Oxidized-LDL Induces Metabolic Dysfunction in Retinal Pigment Epithelial Cells.

Recently, mitochondrial dysfunction has gained attention as a causative factor in the pathogenesis and progression of age-related macular degeneration (AMD). Mitochondrial damage plays a key role in metabolism and disrupts the balance of intracellular metabolic pathways, such as oxidative phosphorylation (OXPHOS) and glycolysis. In this study, we focused on oxidized low-density lipoprotein (ox-LDL), a major constituent of drusen that accumulates in the retina of patients with AMD, and investigated whether it could be a causative factor for metabolic alterations in retinal pigment epithelial (RPE) cells. We found that prolonged exposure to ox-LDL induced changes in fatty acid ß-oxidation (FAO), OXPHOS, and glycolytic activity and increased the mitochondrial reactive oxygen species production in RPE cells. Notably, the effects on metabolic alterations varied with the concentration and duration of ox-LDL treatment. In addition, we addressed the limitations of using ARPE-19 cells for retinal disease research by highlighting their lower barrier function and FAO activity compared to those of induced pluripotent stem cell-derived RPE cells. Our findings can aid in the elucidation of mechanisms underlying the metabolic alterations in AMD.

Clinical Evaluation Based on a New Approach to Improve the Accuracy of 4ß-Hydroxycholesterol Measurement as a Biomarker of CYP3A4 Activity.

This study examines 4ß-Hydroxycholesterol (4ß-HC), which is considered to be a potential marker for the CYP3A4 induction of new chemical entities (NCEs) in drug development. To ensure the use of 4ß-HC as a practical biomarker, it is necessary to accurately measure 4ß-HC and demonstrate that CYP3A4 induction can be appropriately assessed, even for weak inducers. In clinical trials of NCEs, plasma is often collected with various anticoagulants, in some cases, the plasma is acidified, then stored for an extended period. In this study, we examined the effects of these manipulations on the measurement of 4ß-HC, and based on the results, we optimized the plasma collection and storage protocols. We also found that a cholesterol oxidation product is formed when plasma is stored, and by monitoring the compound, we were able to identify when plasma was stored inappropriately. After evaluating the above, clinical drug-drug interaction (DDI) studies were conducted using two NCEs (novel retinoid-related orphan receptor γ antagonists). The weak CYP3A4 induction by the NCEs (which were determined based on a slight decline in the systemic exposure of a probe substrate (midazolam)), was detected by the significant increase in 4ß-HC levels (more specifically, 4ß-HC/total cholesterol ratios). Our new approach, based on monitoring a cholesterol oxidation product to identify plasma that is stored inappropriately, allowed for the accurate measurement of 4ß-HC, and thus, it enabled the evaluation of weak CYP3A4 inducers in clinical studies without using a probe substrate.

LC-MS/MS analysis of milk triacylglycerol hydroperoxide isomers which are generated corresponding to the photo- and thermal-oxidation.

Milk is a rich source of essential nutrients such as lipids. However, lipid oxidation can be considered a crucial factor in determining the initial stage of milk deterioration. Therefore, it is essential to identify the mechanisms of lipid oxidation, such as photo-oxidation or thermal oxidation, to efficiently prevent it by selecting proper antioxidants. In this study, the oxidation mechanisms of long-life (LL) milk were investigated, and triacylglycerol hydroperoxide isomers generated corresponding to the oxidation mechanisms were analyzed by LC-MS/MS. This study first prepared the standard of TG 4:0_16:0_18:1;OOH isomers, which are the appropriate target for evaluating LL milk's oxidation mechanism. The authentic standards provided the robust analysis of TG 4:0_16:0_18:1;OOH isomers and suggested that LL milk was susceptible to photo-oxidation rather than thermal-oxidation. Furthermore, it was discovered that radicals play a role in the oxidation of LL milk during photo-oxidation. This information could be valuable in effectively preventing photo-oxidation in LL milk. It is important to note that milk is contained in a variety of food products. Hence, these findings would be applicable not only to milk but also to various milk-containing food products.

Analysis of oxidized glucosylceramide and its effects on altering gene expressions of inflammation induced by LPS in intestinal tract cell models.

Glucosylceramide (GlcCer) belongs to sphingolipids and is found naturally in plant foods and other sources that humans consume daily. Our previous studies demonstrated that GlcCer prevents inflammatory bowel disease both in vitro and in vivo, whose patients are increasing alarmingly. Although some lipids are vulnerable to oxidation which changes their structure and activities, it is unknown whether oxidative modification of GlcCer affects its activity. In this research, we oxidized GlcCer in the presence of a photosensitizer, analyzed the oxide by mass spectrometric techniques, and examined its anti-inflammatory activity in lipopolysaccharide (LPS)-treated differentiated Caco-2 cells as in vitro model of intestinal inflammation. The results showed that GlcCer is indeed oxidized, producing GlcCer hydroperoxide (GlcCerOOH) as a primary oxidation product. We also found that oxidized GlcCer preserves beneficial functions of GlcCer, suppressing inflammatory-related gene expressions. These findings suggested that GlcCerOOH may perform as an LPS recognition antagonist to discourage inflammation rather than induce inflammation.

The difference in the cellular uptake of tocopherol and tocotrienol is influenced by their affinities to albumin.

Vitamin E is classified into tocopherol (Toc) and tocotrienol (T3) based on its side chains. T3 generally has higher cellular uptake than Toc, though the responsible mechanism remains unclear. To elucidate this mechanism, we hypothesized and investigated whether serum albumin is a factor that induces such a difference in the cellular uptake of Toc and T3. Adding bovine serum albumin (BSA) to serum-depleted media increased the cellular uptake of T3 and decreased that of Toc, with varying degrees among α-, ß-, γ-, and δ-analogs. Such enhanced uptake of α-T3 was not observed when cells were incubated under low temperature (the uptake of α-Toc was also reduced), suggesting that Toc and T3 bind to albumin to form a complex that results in differential cellular uptake of vitamin E. Fluorescence quenching study confirmed that vitamin E certainly bound to BSA, and that T3 showed a higher affinity than Toc. Molecular docking further indicated that the differential binding energy of Toc or T3 to BSA is due to the Van der Waals interactions via their side chain. Overall, these results suggested that the affinity of Toc and T3 to albumin differs due to their side chains, causing the difference in their albumin-mediated cellular uptake. Our results give a better mechanistic insight into the physiological action of vitamin E.

Structural Analysis and Anti-Inflammatory Effect of a Digalactosyldiacylglycerol-Monoestolide, a Characteristic Glycolipid in Oats.

Digalactosyldiacylglycerol- (DGDG-) monoestolide is a characteristic glycolipid in oats. DGDG-monoestolides possess a unique structure whereby a fatty acid of DGDG is replaced by a fatty acid ester of hydroxy fatty acid (FAHFA). While the physiological effects of DGDG and FAHFA have been reported previously, the effects of DGDG-monoestolides are unknown. Hence, we isolated a major DGDG-monoestolide molecular species from oats, analyzed its structure, and evaluated its anti-inflammatory effect. Based on GC-MS, MS/MS, and NMR analyses, the isolated compound was identified as a DGDG-monoestolide that contains the linoleic acid ester of 15-hydroxy linoleic acid (LAHLA) and linoleic acid (i.e., DGDG-LAHLA). The isolated DGDG-LAHLA was evaluated for its anti-inflammatory effect on LPS-stimulated RAW264 cells. The production of nitric oxide and cytokines (IL-6, TNF-α, and IL-10) were significantly decreased by DGDG-LAHLA, suggesting the anti-inflammatory effect of DGDG-LAHLA for the first time. In addition, our data showed a pronounced uptake of DGDG-LAHLA by cells. Some compounds corresponding to the predicted DGDG-LAHLA metabolites were also detected, suggesting that both intact DGDG-LAHLA and its metabolites may contribute to the above anti-inflammatory activities. These results are expected to expand the availability of oats as a functional food.

Effects of Sphingolipid Fractions from Golden Oyster Mushroom (Pleurotus citrinopileatus) on Apoptosis Induced by Inflammatory Stress in an Intestinal Tract in vitro Model.

Previously, we reported that the polar lipid fraction from the golden oyster mushroom, Pleurotus citrinopileatus, suppresses colon injuries which result from apoptosis induced by inflammatory stresses in vivo and in vitro (Yamashita et al., J. Oleo Sci., 69, 751-757 (2020)). Here, we investigated the use of lipid classes in mushroom polar lipid fraction in alleviating colon injury using differentiated Caco-2 cells as an intestinal tract model. The mushroom polar lipid fraction was separated into four fractions using silica thin layer chromatography. Each mushroom polar lipid fraction suppressed lipopolysaccharide (LPS)-induced decreases in the viability of intestinal cells, and the effects of sphingolipid fractions were significantly stronger than those of fraction that did not contain sphingolipids. Addition of sphingolipid fractions suppressed the expression of apoptosis-related proteins (e.g., death receptors and caspases) in the LPS-treated cells. Mushroom polar lipids, especially sphingolipids suppress intestinal apoptosis induced by inflammatory stress, and highly polar sphingolipids may exert stronger suppressive effects.

Polar Lipid Fraction from Golden Oyster Mushrooms (Pleurotus citrinopileatus) Suppresses Colon Injuries from Inflammatory Stresses in vivo and in vitro.

The rising incidence of inflammatory bowel disease (IBD) in East Asian countries has necessitated the implementation of preventive methods in the form of dietary supplementation and changes in dietary habits. We have previously reported that dietary golden oyster mushroom (Pleurotus citrinopileatus) ethanol extract (GOMEE) suppresses intestinal inflammation in mouse models of IBD induced by dextran sulfate sodium salt (DSS). Here, we investigated the components of GOMEE that exert suppressive effects on colon inflammation in vivo and in vitro. The total lipid fraction was extracted from GOMEE, and the polar and neutral lipid fractions were subsequently separated via solvent fractionation. Mice were assigned to dietary groups-control, 1% total lipid, 1% polar lipid, or 1% neutral lipid diet-and fed the respective diets for one week; mice were administered 1.5% DSS in drinking water ad libitum for 20 days. Dietary supplementation with the total or polar lipid fraction alleviated DSS-induced chorionic crypt injury as determined by morphological observation, while dietary supplementation with the neutral lipid fraction did not produce such effects. In the in vitro study, using differentiated Caco-2 cells as the colon model, treatment with the total or polar lipid fraction suppressed cell decrease by lipopolysaccharide (LPS)-induced apoptosis whereas treatment with the neutral lipid fraction did not. Moreover, accumulation of glucosylceramide (GlcCer), a fungal sphingolipid, was observed in the intestinal cells after treatment with polar lipid fraction. These results suggest that the active components of GOMEE that suppress colon inflammation are polar lipids, especially GlcCer. The structure of mushroom GlcCer differs from that of the plant counterpart and is therefore expected to exert different food functions.