13% ± 7.34% without or with removal

of 25 μM of EFV; each value calculated versus its respective control, n = 5). Furthermore, the effects were specific for EFV, because the presence of NVP (10, 25, and 50 μM) in the gas-tight chambers did not modify the rate of O2 consumption by Hep3B cells (Fig. 1C). Figure 2 shows the effects of EFV on mitochondrial complex I activity in isolated mitochondria of rats. Figure 2A, B, C (traces), and D (rate of O2 consumption) illustrate that the mitochondria incubated with EFV respired poorly in the presence of complex I substrates malate and glutamate. Indeed, the inhibition of respiration with EFV 25 and 50 μM did not differ from that induced by the specific complex I inhibitor rotenone (2 μM). The inhibitory effects of EFV were absent when succinate (5 mM), a complex II electron donor, was added to bypass LY2109761 nmr complex I–dependent respiration. Selleckchem INCB024360 The mitochondria exhibited O2 consumption rates similar to those of controls, thus suggesting that complex I was the main

target of EFV. Incubation with EFV produced a significant and concentration-dependent increase in the fluorescence of DCFH-DA, indicating an augmented production of ROS (Fig. 3A). This effect was rapid, was maintained throughout the 1-hour period evaluated, and was not reproduced when cells were treated with NVP (Fig. 3B). Figure 3C shows that 1 hour incubation with EFV induced a significant and concentration-dependent decrease in intracellular ATP, whereas incubation with NVP did not alter ATP levels (Fig. 3D). Figure 4 shows representative buy Gefitinib western blot analysis of phosphorylated AMPK (P-AMPK), the active form of the enzyme, of cells incubated with EFV. Densitometric analysis revealed that EFV induced a concentration-dependent and time-dependent increase in P-AMPK. EFV 50 μM produced a significant phosphorylation of AMPK during a 1-hour incubation period. The effects of EFV 25 μM were statistically significant after 4 hours, but not after 1 hour, whereas those of 10 μM reached significance only after 8 hours (Fig. 4A, B, and C). The levels of P-AMPK were also significantly (P < 0.01) increased by incubation (4 hours)

with rotenone (10 μM, 281.28% ± 53.59% of control, n = 4) but not with NVP (Fig. 4D). Incubation (4 hours) with EFV 10 or 25 μM did not modify the concentration of glucose in the medium (114.10% ± 24.04% and 105.74% ± 21.58% of control, n = 3) or the expression of GLUT-1 (111.70% ± 19.95% and 136.40% ± 37.20% of control, n = 9), which suggests that the glucose metabolism targets of P-AMPK were not affected. The effects of EFV were reproduced in primary hepatic cells. Figure 5A shows that the inhibitory effect of EFV (10 and 25 μM) on the rate of O2 consumption was similar to that exerted on Hep3B cells. Similarly, densitometric analysis of blots from human tissue revealed that incubation with EFV (25 μM, 4 hours) induced a significant increase in P-AMPK (Fig. 5B).