Novel Fluorescence-Based Method To Characterize the Antioxidative Effects of Food Metabolites on Lipid Droplets in Cultured Hepatocytes

A fluorescence microscopic method for characterizing size, quantity, and oxidation of lipid droplets (LDs) in HepG2 cells was developed. LDs were induced by palmitic (PA), oleic (OA), or linoleic acids (LA) and stained with two fluorescent probes for neutral lipids and lipid peroxides. Each fatty acid increased the number of LDs and oxidized LDs (oxLDs) and the degree of LD oxidation time dependently, as well as increased intracellular triglyceride hydroperoxides. LDs induced by LA without 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) showed the most significant oxidation degree over PA and OA, especially in large LDs (area ≥ 3 μm2, oxLD/LD = 52.3 ± 21.7%). Under this condition, two food-derived antioxidants were evaluated, and both of them significantly improved the LD characteristics. Moreover, chlorogenic acid reduced the quantity of large LDs by 74.0–87.6% in a dose-dependent manner. The proposed method provides a new approach to evaluate the effect of dietary antioxidants on LD characteristics

Food-Derived beta-Carboline Alkaloids Ameliorate Lipid Droplet Accumulation in Human Hepatocytes

Lipid droplet accumulation (LDA) in hepatocytes is the initial stage of nonalcoholic fatty liver disease (NAFLD). In the search for natural compounds for the prevention of NAFLD, a series of beta-carboline alkaloid derivatives, inspired by flazin and its derivative, newly identified in Crassostrea gigas Thunberg. extracts, were examined for LDA inhibition (LDAI) activity in oleic acid-loaded hepatocytes (HepG2). Eight compounds with a piperidine or pyridine C-ring were chemically synthesized (1-8). Among them, compounds 2 and 4 (flazin) with a carboxy group at C-3 and furfuryl alcohol moiety at C-1 showed low cytotoxicity and they exhibited significant LDAI activity. Compound 2 with piperidine C-ring was identified for the first time in C. gigas extract, and ameliorated the lipid accumulation with the LDAI value of 25.4%. Active compounds 2 and 4 significantly inhibited triacylglycerol species accumulation in cells. These compounds upregulated ATGL and downregulated SREBP1, FASN, and SCD1 genes, suggesting that they activated lipolysis and suppressed lipogenesis, respectively. These results suggest that beta-carboline alkaloids, especially compounds 2 and 4, might be potentially useful for preventing NAFLD

Docosahexaenoic Acid Esters of Hydroxy Fatty Acid Is a Novel Activator of NRF2

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with interesting physiological functions in mammals. Despite their structural diversity and links with nuclear factor erythroid 2-related factor 2 (NRF2) biosynthesis, FAHFAs are less explored as NRF2 activators. Herein, we examined for the first time the synthetic docosahexaenoic acid esters of 12-hydroxy stearic acid (12-DHAHSA) or oleic acid (12-DHAHOA) against NRF2 activation in cultured human hepatoma-derived cells (C3A). The effect of DHA-derived FAHFAs on lipid metabolism was explored by the nontargeted lipidomic analysis using liquid chromatography-mass spectrometry. Furthermore, their action on lipid droplet (LD) oxidation was investigated by the fluorescence imaging technique. The DHA-derived FAHFAs showed less cytotoxicity compared to their native fatty acids and activated the NRF2 in a dose-dependent pattern. Treatment of 12-DHAHOA with C3A cells upregulated the cellular triacylglycerol levels by 17-fold compared to the untreated group. Fluorescence imaging analysis also revealed the suppression of the degree of LDs oxidation upon treatment with 12-DHAHSA. Overall, these results suggest that DHA-derived FAHFAs as novel and potent activators of NRF2 with plausible antioxidant function

Oxidative Stress Linked Organ Lipid Hydroperoxidation and Dysregulation in Mouse Model of Nonalcoholic Steatohepatitis: Revealed by Lipidomic Profiling of Liver and Kidney

Nonalcoholic steatohepatitis (NASH) is a prevalent disease related to lipid metabolism disorder and oxidative stress. Lipid hydroperoxidation is known to be a critical driving force of various disorders and diseases. However, the combination of both intact and hydroperoxidized lipids in NASH has not yet been studied. In this work, the liver and kidney samples from NASH-model mice were comprehensively investigated by using the LC/MS-based lipidomic analysis. As a result, triglycerides showed the amount accumulation and the profile alteration for the intact lipids in the NASH group, while phosphatidylethanolamines, lysophosphatidylethanolamines, plasmalogens, and cardiolipins largely depleted, suggesting biomembrane damage and mitochondria dysfunction. Notably, the lipid hydroperoxide species of triglyceride and phosphatidylcholine exhibited a significant elevation in both the liver and the kidney of the NASH group and showed considerable diagnostic ability. Furthermore, the relationship was revealed between the lipid metabolism disturbance and the lipid hydroperoxide accumulation, which played a key role in the vicious circle of NASH. The present study suggested that the omics approach to the lipid hydroperoxide profile might be the potential diagnostic marker of NASH and other oxidative stress-related diseases, as well as the evaluative treatment index of antioxidants.</p