― 262 ―instructions.Persulfide detectionIntracellular persulfides were detected using Sulfane Sulfur Probe 4 (SSP4, Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions.Small interfering RNA (siRNA) transfectionMouse Nfe2l2 (Nrf2) (cat. # L-040766-00-0005), mouse Keap1 (cat. # M-041104-01-0005), mouse Cars2 (cat. # L-060862-01-0005), mouse Sqor (cat. # M-063889-00-0005), mouse Ethe1 (cat. # L-056791-01-0005), mouse Suox (cat. # L-053903-01-0005), human KEAP1 (cat. # L-012453-00-0020), human SLC7A11 (cat. # M-007612-01-0010), human SQOR (cat. # M-008271-01-0010), human ETHE1 (cat. # M-012508-00-0005), and human SUOX (cat. # L-008310-00-0005) siRNAs were purchased from Dharmacon (Lafayette, CO, USA). Human NFE2L2 (Nrf2) siRNAs were purchased from Invitrogen (Waltham, MA, USA, cat. # HSS107128).ATP measurementsThe ADP/ATP ratio measurement was performed according to the manufacturer’s instructions (ADP/ATP Ratio Assay Kit, cat. # MAK135, Sigma‒Aldrich, USA).We first examined how cysteine availability impacted intracellular persulfide levels and mitochondrial function. Because one of the major cysteine sources is extracellular cystine, cells were challenged with cystine restriction for up to 24 hr. Hepa1c1c7 cells exhibited a reduction in intracellular persulfides, as evaluated by the fluorescent probe SSP45) (Fig. 1A), and a decrease in mitochondrial membrane potential (MMP), as evaluated by the fluorescent probe JC-10 (Fig. 1B), after 12 hr of exposure to 20 µM cystine compared with 200 µM cystine. We next examined ATP production in Hepa1c1c7 cells exposed to 20 µM cystine for 24 hr and found that cystine restriction elevated the ADP/ATP ratio (Fig. 1C). These results indicated that cystine restriction reduces intracellular persulfides and suppresses mitochondrial energy metabolism, which is consistent with our previous observation that mitochondrial persulfide production is necessary for the maintenance of mitochondrial activity.Glutathione trisulfide (GSSSG) supplementation promotes mitochondrial activity.We next asked whether exogenous persulfide supplementation activates mitochondrial function. Glutathione trisulfide (GSSSG) was added to the culture medium of Hepa1c1c7 cells as a source of persulfides, and intracellular persulfides and mitochondrial function were evaluated after 24 hr. Supplementation with GSSSG increased persulfides (Fig. 2A) and elevated MMP (Fig. 2B). The increase of MMP was not induced by GSSG supplementation (Fig. 2C). Thus, increased persulfides contributed to the activation of mitochondrial function.Results and DiscussionCystine restriction reduces intracellular persulfides and inhibits mitochondrial activity.
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