Arachidonic acid-derived dihydroxy fatty acids in neonatal cord blood relate symptoms of autism spectrum disorders and social adaptive functioning: Hamamatsu Birth Cohort for Mothers and Children (HBC Study).

AIM: Autism spectrum disorder (ASD) is associated with abnormal lipid metabolism, such as a high total ratio of omega-6 to omega-3 in polyunsaturated fatty acids (PUFAs). PUFAs are metabolized to epoxy fatty acids by cytochrome P450 (CYP); then, dihydroxy fatty acid is produced by soluble epoxide hydrolase. This study examined the association between PUFA metabolites in the cord blood and ASD symptoms and adaptive functioning in children. METHODS: This prospective cohort study utilized cord blood to quantify PUFA metabolites of the CYP pathway. The Autism Diagnostic Observation Schedule (ADOS-2) and Vineland Adaptive Behaviors Scales, Second Edition (VABS-II) were used to assess subsequent ASD symptoms and adaptive functioning in children at 6 years. The analysis included 200 children and their mothers. RESULTS: Arachidonic acid-derived diols, 11,12-diHETrE was found to impact ASD symptom severity on the ADOS-2-calibrated severity scores and impairment in the socialization domain as assessed by the VABS-II (P = 0.0003; P = 0.004, respectively). High levels of 11,12-diHETrE impact social affect in ASD symptoms (P = 0.002), while low levels of 8,9-diHETrE impact repetitive/restrictive behavior (P = 0.003). Notably, there was specificity in the association between diHETrE and ASD symptoms, especially in girls. CONCLUSION: These findings suggest that the dynamics of diHETrE during the fetal period is important in the developmental trajectory of children after birth. Given that the role of diol metabolites in neurodevelopment in vivo is completely uncharacterized, the results of this study provide important insight into the role of diHETrE and ASD pathophysiology.

Linoleic acid and linoleate diols in neonatal cord blood influence birth weight.

Background: Low-birth-weight infants exhibit a high risk for postnatal morbidity. Cytochrome P450 (CYP) and epoxide hydrolase (EH) are involved in the metabolism of factors responsible for low-birth-weight in infants. Both CYPs and EHs have high substrate specificity and are involved in polyunsaturated fatty acid (PUFA) metabolism. The CYP pathway produces epoxy fatty acids (EpFAs), which are further degraded by soluble EH (sEH). Additionally, sEH inhibition enhances the action of EpFAs and suppresses inflammatory responses. During pregnancy, excessive activation of maternal inflammatory response is a significant factor associated with low-birth-weight. However, the association of EpFAs, which have potential anti-inflammatory properties, with the low-birth-weight of infants remains uninvestigated. This study aimed to clarify the association between the umbilical cord serum EpFA and low-birth-weight using data obtained from the Hamamatsu Birth Cohort for Mothers and Children (HBC Study) by analyzing the umbilical cord blood samples. Method: We selected a subgroup of 200 infants (106 boys and 94 girls), quantified EpFA concentration in their cord blood samples collected at birth, and examined its correlation with birth weight. Results: The comparison between the low-birth-weight and normal-birth-weight groups revealed no significant correlation between PUFA and EpFA concentrations, but a significant correlation was observed in the linoleate diol concentrations of the two groups. Furthermore, birth weight did not significantly correlate with PUFA, EpFA, and diol concentrations in cord blood; however, multiple regression analysis showed a significant negative correlation of birth weight with the concentration of linoleic acid (LA) (r = -0.101, p = 0.016) as well as LA-derived dihydroxyoctadecenoic acid (diHOME) (r = -0.126, p = 0.007), 9,10-diHOME (r = -0.115, p = 0.014), and 12,13-diHOME (r = -0.126, p = 0.007) after adjusting for obstetric factors, including gestational age, infant's sex, childbirth history, delivery method, and maternal height. Conclusions: Birth weight was significantly correlated with the concentration of LA and linoleate diol diHOME after adjusting for obstetric confounders. Our results show that CYP and sEH involved in PUFA metabolism may influence the birth weight of infants. Further validation is needed to provide insights regarding maternal intervention strategies required to avoid low-birth-weight in infants in the future.

Production of Hydroxy Fatty Acids, Precursors of γ-Hexalactone, Contributes to the Characteristic Sweet Aroma of Beef.

Aroma is an essential factor for meat quality. The meat of Japanese Black cattle exhibits fine marbling and a rich and sweet aroma with a characteristic lactone composition. The mechanism of lactone formation associated with beef aroma has not been elucidated. In this study, we examined the precursors of γ-hexalactone, an indicator of the sweet aroma of beef and identified the mechanism underlying γ-hexalactone production. A low-temperature vacuum system was used to prepare beef tallow from Japanese Black cattle and Holstein cattle. The odor components were identified using headspace-gas chromatography. The analysis revealed that γ-hexalactone, γ-dodecalactone, δ-tetradecalactone, and δ-hexadecalactone were present as sweet aroma components of beef tallow prepared from marbling and muscle. Since we previously reported that γ-hexalactone formation correlates with linoleic acid content in beef, we analyzed ten oxidized fatty acids derived from linoleic acid by liquid chromatography-triple quadrupole mass spectrometry and detected two hydroxy-octadecadienoic acids (9S-HODE and 13S-HODE) in beef tallow. Significant differences in arachidonic acid 15-lipoxygenase and cyclooxygenase protein expression levels among subcutaneous fat, intramuscular fat, and muscle tissue were observed. Our results suggest that the combination of linoleic acid and the expression of lipid oxidase derived from beef muscle and intramuscular fat produce hydroxy fatty acids that result in a sweet aroma.

Fatty Acid Synthesis Is Indispensable for Survival of Human Pluripotent Stem Cells.

The role of lipid metabolism in human pluripotent stem cells (hPSCs) is poorly understood. We have used large-scale targeted proteomics to demonstrate that undifferentiated hPSCs express different fatty acid (FA) biosynthesis-related enzymes, including ATP citrate lyase and FA synthase (FASN), than those expressed in hPSC-derived cardiomyocytes (hPSC-CMs). Detailed lipid profiling revealed that inhibition of FASN resulted in significant reduction of sphingolipids and phosphatidylcholine (PC); moreover, we found that PC was the key metabolite for cell survival in hPSCs. Inhibition of FASN induced cell death in undifferentiated hPSCs via mitochondria-mediated apoptosis; however, it did not affect cell survival in hPSC-CMs, neurons, or hepatocytes as there was no significant reduction of PC. Furthermore, we did not observe tumor formation following transplantation of FASN inhibitor-treated cells. Our findings demonstrate the importance of de novo FA synthesis in the survival of undifferentiated hPSCs and suggest applications for FASN inhibition in regenerative medicine.

Microglia-released leukotriene B4 promotes neutrophil infiltration and microglial activation following intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) from blood vessel rupture results in parenchymal hematoma formation and neuroinflammation, ultimately leading to neurodegeneration. Several lines of evidence suggest that the severity of ICH-induced neural damage is exacerbated by infiltration of T-cells, monocytes, and especially neutrophils into the hematoma. Neutrophil migration is regulated by chemokines, formyl peptides, and leukotriene B4 (LTB4), a metabolite of arachidonic acid. In this study, we demonstrate that LTB4 is a key signaling factor promoting microglial activity and leukocyte infiltration into hematoma and thus a potentially critical determinant of ICH pathogenesis and clinical outcome. Lipidomic analysis revealed markedly increased LTB4 concentration in the hematoma-containing brain tissues 6-24 h after experimental ICH in mice. Expression of 5-lipoxygenase, a rate-limiting enzyme for LTB4 production, was upregulated in activated microglia and neutrophils within the hematoma following ICH. Treatment of cultured BV-2 microglia with thrombin, which is abundant in hematoma, promoted activation, proinflammatory cytokine expression, and LTB4 secretion. Further, conditioned medium from thrombin-stimulated BV-2 cells potentiated the transwell migration of neutrophil-like cells, a response blocked by a LTB4 receptor antagonist. These results suggest that arachidonic acid conversion to LTB4 following ICH contributes to neuroinflammation and ensuing neural tissue damage by inducing microglial activation and neutrophil recruitment.

Lysophosphatidic acid-induced YAP/TAZ activation promotes developmental angiogenesis by repressing Notch ligand Dll4.

Lysophosphatidic acid (LPA) is a potent lipid mediator with various biological functions mediated through six G protein-coupled receptors (GPCRs), LPA1-6. Previous studies have demonstrated that LPA-Gα12/Gα13 signaling plays an important role in embryonic vascular development. However, the responsible LPA receptors and underlying mechanisms are poorly understood. Here, we show a critical role of LPA4 and LPA6 in developmental angiogenesis. In mice, Lpa4;Lpa6 double knockout (DKO) embryos were lethal due to global vascular deficiencies, and endothelial cell (EC)-specific Lpa4;Lpa6 DKO retinas had impaired sprouting angiogenesis. Mechanistically, LPA activated the transcriptional regulators YAP and TAZ through LPA4/LPA6-mediated Gα12/Gα13-Rho-ROCK signaling in ECs. YAP/TAZ knockdown increased ß-catenin- and Notch intracellular domain (NICD)-mediated endothelial expression of the Notch ligand delta-like 4 (DLL4). Fibrin gel sprouting assay revealed that LPA4/LPA6, Gα12/Gα13, or YAP/TAZ knockdown consistently blocked EC sprouting, which was rescued by a Notch inhibitor. Of note, the inhibition of Notch signaling also ameliorated impaired retinal angiogenesis in EC-specific Lpa4;Lpa6 DKO mice. Overall, these results suggest that the Gα12/Gα13-coupled receptors LPA4 and LPA6 synergistically regulate endothelial Dll4 expression through YAP/TAZ activation. This could in part account for the mechanism of YAP/TAZ-mediated developmental angiogenesis. Our findings provide a novel insight into the biology of GPCR-activated YAP/TAZ.

Sirt1 counteracts decrease in membrane phospholipid unsaturation and diastolic dysfunction during saturated fatty acid overload.

BACKGROUND: The fatty acid (FA) composition of membrane phospholipid reflects at least in part dietary fat composition. Saturated FA (SFA) suppress Sirt1 activity, while monounsaturated FA (MUFA) counteract this effect. OBJECTIVE: We explored a role of Sirt1 in homeostatic control of the fatty acid composition of membrane phospholipid in the presence of SFA overload. METHODS AND RESULTS: Sirt1 deficiency in cardiomyocytes decreased the expression levels of liver X receptor (LXR)-target genes, particularly stearoyl-CoA desaturase-1 (Scd1), a rate-limiting enzyme in the cellular synthesis of MUFA from SFA, increased membrane SFA/MUFA ratio, and worsened left ventricular (LV) diastolic function in mice fed an SFA-rich high fat diet. In cultured cardiomyocytes, Sirt1 knockdown (KD) exacerbated the palmitate overload-induced increase in membrane SFA/MUFA ratio, which was associated with decrease in the expression of LXR-target genes, including Scd1. Forced overexpression of Scd1 in palmitate-overloaded Sirt1KD cardiomyocytes lowered the SFA/MUFA ratio. Nicotinamide mononucleotide (NMN) increased Sirt1 activity and Scd1 expression, thereby lowering membrane SFA/MUFA ratio in palmitate-overloaded cardiomyocytes. These effects of NMN were not observed for Scd1KD cardiomyocytes. LXRα/ßKD exacerbated palmitate overload-induced increase in membrane SFA/MUFA ratio, while LXR agonist T0901317 alleviated it. NMN failed to rescue Scd1 protein expression and membrane SFA/MUFA ratio in palmitate-overloaded LXRα/ßKD cardiomyocytes. The administration of NMN or T0901317 showed a dramatic reversal in membrane SFA/MUFA ratio and LV diastolic function in SFA-rich HFD-fed mice. CONCLUSION: Cardiac Sirt1 counteracted SFA overload-induced decrease in membrane phospholipid unsaturation and diastolic dysfunction via regulating LXR-mediated transcription of the Scd1 gene.

Basal expression of interferon regulatory factor 1 drives intrinsic hepatocyte resistance to multiple RNA viruses.

Current models of cell-intrinsic immunity to RNA viruses centre on virus-triggered inducible antiviral responses initiated by RIG-I-like receptors or Toll-like receptors that sense pathogen-associated molecular patterns, and signal downstream through interferon regulatory factors (IRFs), transcription factors that induce synthesis of type I and type III interferons1. RNA viruses have evolved sophisticated strategies to disrupt these signalling pathways and evade elimination by cells, attesting to their importance2. Less attention has been paid to how IRFs maintain basal levels of protection against viruses. Here, we depleted antiviral factors linked to RIG-I-like receptor and Toll-like receptor signalling to map critical host pathways restricting positive-strand RNA virus replication in immortalized hepatocytes and identified an unexpected role for IRF1. We show that constitutively expressed IRF1 acts independently of mitochondrial antiviral signalling (MAVS) protein, IRF3 and signal transducer and activator of transcription 1 (STAT1)-dependent signalling to provide intrinsic antiviral protection in actinomycin D-treated cells. IRF1 localizes to the nucleus, where it maintains the basal transcription of a suite of antiviral genes that protect against multiple pathogenic RNA viruses, including hepatitis A and C viruses, dengue virus and Zika virus. Our findings reveal an unappreciated layer of hepatocyte-intrinsic immunity to these positive-strand RNA viruses and identify previously unrecognized antiviral effector genes.

The Gα12/13-coupled receptor LPA4 limits proper adipose tissue expansion and remodeling in diet-induced obesity.

White adipose tissue (WAT) can dynamically expand and remodel through adipocyte hypertrophy and hyperplasia. The relative contribution of these 2 mechanisms to WAT expansion is a critical determinant of WAT function and dysfunction in obesity. However, little is known about the signaling systems that determine the mechanisms of WAT expansion. Here, we show that the GPCR LPA4 selectively activates Gα12/13 proteins in adipocytes and limits continuous remodeling and healthy expansion of WAT. LPA4-KO mice showed enhanced expression of mitochondrial and adipogenesis genes and reduced levels of inhibitory phosphorylation of PPARγ in WAT, along with increased production of adiponectin. Furthermore, LPA4-KO mice showed metabolically healthy obese phenotypes in a diet-induced obesity model, with continuous WAT expansion, as well as protection from WAT inflammation, hepatosteatosis, and insulin resistance. These findings unravel a potentially new signaling system that underlies WAT plasticity and expandability, providing a promising therapeutic approach for obesity-related metabolic disorders.

Loss of DDHD2, whose mutation causes spastic paraplegia, promotes reactive oxygen species generation and apoptosis.

DDHD2/KIAA0725p is a mammalian intracellular phospholipase A1 that exhibits phospholipase and lipase activities. Mutation of the DDHD2 gene causes hereditary spastic paraplegia (SPG54), an inherited neurological disorder characterized by lower limb spasticity and weakness. Although previous studies demonstrated lipid droplet accumulation in the brains of SPG54 patients and DDHD2 knockout mice, the cause of SPG54 remains elusive. Here, we show that ablation of DDHD2 in mice induces age-dependent apoptosis of motor neurons in the spinal cord. In vitro, motor neurons and embryonic fibroblasts from DDHD2 knockout mice fail to survive and are susceptible to apoptotic stimuli. Chemical and probe-based analysis revealed a substantial decrease in cardiolipin content and an increase in reactive oxygen species generation in DDHD2 knockout cells. Reactive oxygen species production in DDHD2 knockout cells was reversed by the expression of wild-type DDHD2, but not by an active-site DDHD2 mutant, DDHD2 mutants related to hereditary spastic paraplegia, or DDHD1, another member of the intracellular phospholipase A1 family whose mutation also causes spastic paraplegia (SPG28). Our results demonstrate the protective role of DDHD2 for mitochondrial integrity and provide a clue to the pathogenic mechanism of SPG54.

The lysophosphatidic acid receptor LPA4 regulates hematopoiesis-supporting activity of bone marrow stromal cells.

Lysophosphatidic acid (LPA) is a pleiotropic lipid mediator that acts through G protein-coupled receptors (LPA1-6). Although several biological roles of LPA4 are becoming apparent, its role in hematopoiesis has remained unknown. Here, we show a novel regulatory role for LPA4 in hematopoiesis. Lpar4 mRNA was predominantly expressed in mouse bone marrow (BM) PDGFRα(+) stromal cells, known as the components of the hematopoietic stem/progenitor cell (HSPC) niche. Compared with wild-type mice, LPA4-deficient mice had reduced HSPC numbers in the BM and spleen and were hypersusceptible to myelosuppression, most likely due to impairments in HSPC recovery and stem cell factor production in the BM. Analysis of reciprocal BM chimeras (LPA4-deficient BM into wild-type recipients and vice versa) indicated that stromal cells likely account for these phenotypes. Consistently, LPA4-deficient BM stromal cells showed downregulated mRNA expression of stem cell factor and tenascin-c in vitro. Taken together, these results suggest a critical and novel role for the LPA/LPA4 axis in regulating BM stromal cells.

Trilogy of ACE2: a peptidase in the renin-angiotensin system, a SARS receptor, and a partner for amino acid transporters.

Angiotensin-converting enzyme (ACE) 2 is a homolog to the carboxypeptidase ACE, which generates angiotensin II, the main active peptide of renin-angiotensin system (RAS). After the cloning of ACE2 in 2000, three major ACE2 functions have been described so far. First ACE2 has emerged as a potent negative regulator of the RAS counterbalancing the multiple functions of ACE. By targeting angiotensin II ACE2 exhibits a protective role in the cardiovascular system and many other organs. Second ACE2 was identified as an essential receptor for the SARS coronavirus that causes severe acute lung failure. Downregulation of ACE2 strongly contributes to the pathogenesis of severe lung failure. Third, both ACE2 and its homologue Collectrin can associate with amino acid transporters and play essential role in the absorption of amino acids in the kidney and gut. In this review, we will discuss the multiple biological functions of ACE2.

Angiotensin-converting enzyme 2 (ACE2) in disease pathogenesis.

Angiotensin-converting enzyme 2 (ACE2), a first homolog of ACE, regulates the renin-angiotensin system by counterbalancing ACE activity. Accumulating evidence in recent years has demonstrated a physiological and pathological role of ACE2 in the cardiovascular, renal and respiratory systems. For instance, in the acute respiratory distress syndrome (ARDS), ACE, AngII, and AT1R promote the disease pathogenesis, whereas ACE2 and the AT2R protect from ARDS. Importantly, ACE2 has been identified as a key SARS-coronavirus receptor and plays a protective role in SARS pathogenesis. Furthermore, the recent explosion of research into the ACE2 homolog, collectrin, has revealed a new physiological function of ACE2 as an amino acid transporter, which explains the pathogenic role of gene mutations in Hartnup disorder. This review summarizes and discusses the recently unveiled roles for ACE2 in disease pathogenesis.