Talk:Pavlovic 2022 Abstract Bioblast

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Comments by the Reviewer

Moreno-Sanchez Rafael 2022-05-31

  • This abstract describes work that analyzes a highly important issue with clinical relevance. The elucidation of the biochemical mechanisms involved in the hypoglucemia-induced drug Metformin is certainly needed for improving clinical protocols of dosing for diabetic patients. Metformin has also been proposed as an anti-cancer drug. A few observations are listed below intended to gain strength in delivering the message.
  • It is stated that metformin treatment decreased the ROUTINE respiration and the “OXPHOS state” (the authors may be referring to the OXPHOS capacity), and increased “depolarization of the mitochondrial inner membrane” in mouse skeletal muscle cells. These are interesting results, but the results on ET capacity, LEAK respiration and residual O2 consumption should be also disclosed, as they may have key mechanistic clues.
  • The metformin effects on ROUTINE respiration and OXPHOS capacity “were more pronounced in the low glucose medium”. An effort should be made to elaborate a mechanistic explanation for this observation. Did gene transcription change when cells were cultured in low glucose vs. high glucose? Was the phospholipid membrane composition altered, in which a higher membrane permeability developed as a result of increased unsaturated fatty acid content and decreased cardiolipin level (this phospholipid regulates activity of several respiratory complexes and ATP/ADP translocase)?
  • Erase the sentence in the 3rd paragraph “5 mM metformin…”, because it repeats information stated in the preceding text.

Version 1

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 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. 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.