Metformin impairs mitochondrial function in skeletal muscle of both lean and diabetic rats in a dose-dependent manner

Abstract

Metformin is a widely prescribed drug for the treatment of type 2 diabetes. Previous studies have demonstrated in vitro that metformin specifically inhibits Complex I of the mitochondrial respiratory chain. This seems contraindicative since muscle mitochondrial dysfunction has been linked to the pathogenesis of type 2 diabetes. However, its significance for in vivo skeletal muscle mitochondrial function has yet to be elucidated. The aim of this study was to assess the effects of metformin on in vivo and ex vivo skeletal muscle mitochondrial function in a rat model of diabetes. Healthy (fa/+) and diabetic (fa/fa) Zucker diabetic fatty rats were treated by oral gavage with metformin dissolved in water (30, 100 or 300 mg/kg bodyweight/day) or water as a control for 2 weeks. After 2 weeks of treatment, muscle oxidative capacity was assessed in vivo using 31P magnetic resonance spectroscopy and ex vivo by measuring oxygen consumption in isolated mitochondria using high-resolution respirometry. Two weeks of treatment with metformin impaired in vivo muscle oxidative capacity in a dose-dependent manner, both in healthy and diabetic rats. Whereas a dosage of 30 mg/kg/day had no significant effect, in vivo oxidative capacity was 21% and 48% lower after metformin treatment at 100 and 300 mg/kg/day, respectively, independent of genotype. High-resolution respirometry measurements demonstrated a similar dose-dependent effect of metformin on ex vivo mitochondrial function. In conclusion, metformin compromises in vivo and ex vivo muscle oxidative capacity in Zucker diabetic fatty rats in a dose-dependent manner.

Figure 1. In vivo oxidative capacity of tibialis anterior (TA) muscle, assessed by ³¹P MRS.

Representative examples of ³¹P MR spectra obtained during rest with 32 averages (A) and at the end of the electrical-stimulation protocol with 4 averages (B). (C) Representative examples of relative PCr concentrations during rest, muscle stimulation and recovery (time resolution  =  20 s) for a water-treated diabetic rat (open symbols) and a diabetic rat treated with metformin at 300 mg/kg body weight/day (filled symbols). PCr concentrations are expressed as a percentage of the resting PCr concentration. Mono-exponential functions (dark lines) were fit to the recovery data and the PCr recovery rate constants were 0.63 and 0.21 min^-1 for the water-treated and metformin-treated animal, respectively. (D) Rate constants of PCr recovery, k _PCr, after electrical stimulation in TA muscle of lean and diabetic rats treated with water or 30, 100 or 300 mg/kg body weight/day metformin (MET30, MET100 and MET300 respectively). Data is represented as mean ± SD (n = 6 per group). k _PCr was significantly lower in diabetic rats compared with lean rats, independent of treatment regimen (ANOVA: P<0.001). In addition, treatment had a significant effect on k _PCr, independent of genotype, and a pairwise analysis of differences is provided by Bonferroni-corrected post-hoc tests: ^* P<0.001 when compared with water-treated animals, ^† P<0.001 when compared with MET30-treated animals, ^‡ P<0.001 when compared with MET100-treated animals.

Figure 2. Relative mitochondrial-DNA copy number of lean and diabetic rats after 2 weeks of treatment with either water or metformin (300 mg/kg bodyweight/day).

Data is represented as mean ± SD (n = 6 per group).

Figure 3. O₂ consumption rates determined in mitochondria isolated from TA muscle of lean and diabetic rats treated with water or 30, 100 or 300 mg/kg body weight/day metformin (MET30, MET100 and MET300, respectively) for 2 weeks, fueled by pyruvate plus malate (Complex I-dependent substrate).

Respiratory capacity was determined in the OXPHOS state, when mitochondrial respiration is coupled to ATP synthesis; the LEAK-state, when the system is limited by ADP; and the ETS state, after uncoupling of the ETS from ATP synthesis. Data is represented as mean ± SD (n = 6 per group). For the OXPHOS state, the interaction between genotype and treatment was borderline significant and for the LEAK and ETS state, the interaction between genotype and treatment was significant. A pairwise analysis of differences is provided by Bonferroni-corrected two-sided unpaired t-tests: ^* P<0.05 when compared with water-treated animals of the same genotype, ^† P<0.05 when compared with MET30-treated animals of the same genotype, ^‡ P<0.05 when compared with MET100-treated animals of the same genotype, ^# P<0.05 when compared with lean animals of the same treatment regimen.

Figure 4. O₂ consumption rates determined in mitochondria isolated from TA muscle of lean and diabetic rats treated with water or 30, 100 or 300 mg/kg body weight/day metformin (MET30, MET100 and MET300 respectively) for 2 weeks, fueled by succinate plus rotenone (Complex II-dependent substrate).

Respiratory capacity was determined in the OXPHOS state, when mitochondrial respiration is coupled to ATP synthesis; and the LEAK-state, when the system is limited by ADP. Data is represented as mean ± SD (n = 6 per group). For the OXPHOS state, the interaction between genotype and treatment was significant and a pairwise analysis of differences is provided by Bonferroni-corrected two-sided unpaired t-tests: ^‡ P<0.05 when compared with MET100-treated animals of the same genotype.

Figure 5. O₂ flux measured in mitochondria isolated from TA muscle of lean and diabetic ZDF rats after 5 min of incubation with metformin (1 mM), normalized to O₂ flux measured in isolated mitochondria without addition of metformin.

Respiratory capacity was determined in the OXPHOS state, when mitochondrial respiration is coupled to ATP synthesis, fueled with either pyruvate plus malate (Complex I respiration) or succinate plus rotenone (Complex II respiration). Data is represented as mean ± SD (n = 6 per group). Incubation with metformin significantly lowered OXPHOS respiration fueled with pyruvate plus malate, independent of genotype (ANOVA: * P<0.001). Metformin did not affect Complex II respiration.