah, v katerih območjih je signal linearen, in kako redčitve vzorca vplivajo na zanesljivost ocenjevanja aktivacije AMPK. Predvidevali smo, da je območje linearnosti signala za pAMPK in AMPK oziroma pACC in ACC pri različnih redčitvah različno ter, da bi z nanosom manjše mase proteinov lahko izboljšali zaznavanje z AICAR spodbujene aktivacije AMPK s prenosom western. Poskuse smo razdelili na dva dela, in sicer smo najprej testirali učinek AICAR in MTX na aktivacijo AMPK v podganjih skeletnomišičnih celicah celične linije L6, nato pa še zanesljivost spremljanja farmakološke aktivacije AICAR in MTX na aktivacijo AMPK pri različnih redčitvah vzorcev celičnih lizatov L6. Potrdili smo, da kombinacija AICAR in MTX statistično značilno (glede na kontrolo in tretma samo z AICAR ali z MTX) poveča fosforilacijo AMPK. Ugotovili smo tudi, da je bil signal linearen za vzorce pAMPK in pACC z 20-% do 80-% koncentracijo. Od 80-% koncentracije naprej se je strmina grafa zmanjšala in ni bila več v linearnem območju. Ugotovili smo tudi, da spremljanje farmakološke aktivacije AMPK s prenosom western lahko optimiziramo z nanosom manjših koncentracij proteina na gel (80 % običajne koncentracije proteina), saj tako dobimo boljše rezultate. Signal je tako bolj v linearnem območju, obenem pa porabimo manj vzorca.

• Language: Slovenian
• Keywords: AMPK, fosforilacija, AICAR, MTX, skeletna mišica
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-139825
• COBISS.SI-ID: 126798851
• Publication date in RUL: 07.09.2022

Secondary language

• Language: English
• Title: Pharmacological regulation of phosphorylation of AMP-activated protein kinase in cultured skeletal muscle cells

Abstract

AMP-activated protein kinase (AMPK) is an important sensor of the energy state of the cell. It is activated in skeletal muscle during physical activity and contributes to the regulation of metabolic processes. Since the activation of AMPK improves glucose homeostasis of insulin action, AMPK is an important therapeutic target for the development of new treatments for metabolic diseases, including type 2 diabetes. Development of new skeletal muscle pharmacological agents requires reliable in vitro experimental models. The aim of the thesis was to check the reliability of monitoring the pharmacological activation of AMPK in skeletal muscle cells, in which range the signal is linear and how the dilution of samples affects the reliability of the assessment of AMPK activation. We assumed that the range of linearity of the signal for pAMPK and AMPK, or pACC and ACC is different at different dilutions and that by applying a lower protein mass, we could improve the knowledge of AICAR-stimulated activation of AMPK by western blotting. We divided the experiments into two parts, so we first tested the action of AICAR and MTX on AMPK activation in rat skeletal muscle cells of the L6 cell line, and then the reliability of monitoring the pharmacological activation of AICAR and MTX on AMPK activation. We confirmed that the combination of AICAR and MTX significantly (compared to control and treatment with AICAR or MTX alone) increases AMPK phosphorylation. We also found that the signal was linear for pAMPK and pACC samples with concentration from 20 % to 80 %. From 80 % concentration onwards, the slope of the graph decreased and was no longer in the linear range. We have also established that the monitoring of pharmacological activation of AMPK by western blotting can be optimized by applying lower protein concentrations in the gel (80 % of the normal protein concentration), as this way we get better results. The signal is thus better in the linear range, while at the same time we use less sample.

• Keywords: AMPK, phosphorylation, AICAR, MTX, skeletal muscle