Regulation of reactive oxygen species production and uncoupling protein 2 expression in mice hepatocytes by lopimune

Abstract

OBJECTIVES: Lopimune, a protease inhibitor, is the first combination pill containing both lopinavir and ritonavir to be introduced as an HIV therapy providing virologic and immunologic improvements (100/25 mg of lopinavir/ritonavir, UNIPHARM Company). Though treatment with lopimune has proven to be effective, no studies have introduced the side effects of lopimune on mitochondrial bioenergetics in hepatocytes. The objective of this study is to demonstrate the effect of lopimune on mitochondria of mice hepatic cells, by evaluating mitochondrial respiration, production of reactive oxygen species (ROS) and expression of uncoupling protein-2 (UCP2). METHODS: Lopimune was administered intra-peritoneally to BALB/c mice at a dosage of 125 mg/kg/day. Mitochondria were isolated from liver using differential centrifugation and hepaocytes were isolated by collagenase perfusion procedure. Mitochondrial respiration was measured using Rank Brothers oxygen electrode. ROS production in hepatocytes was monitored by flow cytometer using 2ʹ,7ʹ-Dichlorofluorescin diacetate probe. UCP2 protein expression was detected by western blot. RESULTS: Lopimune induced a significant decrease of approximately 30% in respiratory control ratio (RCR) starting from day 4 till day 9 of treatment, indicating mitochondrial inefficiency in the production of ATP. This decrease in RCR was due to an increase in state 4 respiration, i.e an increase in mitochondrial proton leak. State 2 and state 3 respirations were not affected. The proton leak induced in mitochondria of lopimune treated mice was sensitive to guanidine diphosphate (GDP), a UCP specific inhibitor. Moreover, ROS production significantly increased by about 3 folds in hepatocytes after day 1 of lopimune treatment and started decreasing from day 3 forward returning to base line levels on day 5 and 7. Importantly, UCP2 which is absent in normal hepatocytes, was expressed starting from day 4 of lopimune administration. CONCLUSION: Lopimune impairs mitochondrial functions and facilitates the expression and function of UCP2 in hepatocytes, in line with a decrease in ROS production. The induction of the proton leak by UCP2 limits mitochondrial ROS production as a feedback response to over production of ROS by the respiratory chain in lopimune treated mice.