Diverse actions of 15 structurally unrelated mitochondrial uncouplers in cells and mice

Abstract

Mitochondrial uncouplers are used as chemical tools to study mitochondrial function in vitro and in vivo, and some molecules are in development for the treatment of metabolic diseases. One problem in the field is that any molecule that increases proton transport into the mitochondrial matrix independent of ATP production can be classified as an uncoupler regardless of off-target activities. Therefore, there are dozens of classes of molecules that exhibit a wide spectrum of phenotypes. Herein we directly compared 15 mitochondrial uncouplers side-by-side in a well-defined cell system to better understand their in vitro dose response profiles and the top molecules with suitable pharmacology and safety profiles were compared in db/db mice. Fifteen mitochondrial uncouplers were characterised in vitro in CHO-K1 cells. The top five candidates were selected for further characterisation in male db/db mice based on their in vitro dose response and/or tolerability. We tested two doses of each mitochondrial uncoupler in mice and benchmarked their efficacy to a lifestyle intervention of 35% calorie restriction as well as to lean db/+ metabolically healthy mice. Eleven groups of mice were fed ad libitum either; 1) chow (control), 2) chow with 0.15% BAM15 (w/w), 3) chow with 0.2% BAM15 (w/w), 4) chow with 0.1% NEN (w/w), 5) chow with 0.25% NEN (w/w), 6) chow with 0.01% OPC-163493 (w/w), 7) chow with 0.02% OPC-63493 (w/w), 8) chow with 0.015% ES9 (w/w), 9) chow with 0.03% ES9 (w/w), 10) chow with 0.2% NTZ (w/w), and 11) chow with 0.4% NTZ (w/w). Another group of mice was fed chow to receive ∼65% of the average daily food intake of control mice as a model of calorie restriction (CR). Mice were metabolically phenotyped over 4 weeks of treatment with assessment of key readouts including body weight, HbA1c, blood glucose and glucose tolerance tests. At termination, key tissues were collected and plasma was analysed for markers of toxicity. Few mitochondrial uncouplers behaved similarly in vitro, with 11 molecules impairing maximal mitochondrial capacity. In vivo, BAM15 dose-dependently improved body weight and metabolic parameters in db/db mice, with 0.2% BAM15 treatment yielding statistically significant improvements in body weight, fat pad weight, glucose tolerance, blood glucose, HbA1c, liver weight and triglyceride content. The next-best treatment was 0.03% ES9 which significantly improved glucose tolerance, blood glucose levels, and HbA1c, but increased body weight, liver size and steatosis relative to db/db controls. Mitochondrial uncouplers BAM15 and ES9 had the greatest dose tolerance range in vitro, while BAM15 had the best overall effects on body weight, glucose control and liver steatosis in db/db mice. This study reveals diverse phenotypes across 15 classes of mitochondrial uncouplers and underscores the need for rigorous evaluation to identify molecules that drive stable mitochondrial respiration without unwanted mitochondrial inhibition or off-target effects. Ultimately, mitochondrial uncouplers should not be generalized and each uncoupler molecule needs to be considered by its own actions in well-defined experimental conditions.