Effects of metformin on energy status and mitochondrial function of muscle cells in vitro

[K. Pavlović]

104, [16] str.

medicina - molekularna medicina / medicine - molecular medicine

Metformin je oralni antidijabetik i lek prvog izbora u terapiji dijabetesa tipa 2. Iako se koristi duže od 60 godina, molekularni mehanizmi dejstva metformina nisu u potpunosti razjašnjeni. Istraživanja pokazuju da metformin dovodi do inhibicije kompleksa I respiratornog lanca i aktivacije AMP-om aktivirane protein kinaze (AMPK), međutim koncentracije metformina korišćene u ovim studijama su sporne, baš kao i njihova farmakološka relevantnost. Cilj ove studije bio je da ispita efekte različitih koncentracija metformina na energetski metabolizam i aktivnost najznačajnijih signalnih puteva, u mišićnim ćelijama in vitro. Svi eksperimenti rađeni su na ćelijskoj liniji C2C12, a izvođeni su u uslovima visoke i niske koncentracije glukoze. Ćelije su tretirane metforminom terapijske (50 μM) ili suprafarmakološke (5 mM) koncentracije. Mitohondrijska funkcija merena je respirometrijom visoke rezolucije, a produkcija reaktivnih kiseoničnih vrsta i mitohondrijski membranski potencijal protočnom citofluorimetrijom. Aktivnost signalnih puteva AMPK i PI3K/Akt merena je kvantifikacijom fosforilisanih formi ispitivanih proteina imunoblotom. Metformin suprafarmakološke koncentracije inhibirao je mitohondrijsku funkciju intaktnih i permeabilizovanih ćelija, delovanjem na kompleks I respiratornog lanca, dok terapijska koncentracija metformina nije uticala na respiraciju. Ni jedna od ispitivanih koncentracija metformina nije dovela do promene energetskog i redoks statusa ćelija. Suprafarmakološka, ali ne i terapijska koncentracija metformina, dovela je do depolarizacije unutrašnje membrane mitohondrija, povećanja produkcije reaktivnih kiseoničnih vrsta i aktivacije signalnih puteva AMPK i PI3K/Akt. Zaključujemo da terapijska koncentracija metformina u mišićnim ćelijama ne dovodi do efekata na energetski metabolizam koji su pokazani korišćenjem suprafarmakoloških koncentracija.

Metformin is an oral antidiabetic agent and first-line drug in type 2 diabetes treatment. Even though it has been used for over 60 years, the molecular mechanisms of metformin action are not completely understood. Metformin-induced inhibition of mitochondrial respiratory chain complex I and activation of AMP-activated protein kinase (AMPK) have been confirmed by studies, but metformin concentrations used in these studies and their pharmacological relevance are a common point of debate. The aim of this study was to explore the effects of different metformin concentrations on energy metabolism and activity of relevant signaling pathways in C2C12 muscle cells in vitro. All experiments were performed on C2C12 cell line, and conducted in conditions of high and low glucose concentration. Cells were treated with therapeutic (50 μM) or suprapharmacological (5 mM) metformin concentrations. Mitochondrial function was measured by high-resolution respirometry, ROS production and mitochondrial membrane potential by flow cytometry. AMPK and PI3K/Akt signalling pathway activity was studied by quantification of phosphorylated forms of proteins of interest. Suprapharmacological metformin concentration caused an inhibition of mitchondrial function of intact and permeabilised cells by affecting complex I, while therapeutic metformin concentration had no such effect. None of the studied treatment concentrations affected energy or redox status of the cells. Suprapharmacological, but not therapeutic metformin concentration, lead to mitochondrial inner membrane depolarisation, increased ROS production and AMPK and PI3K/Akt signalling pathway activation. In conclusion, therapeutic metformin treatment does not affect energy metabolism in muscle cells in the manner which was shown using suprapharmacological metformin concentrations.

metformin, mitohondrije, respiratorni lanac, AMPK, PI3K/Akt, glukoza

metformin, mitochondria, respiratory chain, AMPK, PI3K/Akt, glucose

612:577.1(043.3)

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