Metformin directly acts on mitochondria to inhibit respiration. (A-B) Design of experiments with isolated mitochondria from murine skeletal muscle. Mitochondria were incubated with either complex I (malate and pyruvate) or complex II (succinate and rotenone) substrates. Typical respiratory control ratio (RCR) values are shown for mitochondria respiring on either complex I or II substrates. Respiration in the presence of substrates is called state 2. Respiration in the presence of ADP where mitochondria are using ADP to make ATP is called state 3. Respiration in the presence of oligomycin where mitochondria are driving proton leak reactions is called state 4. FCCP stimulates uncoupled respiration and represents the maximal respiratory capacity. RCR values are calculated by dividing the rate of respiration in state 3 by that in state 4 and are indicative of the integrity of the mitochondrial suspensions. (C-F) Mitochondria isolated from murine skeletal muscle were incubated with complex I (C,E) or complex II (D,F) substrates and treated with ddH2O (control) or metformin (2 mM) (E-F). Respiration rates are expressed as the fold difference from untreated mitochondria. Data are presented as means ± SEM. n = 3. *P <0.05, Student’s t- test.