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. 2018 Apr 4;8(1):5646.
doi: 10.1038/s41598-018-24017-7.

Metformin adapts its cellular effects to bioenergetic status in a model of metabolic dysfunction

Christopher Auger  1 , Thibacg Sivayoganathan  2 , Abdikarim Abdullahi  2 , Alexandra Parousis  1 , Bo Wen Pang  2 , Marc G Jeschke  3   4
Affiliations

Metformin adapts its cellular effects to bioenergetic status in a model of metabolic dysfunction

Christopher Auger et al. Sci Rep. .

Abstract

Thermal injury induces a complex immunometabolic response, characterized by hyperglycemia, extensive inflammation and persistent hypermetabolism. It has been suggested that attenuation of the hypermetabolic response is beneficial for patient wellbeing. To that effect, metformin represents an attractive therapeutic agent, as its effects on glycemia, inflammation and bioenergetics can improve outcomes in burn patients. Therefore, we studied metformin and its effects on mitochondrial bioenergetics in a murine model of thermal injury. We set out to determine the impact of this agent on mitochondrial hypermetabolism (adult mice) and mitochondrial dysfunction (aged mice). Seahorse respirometry complimented by in-gel activity assays were used to elucidate metformin's cellular mechanism. We found that metformin exerts distinctly different effects, attenuating the hypermetabolic mitochondria of adult mice while significantly improving mitochondrial bioenergetics in the aged mice. Furthermore, we observed that these changes occur both with and without adenosine monophosphate kinase (AMPK) activation, respectively, and analyzed damage markers to provide further context for metformin's beneficial actions. We suggest that metformin has a dual role following trauma, acting via both AMPK-dependent and independent pathways depending on bioenergetic status. These findings help further our understanding of metformin's biomolecular effects and support the continued use of this drug in patients.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Timeline and study design. Adult (8 week old) and aged (50 week old) mice were divided into the following groups: control; control + metformin; burn; burn + metformin. Mice not receiving metformin (100 mg/kg) were given saline via intraperitoneal injection. TBSA: total body surface area.
Figure 2
Figure 2
Seahorse XF96 respirometry analysis of adult mice at day 7 post injury. (a) Respiration profiles of liver mitochondria from control mice (grey), burned mice (black) and burned mice with metformin treatment (100 mg/kg; green). (b) Production of reactive oxygen species as measured by oxidation of 2,7-dichlorodihydrofluorescein-diacetate (DCFDA) in isolated mitochondria given substrate (5 mM pyruvate, 3 mM malate, 4 mM ADP); λ = 495 nm, λ’ = 529 nm. Basal (c), state 3 (d) and state 3 u (e) respiration parameters in isolated mitochondria as measured via Seahorse XF96 extracellular flux assays. formula image: control (n = 5); ■: burn (n = 5); formula image: burn + metformin treatment (n = 6). Values are presented as mean ± standard deviation. *p ≤ 0.05.
Figure 3
Figure 3
In-gel activity assays for electron transport chain (ETC) complexes from adult mice. (a) Representative images of complexes I-IV and ATP synthase of the ETC, assayed following blue native polyacrylamide gel electrophoresis of liver mitochondrial homogenates to demonstrate the effect of metformin in injured adult animals. (b) Densitometric measurements of in-gel activity assay bands using ImageJ for Windows. (c) Bands selected from the gel following staining with Coomassie Blue G-250 served as a loading control. In-gel activity assays from panel a were performed in the absence of key reaction mixture substrates (d) or in the presence of inhibitors (e) to confirm band specificity. (f) Respiration profiles of liver mitochondria from adult control mice (grey; n = 9) and control mice given metformin (green; n = 9). formula image: control (n = 5); ■: burn (n = 5); formula image: burn + metformin treatment (n = 5). Values are presented as mean ± standard deviation. *p ≤ 0.05; **p ≤ 0.01.
Figure 4
Figure 4
Damage markers and metformin signaling in adult mice. (a) Protein oxidation of liver homogenates as measured by derivatization with 2,4-dinitrophenylhydrazine (DNPH). (b) Representative cropped Western blot of 3-nitrotyrosine residues and GADPH in liver homogenates from adult mice. (c) Densitometric measurements of 3-nitrotyrosine blots from control (n = 4), burn (n = 5) and burn + metformin-treated mice (n = 5). (d) Circulating plasma mitochondrial DNA from control, injured and treated mice. (e) Changes in body weight between day 1 and day 7 in all adult cohorts as expressed as % of the original mass. (f) Representative Western blots of phosphorylated adenosine monophosphate-activated protein kinase (pAMPK), total AMPK, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Densitometry was performed to assess the activation of AMPK via its phosphorylation. formula image: control (n = 5); ■: burn (n = 5); formula image: burn + metformin treatment (n = 5). Values are presented as mean ± standard deviation. *p ≤ 0.05; **p ≤ 0.01.
Figure 5
Figure 5
Seahorse XF96 respirometry analysis of aged mice at day 7 post injury. (a) Respiration profiles of liver mitochondria from control mice (grey), burned mice (black) and burned mice with metformin treatment (100 mg/kg; green). (b) Production of reactive oxygen species as measured by oxidation of DCFDA in isolated mitochondria given substrate (5 mM pyruvate, 3 mM malate, 4 mM ADP); λ = 495 nm, λ’ = 529 nm. Basal (c), state 3 (d) and state 3 u (e) respiration parameters in isolated mitochondria as measured via Seahorse XF96 extracellular flux assays. formula image: control (n = 5); ■: burn (n = 5); formula image: burn + metformin treatment (n = 6). Values are presented as mean ± standard deviation. *p ≤ 0.05; **p ≤ 0.01.
Figure 6
Figure 6
Enzyme activity assays and control Seahorse XF96 curves from aged mice. (a) Representative images of complexes I-IV and ATP synthase of the ETC, assayed following blue native polyacrylamide gel electrophoresis of liver mitochondrial homogenates to demonstrate the effect of metformin in injured aged animals. (b) Densitometric measurements of in-gel activity assay bands using ImageJ for Windows. (c) Bands selected from the gel following staining with Coomassie Blue G-250 served as a loading control. (d) Respiration profiles of liver mitochondria from aged control mice (grey) and control mice given metformin (green). formula image: control (n = 5); ■: burn (n = 5); formula image: burn + metformin treatment (n = 6). Values are presented as mean ± standard deviation. *p ≤ 0.05; **p ≤ 0.01.
Figure 7
Figure 7
Damage markers and metformin signaling in aged mice. (a) Protein oxidation of liver homogenates as measured by derivatization with DNPH. (b) Representative cropped Western blot of 3-nitrotyrosine residues and GAPDH in liver homogenates from aged mice. (c) Densitometric measurements of 3-nitrotyrosine blots from control (n = 4), burn (n = 5) and burn + metformin-treated mice (n = 5). (d) Circulating plasma mitochondrial DNA from control, injured and treated mice. (e) Changes in body weight between day 1 and day 7 in all aged cohorts as expressed as % of the original mass. (f) Representative Western blots of pAMPK, total AMPK and GAPDH. Densitometry was performed to assess the activation of AMPK via its phosphorylation. formula image: control (n = 5); ■: burn (n = 5); formula image: burn + metformin treatment (n = 5). Values are presented as mean ± standard deviation. *p ≤ 0.05.
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