Alterations in glucose metabolism by cyclosporine in rat brain slices link to oxidative stress: Interactions with mTOR inhibitors

Abstract

1 Co-administration of the calcineurin inhibitor cyclosporine and the mTOR inhibitors sirolimus or everolimus increases the efficacy of immunosuppression after organ transplantation. However, clinical studies showed enhancement of cyclosporine toxicity. To characterize the biochemical mechanisms involved, we assessed the time-dependent effects of cyclosporine in combination with mTOR inhibitors on energy production (ex vivo 31P-MRS), glucose metabolism (ex vivo 13C-MRS), and reactive oxygen species (ROS) formation (using the fluorescent agent 2′,7′-dichlorofluorescein diacetate) in perfused rat brain slices. 2 Cyclosporine alone inhibited energy production (ATP: 75 ± 9%), the Krebs cycle (4- 13C-glutamate from 1- 13C-glucose: 61 ± 27%), and oxidative phosphorylation (NAD +: 62 ± 25%) after 4h of perfusion. After 10h, activation of anaerobic glycolysis (3- 13C-lactate: 140 ± 17%) compensated for inhibition of mitochondrial energy production and lowered the intracellular pH. ROS formation was increased after 4h (285 ± 55% of untreated control), but not after 10h. mTOR inhibitors alone inhibited lactate production. When combined with cyclosporine, sirolimus enhanced cyclosporine-induced inhibition of energy metabolism (ATP: 64 ± 9%) and ROS formation (367 ± 46%). Most importantly, sirolimus inhibited cytosolic glycolysis and therefore compensation for cyclosporine-induced ATP reduction after 1Oh. In contrast to sirolimus, everolimus antagonized cyclosporine-induced inhibition of mitochondrial energy metabolism (ATP: 91 ± 7%) and ROS formation (170 ± 49%). The antioxidant tocopherol antagonized all cyclosporine effects on cell metabolism. 3 Cyclosporine time-dependently inhibited mitochondrial metabolism and increased ROS, followed by compensation involving anaerobic glycolysis. Everolimus antagonized cyclosporine-induced mitochondrial dysfunction, whereas sirolimus inhibited compensatory anaerobic glycolysis, thus enhancing cyclosporine's negative effects. ROS play the key role in mediating the negative effects of cyclosporine on cell energy metabolism.