Characterization of the Structural Diversity and Structure-Specific Behavior of Oxidized Phospholipids by LC-MS/MS.

Polyunsaturated fatty acids (PUFAs), esterified to phospholipids, are susceptible to oxidation. They form oxidized phospholipids (OxPLs) by oxygenases or reactive oxygen species (ROS), or both. These OxPLs are associated with various diseases, such as atherosclerosis, pulmonary injuries, neurodegenerative diseases, cancer, and diabetes. Since many types of OxPLs seem to be generated in vivo, precise determination of their structural diversity is required to understand their potential structure-specific functions. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful method to quantitatively measure the structural diversity of OxPLs present in biological samples. This review outlines recent advances in analytical methods for OxPLs and their physiological relevance in health and diseases.

Oxidized Phospholipids and Neutrophil Elastase Coordinately Play Critical Roles in NET Formation.

Neutrophil extracellular traps (NETs) are web-like structures consisting of decondensed chromatin DNA and contents of granules, such as myeloperoxidase (MPO) and neutrophil elastase (NE). NETs are usually released from neutrophils undergoing NETosis, a neutrophil-specific cell death mode characterized by the collapse and disappearance of cell membranes and nuclear envelopes. It is well known that production of reactive oxygen species (ROS) triggers NETosis and NET formation. However, details of intracellular signaling downstream of ROS production during NETosis and NET formation remains uncertain. Here, we demonstrated that the peroxidation of phospholipids plays a critical role in NETosis and NET formation induced by phorbol 12-myristate13-acetate (PMA) or immune complex in vitro and by lipopolysaccharide (LPS) in vivo. This phospholipid peroxidation is mediated by the enzymatic activity of MPO. On the other hand, NE, which was previously reported to be released from granules to cytosol by MPO during NET formation, is not required for either the peroxidation of phospholipids or the execution of NETosis, but contributes to chromatin decondensation and nuclear swelling independently of MPO-mediated oxidized phospholipids. Analysis of isolated nuclei clearly demonstrated that oxidized phospholipids and NE differently yet synergistically execute chromatin decondensation and nuclear swelling, and the subsequent release of nuclear contents. These findings indicate the dual roles of MPO in NETosis and NET formation, and provide new insight into the molecular mechanism of these phenomena.

Lipidomics links oxidized phosphatidylcholines and coronary arteritis in Kawasaki disease.

AIMS: Coronary arteritis is a life-threatening complication that may arise in the acute stage of Kawasaki disease (KD), the leading cause of systemic vasculitis in childhood. Various microorganisms and molecular pathogens have been reported to cause KD. However, little is known about the key molecules that contribute to the development of coronary arteritis in KD. METHODS AND RESULTS: To identify causative molecules for coronary arteritis in KD, we prospectively recruited 105 patients with KD and 65 disease controls in four different parts of Japan from 2015 to 2018. During this period, we conducted lipidomics analyses of their sera using liquid chromatography-mass spectrometry (LC-MS). The comprehensive LC-MS system detected a total of 27 776 molecules harbouring the unique retention time and m/z values. In the first cohort of 57 KD patients, we found that a fraction of these molecules showed enrichment patterns that varied with the sampling region and season. Among them, 28 molecules were recurrently identified in KD patients but not in controls. The second and third cohorts of 48 more patients with KD revealed that these molecules were correlated with inflammatory markers (leucocyte counts and C-reactive proteins) in the acute stage. Notably, two of these molecules (m/z values: 822.55 and 834.59) were significantly associated with the development of coronary arteritis in the acute stage of KD. Their fragmentation patterns in the tandem MS/MS analysis were consistent with those of oxidized phosphatidylcholines (PCs). Further LC-MS/MS analysis supported the concept that reactive oxygen species caused the non-selective oxidization of PCs in KD patients. In addition, the concentrations of LOX-1 ligand containing apolipoprotein B in the plasma of KD patients were significantly higher than in controls. CONCLUSION: These data suggest that inflammatory signals activated by oxidized phospholipids are involved in the pathogenesis of coronary arteritis in KD. Because the present study recruited only Japanese patients, further examinations are required to determine whether oxidized PCs might be useful biomarkers for the development of coronary arteritis in broad populations of KD.

Hyperoxidation of ether-linked phospholipids accelerates neutrophil extracellular trap formation.

Because neutrophil extracellular trap (NET) formation is involved in the pathology of a wide variety of diseases, NET-regulating compounds are expected to be useful for the therapies of these diseases. In this study, we identified sulfasalazine (SSZ) as a potent enhancer of NET formation both in vitro and in vivo. Although SSZ did not increase the amount of ROS generated, it accelerated the generation of ether-linked oxidized phospholipids, such as PE (18;1e/15-HETE) and PC (16;0e/13-HODE). Trolox, but not 2-ME, effectively suppressed lipid oxidation and NET formation that were induced by SSZ. SSZ is known as a potent inducer of ferroptosis in cancer cells by inhibiting xCT, a component of the cystine transporter. However, we found that SSZ accelerated NET formation in an xCT-independent manner. Structure-activity relationship studies revealed that the sulfapyridine moiety of SSZ plays a central role in enhancing NET formation. Furthermore, we found that two additional sulfonamide and sulfone derivatives possess NET-inducing activity by accelerating lipid oxidation. These results indicate that the hyperoxidation of ether-linked phospholipids is a key mechanism for accelerating NET formation.

Comprehensive analyses of oxidized phospholipids using a measured MS/MS spectra library.

Oxidized phospholipids (OxPLs) are widely held to be associated with various diseases, such as arteriosclerosis, diabetes, and cancer. To characterize the structure-specific behavior of OxPLs and their physiological relevance, we developed a comprehensive analytical method by establishing a measured MS/MS spectra library of OxPLs. Biogenic OxPLs were prepared by the addition of specific oxidized fatty acids to cultured cells, where they were incorporated into cellular phospholipids, and untargeted lipidomics by LC-quadrupole/TOF-MS was applied to collect MS/MS spectra for the OxPLs. Based on the measured MS/MS spectra for about 400 molecular species of the biogenic OxPLs, we developed a broad-targeted lipidomics system using triple quadrupole MS. Separation precision of structural isomers was optimized by multiple reaction monitoring analysis and this system enabled us to detect OxPLs at levels as low as 10 fmol. When applied to biological samples, i.e., mouse peritoneal macrophages, this system enabled us to monitor a series of OxPLs endogenously produced in a 12/15-lipoxygenase-dependent manner. This advanced analytical method will be useful to elucidate the structure-specific behavior of OxPLs and their physiological relevance in vivo.

Coffee extract inhibits adipogenesis in 3T3-L1 preadipocyes by interrupting insulin signaling through the downregulation of IRS1.

Although epidemiological data have indicated that a strong negative association exists between coffee consumption and the prevalence of obesity-associated diseases, the molecular mechanisms by which coffee intake prevents obesity-associated diseases has not yet been elucidated. In this study, we found that coffee intake significantly suppressed high-fat diet (HFD)-induced metabolic alternations such as increases in body weight and the accumulation of adipose tissue, and up-regulation of glucose, free fatty acid, total cholesterol and insulin levels in the blood. We also found that coffee extract significantly inhibited adipogenesis in 3T3-L1 preadipocytes. In the early phase of adipogenesis, 3T3-L1 cells treated with coffee extract displayed the retardation of cell cycle entry into the G2/M phase called as mitotic clonal expansion (MCE). Coffee extract also inhibited the activation of CCAAT/enhancer-binding protein ß (C/EBPß) by preventing its phosphorylation by ERK. Furthermore, the coffee extract suppressed the adipogenesis-related events such as MCE and C/EBPß activation through the down-regulation of insulin receptor substrate 1 (IRS1). The stability of the IRS1 protein was markedly decreased by the treatment with coffee extract due to proteasomal degradation. These results have revealed an anti-adipogenic function for coffee intake and identified IRS1 as a novel target for coffee extract in adipogenesis.

Coffee inhibits adipocyte differentiation via inactivation of PPARγ.

Recent epidemiological studies showed that coffee consumption is associated with a lower risk of type 2 diabetes, presumably due to suppression of excess fat accumulation in adipocytes. However, the mechanism underlying the effect of coffee on adipocyte differentiation has not been well documented. To elucidate the mechanism, we investigated the effect of coffee on the differentiation of mouse preadipocyte 3T3-L1 cells. Coffee reduced the accumulation of lipids during adipocytic differentiation of 3T3-L1 cells. At 5% coffee, the accumulation of lipids decreased to half that of the control. Coffee also inhibited the expression of the peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor controlling the differentiation of adipocytes. Furthermore, coffee reduced the expression of other differentiation marker genes, aP2, adiponectin, CCAAT-enhancer-binding protein α (C/EBPα), glucose transporter 4 (GLUT4), and lipoprotein lipase (LPL), during adipocyte differentiation. Major bioactive constituents in coffee extracts, such as caffeine, trigonelline, chlorogenic acid, and caffeic acid, showed no effect on PPARγ gene expression. The inhibitory activity was produced by the roasting of the coffee beans.

Dexamethasone suppresses neurosteroid biosynthesis via downregulation of steroidogenic enzyme gene expression in human glioma GI-1 cells.

Emerging evidence indicates that stress hormone glucocorticoids (GC) are an important modulator of brain development and function. To investigate whether GCs modulate neurosteroid biosynthesis in neural cells, we studied the effects of GCs on steroidogenic gene expression in human glioma GI-1 cells. The GC dexamethasone (Dex) reduced steroidogenic acute regulatory protein (StAR), CYP11A1 and 3ß-hydroxysteroid dehydrogenase gene expression in a dose- and GC receptor-dependent manner. In addition to its effects on steroidogenic gene expression, Dex also reduced de novo synthesis of progesterone (PROG). Furthermore, Dex inhibited all-trans retinoic acid (ATRA) and vitamin D3-induced steroidogenic gene expression and PROG production. This suggests that GC regulates steroidogenic gene expression in neural cells via cross-talk with the two fat-soluble vitamins, A and D. The relationship between the effects of GCs on neurosteroid biosynthesis and on cognitive behaviors and hippocampal neural activity is also discussed herein.