We previously showed that H2 acts as a novel antioxidant to protect cells against oxidative stress. Subsequently, numerous studies have indicated the potential applications of H2 in therapeutic and preventive medicine. Moreover, H2 regulates various signal transduction pathways and the expression of many genes. However, the primary targets of H2 in the signal transduction pathways are unknown. Here, we attempted to determine how H2 regulates gene expression. In a pure chemical system, H2 gas (approximately 1%, v/v) suppressed the autoxidation of linoleic acid that proceeds by a free radical chain reaction, and pure 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC), one of the major phospholipids, was autoxidized in the presence or absence of H2. H2 modified the chemical production of the autoxidized phospholipid species in the cell-free system. Exposure of cultured cells to the H2-dependently autoxidized phospholipid species reduced Ca2+ signal transduction and mediated the expression of various genes as revealed by comprehensive microarray analysis. In the cultured cells, H2 suppressed free radical chain reaction-dependent peroxidation and recovered the increased cellular Ca2+, resulting in the regulation of Ca2+-dependent gene expression. Thus, H2 might regulate gene expression via the Ca2+ signal transduction pathway by modifying the free radical-dependent generation of oxidized phospholipid mediators.

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We previously reported that molecular hydrogen (H2) acts as a novel antioxidant to exhibit multiple functions. Moreover, long-term drinking of H2-water (water infused with H2) enhanced energy expenditure to improve obesity and diabetes in db/db mice accompanied by the increased expression of fibroblast growth factor 21 (FGF21) by an unknown mechanism. H2 was ingested by drinking of H2-water or by oral administration of an H2-producing material, MgH2. The comprehensive gene expression profile in the liver of db/db mice was analyzed by DNA microarray. The molecular mechanisms underlying the gene expression profile was investigated using cultured HepG2 cells. Moreover, the effects on lifespan of drinking H2-water were examined using wild-type mice that were fed a fatty diet. Pathway analyses based on comprehensive gene expression revealed the increased expression of various genes involved in fatty acid and steroid metabolism. As a transcription pathway, the PPARα signaling pathway was identified to upregulate their genes by ingesting H2. As an early event, the gene expression of PGC-1α was transiently increased, followed by increased expression of FGF21. The expression of PGC-1α might be regulated indirectly through sequential regulation by H2, 4-hydroxy-2-nonenal, and Akt/FoxO1 signaling, as suggested in cultured cell experiments. In wild-type mice fed the fatty diet, H2-water improved the level of plasma triglycerides and extended their average of lifespan. H2 induces expression of the PGC-1α gene, followed by stimulation of the PPARα pathway that regulates FGF21, and the fatty acid and steroid metabolism.

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Lipid peroxidation of cellular membranes is a complicated cellular event, and it is both the cause and result of various diseases, such as ischemia-reperfusion injury, neurodegenerative diseases, and atherosclerosis. Lipid peroxidation causes non-apoptotic cell death, which is associated with cell fate determination: survival or cell death. During the radical chain reaction of lipid peroxidation, various oxidized lipid products accumulate in cells, followed by organelle dysfunction and the induction of non-apoptotic cell death. Highly reactive oxidized products from unsaturated fatty acids are detected under pathological conditions. Pathological protein aggregation is the general cause of these diseases. The cellular response to misfolded proteins is well-known as the unfolded protein response (UPR) and it is partially concomitant with the response to lipid peroxidation. Moreover, the association between protein aggregation and non-apoptotic cell death by lipid peroxidation is attracting attention. The link between lipid peroxidation and protein aggregation is a matter of concern in biomedical fields. Here, we focus on lethal protein aggregation in non-apoptotic cell death via lipid peroxidation. We reviewed the roles of protein aggregation in the initiation and execution of non-apoptotic cell death. We also considered the relationship between protein aggregation and oxidized lipid production. We provide an overview of non-apoptotic cell death with a focus on lipid peroxidation for therapeutic targeting during protein aggregation diseases.

Polyunsaturated fatty acids are oxidized by non-enzymatic or enzymatic reactions. The oxidized products are multifunctional. In this study, we investigated how oxidized fatty acids inhibit cell proliferation in cultured cells. We used polyunsaturated and saturated fatty acids, docosahexaenoic acid (DHA; 22:6), eicosapentaenoic acid (EPA; 20:5), linoleic acid (LA; 18:2), and palmitic acid (16:0). Oxidized fatty acids were produced by autoxidation of fatty acids for 2 days in the presence of a gas mixture (20% O2 and 80% N2). We found that oxidized polyunsaturated fatty acids (OxDHA, OxEPA and OxLA) inhibited cell proliferation much more effectively compared with un-oxidized fatty acids (DHA, EPA and LA, respectively) in THP-1 (a human monocytic leukemia cell line) and DLD-1 (a human colorectal cancer cell line) cells. In particular, OxDHA markedly inhibited cell proliferation. DHA has the largest number of double bonds and is most susceptible to oxidation among the fatty acids. OxDHA has the largest number of highly active oxidized products. Therefore, the oxidative levels of fatty acids are associated with the anti-proliferative activity. Moreover, caspase-3/7 was activated in the cells treated with OxDHA, but not in those treated with DHA. A pan-caspase inhibitor (zVAD-fmk) reduced the cell death induced by OxDHA. These results indicated that oxidized products from polyunsaturated fatty acids induced apoptosis in cultured cells. Collectively, the switch between cell survival and cell death may be regulated by the activity and/or number of oxidized products from polyunsaturated fatty acids.