title: Alterations in glucose metabolism by cyclosporine in rat brain slices link to oxidative stress: interactions with mTOR inhibitors.
abstract: 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 (31)P-MRS), glucose metabolism (ex vivo (13)C-MRS), and reactive oxygen species (ROS) formation (using the fluorescent agent 2',7'-dichlorofluorescein diacetate) in perfused rat brain slices. Cyclosporine alone inhibited energy production (ATP: 75+/-9%), the Krebs cycle (4-(13)C-glutamate from 1-(13)C-glucose: 61+/-27%), and oxidative phosphorylation (NAD(+): 62+/-25%) after 4 h of perfusion. After 10 h, activation of anaerobic glycolysis (3-(13)C-lactate: 140+/-17%) compensated for inhibition of mitochondrial energy production and lowered the intracellular pH. ROS formation was increased after 4 h (285+/-55% of untreated control), but not after 10 h. 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 10 h. 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. 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.
authors.affiliation: Department of Anesthesiology, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
authors.forename: Uwe
authors.initials: U
authors.lastname: Christians
authors.forename: Sven
authors.initials: S
authors.lastname: Gottschalk
authors.forename: Jelena
authors.initials: J
authors.lastname: Miljus
authors.forename: Carsten
authors.initials: C
authors.lastname: Hainz
authors.forename: Leslie Z
authors.initials: LZ
authors.lastname: Benet
authors.forename: Dieter
authors.initials: D
authors.lastname: Leibfritz
authors.forename: Natalie
authors.initials: N
authors.lastname: Serkova
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D002247:Carbon Isotopes; D016572:Cyclosporine / Q000494:pharmacology*; D004347:Drug Interactions; D004734:Energy Metabolism / Q000187:drug effects; D000068338:Everolimus; D005947:Glucose / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000494:pharmacology; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D009243:NAD / Q000378:metabolism; D010085:Oxidative Phosphorylation / Q000187:drug effects; D018384:Oxidative Stress / Q000502:physiology; D010477:Perfusion; D010725:Phosphocreatine / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D013997:Time Factors
chemical_list: D002247:Carbon Isotopes; D007166:Immunosuppressive Agents; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D010725:Phosphocreatine; D009243:NAD; D016572:Cyclosporine; D000255:Adenosine Triphosphate; D000068338:Everolimus; D011494:Protein Kinases; C546845:mTOR protein, rat; D058570:TOR Serine-Threonine Kinases; D005947:Glucose; D020123:Sirolimus