Short-term exposure to a clinical dose of metformin increases skeletal muscle mitochondrial H2O2 emission and production in healthy, older adults: A randomized controlled trial

Abstract

Background and aims: Metformin is the most commonly prescribed medication to treat diabetes. Emerging evidence suggests that metformin could have off target effects that might help promote healthy muscle aging, but these effects have not been thoroughly studied in glucose tolerant older individuals. The purpose of this study was to investigate the short-term effects of metformin consumption on skeletal muscle mitochondrial bioenergetics in healthy older adults.

Methods: We obtained muscle biopsy samples from 16 healthy older adults previously naïve to metformin and treated with metformin (METF; 3F, 5M), or placebo (CON; 3F, 5M), for two weeks using a randomized and blinded study design. Samples were analyzed using high-resolution respirometry, immunofluorescence, and immunoblotting to assess muscle mitochondrial bioenergetics, satellite cell (SC) content, and associated protein markers.

Results: We found that metformin treatment did not alter maximal mitochondrial respiration rates in muscle compared to CON. In contrast, mitochondrial H2O2 emission and production were elevated in muscle samples from METF versus CON (METF emission: 2.59 ± 0.72 SE Fold, P = 0.04; METF production: 2.29 ± 0.53 SE Fold, P = 0.02). Furthermore, the change in H2O2 emission was positively correlated with the change in type 1 myofiber SC content and this was biased in METF participants (Pooled: R2 = 0.5816, P = 0.0006; METF: R2 = 0.674, P = 0.0125).

Conclusions: These findings suggest that acute exposure to metformin does not impact mitochondrial respiration in aged, glucose-tolerant muscle, but rather, influences mitochondrial-free radical and SC dynamics.

Clinical trial registration: NCT03107884, clinicaltrials.gov.

Keywords: Aging; Complex I respiration; Free radicals; Insulin sensitizers; Metabolism; Mitochondria; Muscle stem cells.

Conflict of interest statement

Declaration of conflict of interest

S.A.S. is a cofounder, consultant, and shareholder for Centaurus Therapeutics.

Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.

Myofiber mitochondrial bioenergetic response to short-term metformin treatment in healthy, older adults. Data are reported as the mean fold-change from PRE ± SE. Red depicts data for CON while blue depicts data points for METF. The dashed line on the Y-axis represents the PRE timepoint. Displayed in panel A: mitochondrial complex-specific, state 3, and fatty acid oxidation respiration (FAO) rates. Malate, pyruvate, ADP and succinate were utilized to simulate respiration. To inhibit mitochondrial complexes 1, 2, 3, and 4 (C1, C2, C3 and C4) rotenone, malonate, antimycin A, and potassium cyanide were utilized, respectively. FAO was assessed with palmitoyl-l-carnitine chloride, l-carnitine hydrochloride, malate, and ADP. Panel B: mitochondrial- H2O2 emission and production rates. To stimulate hydrogen peroxide emission and production, hydrogen peroxide oxidoreductase followed by succinate was administered. Carmustine and auranofin (inhibitors of reactive oxygen species buffers) were then added to maximize hydrogen peroxide emission and production. Panel C: citrate synthase activity of whole muscle. *Significant difference between groups, P<0.05.

Figure 2.

Skeletal muscle immunoblotting for A) oxidative stress (4-HNE), B) endogenous antioxidant (SOD2), C) AMPK signaling (ACC Ser79), and D) metformin transporter content (OCT3) in response to short-term metformin treatment in healthy, older adults. Data are reported as fold-change from PRE ± SE. Red depicts data for CON while blue depicts data for METF. The dashed line on the Y-axis represents the PRE timepoint. Representative blot image for each target protein can be found below each panel.

Figure 3.

Ex vivo myofiber mitochondrial bioenergetic response to incubation in therapeutic metformin levels. Data are reported as the raw values ± SE of fiber bundles collected at PRE, and not incubated with metformin (open squares), connected to the raw values of fiber bundles, also collected at PRE, that were incubated for one hour in 12 μM metformin (dark gray squares). panel A: mitochondrial complex-specific, state 3, and fatty acid oxidation respiration (FAO) rates. Malate, pyruvate, ADP and succinate were utilized to simulate respiration. To inhibit mitochondrial complexes 1, 2, 3, and 4 (C1, C2, C3 and C4) rotenone, malonate, antimycin A, and potassium cyanide were utilized, respectively. FAO was assessed with palmitoyl-l-carnitine chloride, l-carnitine hydrochloride, malate, and ADP. Panel B: mitochondrial- H2O2 emission and production rates. To stimulate hydrogen peroxide emission and production, hydrogen peroxide oxidoreductase followed by succinate was administered. Carmustine and auranofin (inhibitors of reactive oxygen species buffers) were then added to maximize hydrogen peroxide emission and production.

Figure 4.

Myofiber type-specific satellite cell content, size, and capillarity in response to short-term metformin treatment in healthy, older adults. Data are reported as the mean fold-change from PRE ± SE. Red depicts data for CON while blue depicts data for METF. The dashed line on the Y-axis represents the PRE timepoint. Displayed in panel A: satellite cell content; panel B: myofiber size; panel C: myofiber capillarity, defined as capillary-to-fiber perimeter exchange index (CFPE). Panel D: Representative image of the immunofluorescent staining for satellite cell content and capillarity in frozen skeletal muscle cross-sections. Top left panel: depicts a mosaic, merged image of the entire muscle cross-section, captured at 20x magnification. The yellow box in the top left panel represents the image area for the top right panel: depicting satellite cell content, with yellow arrows pointing at Pax7+/DAPI+ cells; bottom left panel: depicting muscle capillaries; and the bottom right panel depicting a close-up, merged image. Satellite cells are shown in green, myonuclei are shown in blue, capillaries are shown in red, and both type 1 myofibers and fiber borders are shown in gray.

Figure 5.

Correlation between changes in type 1 myofiber satellite cell content and changes in mitochondrial H2O2 emission rates in response to short-term metformin treatment in healthy, older adults. Data are reported as individual fold-change data points with type 1 myofiber satellite cell content displayed on the y-axis and H2O2 emission rates displayed on the x-axis. Red depicts data for CON while blue depicts data for METF. Pearson coefficient squared and p-values for both groups pooled, CON, and METF are reported on the figure, and lines of best fit for CON and METF are displayed.