Suzumi M. Tokuoka

Selective ablation of mast cells or basophils reduces peanut-induced anaphylaxis in mice

BACKGROUND Studies with c-kit mutant mast cell (MC)-deficient mice and antibody-mediated depletion of basophils suggest that both MCs and basophils can contribute to peanut-induced anaphylaxis (PIA). However, interpretation of data obtained by using such approaches is complicated because c-kit mutant mice have several phenotypic abnormalities in addition to MC deficiency and because basophil-depleting antibodies can also react with MCs. OBJECTIVE We analyzed (1) the changes in the features of PIA in mice after the selective and inducible ablation of MCs or basophils and (2) the possible importance of effector cells other than MCs and basophils in the PIA response. METHODS Wild-type and various mutant mice were orally sensitized with peanut extract and cholera toxin weekly for 4 weeks and challenged intraperitoneally with peanut extract 2 weeks later. RESULTS Peanut-challenged, MC-deficient Kit(W-sh/W-sh) mice had reduced immediate hypothermia, as well as a late-phase decrease in body temperature that was abrogated by antibody-mediated depletion of neutrophils. Diphtheria toxin-mediated selective depletion of MCs or basophils in Mcpt5-Cre;iDTR and Mcpt8(DTR) mice, respectively, and treatment of wild-type mice with the basophil-depleting antibody Ba103 significantly reduced peanut-induced hypothermia. Non-c-kit mutant MC- and basophil-deficient Cpa3-Cre;Mcl-1(fl/fl) mice had reduced but still significant responses to peanut. CONCLUSION Inducible and selective ablation of MCs or basophils in non-c-kit mutant mice can significantly reduce PIA, but partial responses to peanut can still be observed in the virtual absence of both cell types. The neutrophilia in Kit(W-sh/W-sh) mice might influence the responses of these mice in this PIA model.

Involvement of platelet‐activating factor and LIS1 in neuronal migration

Platelet‐activating factor (PAF, 1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine) is a biologically active lipid mediator. We have previously shown the expression of PAF receptor in neurons and microglia. PAF is produced in the brain from its precursor, and degraded by the enzyme PAF acetylhydrolase. LIS1 is a regulatory subunit of PAF acetylhydrolase, and is identical to a gene whose deletion causes the human neuronal migration disorder, type I lissencephaly. Indeed, Lis1 mutant mice display defects in neuronal migration and layering in vivo, and also in cerebellar granule cell migration in vitro. However, the roles of PAF and the PAF receptor in the neuronal migration remain to be determined. Here, we show that PAF receptor‐deficient mice exhibited histological abnormalities in the embryonic cerebellum. PAF receptor‐deficient cerebellar granule neurons migrated more slowly in vitro than wild‐type neurons, consistent with the observation that a PAF receptor antagonist reduced the migration of wild‐type neurons in vitro. Synergistic reduction of neuronal migration was observed in a double mutant of PAF receptor and LIS1. Unexpectedly, PAF affected the migration of PAF receptor‐deficient neurons, suggesting a receptor‐independent pathway for PAF action. The PAF receptor‐independent response to PAF was abolished in granule neurons derived from the double mutant mice. Thus, our results suggest that the migration of cerebellar granule cells is regulated by PAF through receptor‐dependent and receptor‐independent pathways, and that LIS1 is a pivotal molecule that links PAF action and neuronal cell migration both in vivo and in vitro.

Fatty acid remodeling by LPCAT3 enriches arachidonate in phospholipid membranes and regulates triglyceride transport

Polyunsaturated fatty acids (PUFAs) in phospholipids affect the physical properties of membranes, but it is unclear which biological processes are influenced by their regulation. For example, the functions of membrane arachidonate that are independent of a precursor role for eicosanoid synthesis remain largely unknown. Here, we show that the lack of lysophosphatidylcholine acyltransferase 3 (LPCAT3) leads to drastic reductions in membrane arachidonate levels, and that LPCAT3-deficient mice are neonatally lethal due to an extensive triacylglycerol (TG) accumulation and dysfunction in enterocytes. We found that high levels of PUFAs in membranes enable TGs to locally cluster in high density, and that this clustering promotes efficient TG transfer. We propose a model of local arachidonate enrichment by LPCAT3 to generate a distinct pool of TG in membranes, which is required for normal directionality of TG transfer and lipoprotein assembly in the liver and enterocytes. DOI: http://dx.doi.org/10.7554/eLife.06328.001

A comprehensive quantification method for eicosanoids and related compounds by using liquid chromatography/mass spectrometry with high speed continuous ionization polarity switching

Fatty acids and related metabolites, comprising several hundreds of molecular species, are an important target in disease metabolomics, as they are involved in various mammalian pathologies and physiologies. Selected reaction monitoring (SRM) analysis, which is capable of monitoring hundreds of compounds in a single run, has been widely used for comprehensive quantification. However, it is difficult to monitor a large number of compounds with different ionization polarity, as polarity switching requires a sub-second period per cycle in classical mass spectrometers. In the present study, we developed and evaluated a comprehensive quantification method for eicosanoids and related compounds by using LC/MS with high-speed continuous ionization polarity switching. The new method employs a fast (30ms/cycle) continuous ionization polarity switching, and differentiates 137 targets either by chromatography or by SRM transition. Polarity switching did not affect the lower limits of quantification, which ranged similarly from 0.5 to 200pg on column. Lipid extracts from mouse tissues were analyzed by this method, and 65 targets were quantitatively detected in the brain, including 6 compounds analyzed in the positive ion mode. We demonstrated that a fast continuous ionization polarity switching enables the quantification of a wide variety of lipid mediator species without compromising the sensitivity and reliability.

Fever Is Mediated by Conversion of Endocannabinoid 2-Arachidonoylglycerol to Prostaglandin E2.

Fever is a common response to inflammation and infection. The mechanism involves prostaglandin E2 (PGE2)-EP3 receptor signaling in the hypothalamus, which raises the set point of hypothalamic thermostat for body temperature, but the lipid metabolic pathway for pyretic PGE2 production remains unknown. To reveal the molecular basis of fever initiation, we examined lipopolysaccharides (LPS)-induced fever model in monoacylglycerol lipase (MGL)-deficient (Mgll −/−) mice, CB1 receptor-MGL compound-deficient (Cnr1 −/− Mgll −/−) mice, cytosolic phospholipase A2α (cPLA2α)-deficient (Pla2g4a −/−) mice, and diacylglycerol lipase α (DGLα)-deficient (Dagla −/−) mice. Febrile reactions were abolished in Mgll −/− and Cnr1 −/− Mgll −/− mice, whereas Cnr1 −/− Mgll +/+, Pla2g4a −/− and Dagla −/− mice responded normally, demonstrating that MGL is a critical enzyme for fever, which functions independently of endocannabinoid signals. Intracerebroventricular administration of PGE2 caused fever similarly in Mgll −/− and wild-type control mice, suggesting a lack of pyretic PGE2 production in Mgll −/− hypothalamus, which was confirmed by lipidomics analysis. Normal blood cytokine responses after LPS administration suggested that MGL-deficiency does not affect pyretic cytokine productions. Diurnal body temperature profiles were normal in Mgll −/− mice, demonstrating that MGL is unrelated to physiological thermoregulation. In conclusion, MGL-dependent hydrolysis of endocannabinoid 2-arachidonoylglycerol is necessary for pyretic PGE2 production in the hypothalamus.

Alkylglycerol monooxygenase as a potential modulator for PAF synthesis in macrophages.

Alkylglycerol monooxygenase (AGMO, glyceryl ether monooxygenase) is an enzyme known to catalyze the cleavage of the O-alkyl bond of glyceryl ether lipids. Identification of the gene encoding AGMO was reported recently, however, the involvement of AGMO in modulating cellular lipids has not been reported until now. In this report, we investigate a possible role for AGMO in macrophage platelet-activating factor (PAF) production. AGMO mRNA expression levels decreased with lipopolysaccharide (LPS) treatments in mouse peritoneal macrophages and RAW264.7 cells. Tetrahydrobiopterin-dependent conversion of lyso-PAF to glycerophosphocholine in the microsomal fraction was also reduced in LPS-treated RAW264.7 cells. In the LPS-treated cells, both lyso-PAF and PAF levels increased. Moreover, exogenously expressed AGMO caused a reduction in cellular lyso-PAF and PAF levels in HEK293 cells. Collectively, our results suggest a possible mechanism for AGMO in modulating macrophage PAF production by regulating cellular lyso-PAF levels.

Relief from neuropathic pain by blocking of the platelet-activating factor–pain loop

Neuropathic pain resulting from peripheral neuronal damage is largely resistant to treatment with currently available analgesic drugs. Recently, ATP, lysophosphatidic acid, and platelet‐activating factor (PAF) have been reported to play important inductive roles in neuropathic pain. In the present study, we found that pain‐like behaviors resulting from partial sciatic nerve ligation (PSL) were largely attenuated by deficiency of lysophosphatidylcholine acyltransferase (LPCAT)2, which is one of the PAF biosynthetic enzymes. By contrast, deficiency of the other PAF biosynthetic enzyme, LPCAT1, did not ameliorate neuropathic pain. With regard to the mechanism of the observed effects, LPCAT2 was detected in wild‐type spinal cord microglia, and the absence of LPCAT2 expression precluded spinal PAF expression in LPCAT2‐knockout mice. Furthermore, ATP‐stimulated PAF biosynthesis in macrophages was decreased by pretreatment with the PAF receptor antagonist ABT‐491, indicating the existence of a positive feedback loop of PAF biosynthesis, which we designated the PAF–pain loop. In conclusion, LPCAT2 is a novel therapeutic target for newly categorized analgesic drugs; in addition, our data call for the reevaluation of the clinical utility of PAF receptor antagonists.—Shindou, H., Shiraishi, S., Tokuoka, S. M., Takahashi Y., Harayama, T., Abe, T., Bando, K., Miyano, K., Kita, Y., Uezono, Y., Shimizu, T. Relief from neuropathic pain by blocking of the platelet‐activating factor–pain loop. FASEB J. 31, 2973–2980 (2017). www.fasebj.org

Multiplex quantitative analysis of eicosanoid mediators in human plasma and serum: Possible introduction into clinical testing

Eicosanoid mediators play important roles in maintaining the physiological and pathophysiological homeostasis in the body. Their measurements, however, are rarely performed in clinical practice. In the present study, we analyzed 30 varieties of eicosanoid mediators that were detectable in human plasma and serum collected from healthy donors, using liquid chromatography-tandem mass spectrometry from the viewpoint of the clinical application of the multiplex quantitation of eicosanoid mediators. Wider variety of eicosanoid mediators were detected in serum (27 out of 30) than in plasma (14 out of 30), since the serum was thought to contain lipid mediators released from activated platelets. Larger inter-individual variations were observed in the plasma and serum eicosanoid levels. On the other hand, the concentrations of eicosanoids were not affected by the platelet count but were affected by the concentration of arachidonic acid (AA) within the reference interval (17.4-40.5×1010/L). When serum samples from patients with hematological disorders were analyzed, the concentrations of AA were positively correlated with the platelet count. When the patients underwent ASA therapy, a marked decrease in the concentrations of thromboxane B2 (TXB2) and 12-hydroxyl-heptadecatrienoic acid (12-HHT) was observed. Considering the availability of serum samples in clinical settings, the serum analysis of eicosanoids may be clinically useful.

Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy

Fatty acids are taken up by cells and incorporated into complex lipids such as neutral lipids and glycerophospholipids. Glycerophospholipids are major constituents of cellular membranes. More than 1000 molecular species of glycerophospholipids differ in their polar head groups and fatty acid compositions. They are related to cellular functions and diseases and have beenwell analyzed by mass spectrometry. However, intracellular imaging of fatty acids and glycerophospholipids has not been successful due to insufficient resolution using conventional methods. Here, we developed a method for labeling fatty acids with bromine (Br) and applied scanning X‐ray fluorescence microscopy (SXFM) to obtain intracellular Br mapping data with submicrometer resolution. Mass spectrometry showed that cells took up Br‐labeled fatty acids andmetabolized them mainly into glycerophospholipids in CHO cells. Most Br signals observed by SXFM were in the perinuclear region. Higher resolution revealed a spot‐like distribution of Br inthe cytoplasm. Thecurrentmethodenabledsuccessful visualizationof intracellular Br‐labeledfatty acids. Single‐element labeling combined with SXFM technology facilitates the intracellular imaging of fatty acids, whichprovides anewtool todeterminedynamic changes infattyacids andtheirderivatives at the single‐cell level.—A., Kita, Y., Ishizaka, Y., Yamauchi, K., Kohmura, Y., Lobinski, R., Shimizu, I., Shimizu, T. Imaging of intracellular fatty acids by scanning X‐ray fluorescence microscopy. FASEB J. 30, 4149–4158 (2016). www.fasebj.org

Monoacylglycerol lipase deficiency affects diet-induced obesity, fat absorption, and feeding behavior in CB1 cannabinoid receptor–deficient mice

Excess energy intake causes obesity, which leads to insulin resistance and various other complications of metabolic syndrome, including diabetes, atherosclerosis, dyslipidemia, and nonalcoholic fatty liver disease. Although recent studies have depicted altered lipid metabolism as an underlying feature, the detailed mechanisms are still unclear. Here we describe a possible role in high‐fat diet (HFD)‐induced obesity for monoacylglycerol lipase (MGL), an enzyme that is also known to hydrolyze the endocannabinoid 2‐arachidonoylglycerol in brain. MGL‐deficient [MGL‐knockout (KO)] mice fed a HFD gained less body weight than wild‐type mice and were protected from insulin resistance and hepatic steatosis. Food intake and energy expenditure were not altered in MGL‐KO mice, but blood triglyceride levels after oral olive oil gavage were suppressed, indicating a role for MGL in intestinal fat absorption. Experiments with cannabinoid receptor type 1 (CB1)/MGL double‐KO mice revealed that these phenotypes may include mechanisms that are independent of CB1‐receptor–mediated endocannabinoid functions. We also noted that MGL‐KO mice had less preference for HFD over normal chow diet. Oral but not intraperitoneal lipid administration strongly suppressed the appetites of MGL‐KO and CB1/MGL double‐KO mice, but not of wild‐type and CB1‐KO mice. Appetite suppression was reversed by vagotomy, suggesting involvement of MGL in the gut–brain axis regulation of appetite. Our results provide mechanistic insights of MGLs role in diet‐induced obesity, lipid metabolic disorder, and regulation of appetite.—Yoshida, K., Kita, Y., Tokuoka, S. M., Hamano, F., Yamazaki, M., Sakimura, K., Kano, M., Shimizu, T. Monoacylglycerol lipase deficiency affects diet‐induced obesity, fat absorption, and feeding behavior in CB1 cannabinoid receptor–deficient mice. FASEB J. 33, 2484–2497 (2019). www.fasebj.org