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Difference between revisions of "Pavlovic 2022 Abstract Bioblast"

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|year=2022
|year=2022
|event=[[Bioblast 2022]]
|event=[[Bioblast 2022]]
|abstract=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 chain 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 constant point of debate, as concentrations required to cause mitochondrial Complex I inhibition are significantly higher than the plasma concentrations detected in patients on oral therapy. The aim of this study was to explore the effects of therapeutic metformin concenctrations 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.
|abstract=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 constant point of debate, as concentrations required to cause mitochondrial Complex I inhibition are significantly higher than the plasma concentrations detected in patients on oral therapy. The aim of this study was to explore the effects of therapeutic metformin concenctrations 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 (4.5 mM). Mitochondrial respiration was measured by high-resolution respirometry (Oroboros O2k) while total ROS, superoxide production and mitochondrial membrane depolarization were measured by flow cytometry (FACS Calibur).
C2C12 mouse skeletal muscle cells were cultured in either high (25 mM) or low glucose DMEM (4.5 mM). Mitochondrial respiration was measured by high-resolution respirometry (Oroboros O2k) while total ROS, superoxide production and mitochondrial membrane depolarization were measured by flow cytometry (FACS Calibur).


Mitochondrial respiration of permeabilized cells treated with metformin for 24 h was decreased in ROUTINE state, only in cells treated with the highest concentration (5 mM), while in OXPHOS state (N-pathway) a decrease was observed both in cells treated with 1 mM and 5 mM metformin. This was observed in both cell culture media, but the decrease was more pronounced in low glucose medium. We observed no changes in mitochondrial respiration of differentiated and undifferentiated cells treated with 50 Β΅M 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. Superoxide production was increased by 5 mM metformin treatment in both cell media, which was more pronounced for high glucose compared to low glucose-cultured cells. 5 mM metformin treatment also increased ROS production in high glucose medium-grown cells. 5 mM metformin caused depolarization of inner mitochondrial membrane in both media, the effect being more pronounced in low glucose medium-grown cells.
Mitochondrial respiration of permeabilized cells treated with metformin for 24 h was decreased in the ROUTINE state, only in cells treated with the highest concentration (5 mM), while in the OXPHOS state (N-pathway) a decrease was observed both in cells treated with 1 mM and 5 mM metformin. This was observed in both cell culture media, but the decrease was more pronounced in low glucose medium. We observed no changes in mitochondrial respiration of differentiated and undifferentiated cells treated with 50 Β΅M 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. Superoxide production was increased by 5 mM metformin treatment in both cell media, which was more pronounced for high glucose compared to low glucose-cultured cells. 5 mM metformin treatment also increased ROS production in high glucose medium-grown 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. 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.
According to our results, micromolar, therapeutic metformin treatment did not cause changes in mitochondrial respiration, ROS production or mitochondrial membrane potential. 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.

Revision as of 23:53, 23 May 2022

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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.

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 constant point of debate, as concentrations required to cause mitochondrial Complex I inhibition are significantly higher than the plasma concentrations detected in patients on oral therapy. The aim of this study was to explore the effects of therapeutic metformin concenctrations 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 (4.5 mM). Mitochondrial respiration was measured by high-resolution respirometry (Oroboros O2k) while total ROS, superoxide production and mitochondrial membrane depolarization were measured by flow cytometry (FACS Calibur).

Mitochondrial respiration of permeabilized cells treated with metformin for 24 h was decreased in the ROUTINE state, only in cells treated with the highest concentration (5 mM), while in the OXPHOS state (N-pathway) a decrease was observed both in cells treated with 1 mM and 5 mM metformin. This was observed in both cell culture media, but the decrease was more pronounced in low glucose medium. We observed no changes in mitochondrial respiration of differentiated and undifferentiated cells treated with 50 Β΅M 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. Superoxide production was increased by 5 mM metformin treatment in both cell media, which was more pronounced for high glucose compared to low glucose-cultured cells. 5 mM metformin treatment also increased ROS production in high glucose medium-grown 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. 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.

β€’ Keywords: metformin, concentration, glucose, Complex I, respiration

β€’ 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 – [email protected]
  2. Inst of Medical and Clinical Biochemistry, Faculty of Medicine, Univ of Belgrade
  3. Faculty of Medicine, Univ of Belgrade

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