Pavlovic 2022 Abstract Bioblast

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P15.
Pavlovic Kasja
Pavlovic Kasja, Krako Jakovljevic N, Isakovic AM, Ivanovic T, Markovic I, Lalic NM (2022) Effects of metformin on mitochondrial function in skeletal muscle cells: differences between therapeutic and suprapharmacological concentrations. Bioblast 2022: BEC Inaugural Conference. In: https://doi.org/10.26124/bec:2022-0001

Link: Bioblast 2022: BEC Inaugural Conference

Pavlovic Kasja, Krako Jakovljevic Nina, Isakovic AM, Ivanovic Tijana, Markovic Ivanka, Lalic Nebojsa M (2022)

Event: Bioblast 2022

Metformin is an oral antidiabetic drug that has been widely used in clinical practice for over 60 years. Despite of this, the molecular mechanisms of metformin action are still not completely understood. Although metformin-induced inhibition of mitochondrial respiratory Complex I has been observed in many studies, published data is inconsistent. Furthermore, metformin concentrations used for in vitro studies and their pharmacological relevance are a continuing topic of debate, as concentrations required to cause mitochondrial Complex I inhibition are significantly higher than the plasma concentrations detected in patients on oral therapy [1]. The aim of this study was to explore the effects of therapeutic metformin concentrations on mitochondrial function in muscle cells in vitro, and compare the effects with those of higher concentrations, that have already been established to affect mitochondrial function. We conducted all experiments in conditions of high and low glucose, in order to evaluate the role of glucose availability on metformin action.

C2C12 mouse skeletal muscle cells were cultured in either high (25 mM) or low glucose DMEM (5.5 mM). Mitochondrial respiration was measured by high-resolution respirometry (Oroboros O2k) while total ROS, superoxide production and mitochondrial membrane potential were measured by flow cytometry (FACS Calibur).

Mitochondrial respiration was measured in permeabilized cells treated with growing concentrations of metformin for 24 h. ROUTINE respiration decreased only in cells treated with the highest concentration (5 mM), while OXPHOS capacity (N-pathway) decreased both in cells treated with 1 mM and 5 mM metformin. Cells cultured in low glucose medium were more sensitive to metformin treatment – untreated cells had significantly higher OXPHOS capacity than cells grown in high glucose medium, and when treated with 5 mM metformin the decrease in respiration was more pronounced (68 % for high glucose and 85 % for low glucose). No differences were observed in LEAK respiration, OXPHOS capacity (S-pathway) or residual oxygen consumption (Rox). Measuring respiration of living cells, we observed a decrease in ROUTINE and LEAK respiration and ET capacity in cells treated with 5 mM (but not 50 µM) metformin. We observed no changes in mitochondrial respiration of differentiated and undifferentiated cells treated with 50 µM metformin for 5 days, in any of the respiratory states or either cell culture medium. There was no difference between citrate synthase activity of untreated and cells treated with metformin for 5 days. 5 mM metformin increased total ROS (DHR) and superoxide (DHE) production, which was more pronounced for high glucose compared to low glucose-cultured cells. 5 mM metformin caused depolarization of the mitochondrial inner membrane in both media, the effect being more pronounced in low glucose medium-grown cells. According to our results, micromolar, therapeutic metformin treatment did not cause changes in mitochondrial respiration, ROS production or mitochondrial membrane potential. OXPHOS capacity was higher in untreated low glucose, compared to high glucose medium-cultured cells, which can be explained by the Crabtree effect [2], and was previously shown for C2C12 cells [3]. Higher sensitivity of low glucose-cultured cells to metformin treatment could be a consequence of circumventing the Crabtree effect, by lowering the glucose concentration in cell medium [4]. In conclusion, while suprapharmacological metformin concentrations cause Complex I inhibition in skeletal muscle cells in vitro, therapeutic concentrations cause no such effect in these cells. This suggests the need to further clarify the mechanisms that are relevant for therapeutic effects of metformin in skeletal muscle.

  1. Vial G, Detaille D, Guigas B (2019) Role of mitochondria in the mechanism(s) of action of metformin. https://doi.org/10.3389/fendo.2019.00294
  2. de Kok M, Schaapherder AF, Wüst R, Zuiderwijk M, Bakker JA, Lindeman J, Le Dévédec SE (2021) Circumventing the Crabtree effect in cell culture: A systematic review. https://doi.org/10.1016/j.mito.2021.03.014
  3. Elkalaf M, Anděl M, Trnka J (2013) Low glucose but not galactose enhances oxidative mitochondrial metabolism in C2C12 myoblasts and myotubes. https://doi.org/10.1371/journal.pone.0070772
  4. Marroquin LD, Hynes J, Dykens JA, Jamieson JD, Will Y (2007) Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of hepG2 cells to mitochondrial toxicants. https://doi.org/10.1093/toxsci/kfm052

Keywords: metformin, concentration, glucose, Complex I, muscle

O2k-Network Lab: RS Belgrade Lalic NM


Affiliations

Pavlovic K1, Krako Jakovljevic N1, Isakovic AM2, Ivanovic T1,3, Markovic I2, Lalic NM1,3
  1. Clinic for Endocrinology, Diabetes and Metabolic Diseases, University Clinical Center of Serbia – kasja.pavlovic@med.bg.ac.rs
  2. Inst of Medical and Clinical Biochemistry, Faculty of Medicine, Univ of Belgrade
  3. Faculty of Medicine, Univ of Belgrade

Discussion

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