PLAAT1 deficiency alleviates high-fat diet-induced hepatic lipid accumulation in mice.

The phospholipase A and acyltransferase (PLAAT) family is composed of three isoforms in mice (PLAAT1, 3, and 5), all of which function as phospholipid-metabolizing enzymes exhibiting phospholipase A1 /A2 and acyltransferase activities. Plaat3-deficient (Plaat3-/- ) mice were previously reported to show lean phenotype and remarkable hepatic fat accumulation under high-fat diet (HFD) feeding, while Plaat1-/- mice have not been analyzed. In the present study, we generated Plaat1-/- mice and investigated the effects of PLAAT1 deficiency on HFD-induced obesity, hepatic lipid accumulation, and insulin resistance. After HFD treatment, PLAAT1 deficiency caused a lower body weight gain compared to wild-type mice. Plaat1-/- mice also showed reduced liver weight with negligible hepatic lipid accumulation. In accordance with these findings, PLAAT1 deficiency improved HFD-induced hepatic dysfunction and lipid metabolism disorders. Lipidomics analysis in the liver revealed that in Plaat1-/- mice, the levels of various glycerophospholipids tended to increase, while all classes of lysophospholipids examined tended to decrease, suggesting that PLAAT1 functions as phospholipase A1 /A2 in the liver. Interestingly, the HFD treatment of wild-type mice significantly increased the mRNA level of PLAAT1 in the liver. Furthermore, the deficiency did not appear to elevate the risk of insulin resistance in contrast to PLAAT3 deficiency. These results suggested that the suppression of PLAAT1 improves HFD-induced overweight and concomitant hepatic lipid accumulation.

Roles of Endocannabinoids and Endocannabinoid-Like Molecules in Energy Homeostasis and Metabolic Regulation: A Nutritional Perspective.

The endocannabinoid system is involved in signal transduction in mammals. It comprises principally G protein-coupled cannabinoid receptors and their endogenous agonists, called endocannabinoids, as well as the enzymes and transporters responsible for the metabolism of endocannabinoids. Two arachidonic acid-containing lipid molecules, arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol, function as endocannabinoids. N-acylethanolamines and monoacylglycerols, in which the arachidonic acid chain is replaced with a saturated or monounsaturated fatty acid, are not directly involved in the endocannabinoid system but exhibit agonistic activities for other receptors. These endocannabinoid-like moleculesinclude palmitoylethanolamide, oleoylethanolamide (OEA), and 2-oleoylglycerol. Endocannabinoids stimulate feeding behavior and the anabolism of lipids and glucose, while OEA suppresses appetite. Both central and peripheral systems are included in these nutritional and metabolic contexts. Therefore, they have potential in the treatment and prevention of obesity. We outline the structure, metabolism, and biological activities of endocannabinoids and related molecules, and focus on their involvement in energy homeostasis and metabolic regulation.

Involvement of the γ Isoform of cPLA2 in the Biosynthesis of Bioactive N-Acylethanolamines.

Arachidonylethanolamide (anandamide) acts as an endogenous ligand of cannabinoid receptors, while other N-acylethanolamines (NAEs), such as palmitylethanolamide and oleylethanolamide, show analgesic, anti-inflammatory, and appetite-suppressing effects through other receptors. In mammalian tissues, NAEs, including anandamide, are produced from glycerophospholipid via N-acyl-phosphatidylethanolamine (NAPE). The ɛ isoform of cytosolic phospholipase A2 (cPLA2) functions as an N-acyltransferase to form NAPE. Since the cPLA2 family consists of six isoforms (α, ß, γ, δ, ɛ, and ζ), the present study investigated a possible involvement of isoforms other than ɛ in the NAE biosynthesis. Firstly, when the cells overexpressing one of the cPLA2 isoforms were labeled with [14C]ethanolamine, the increase in the production of [14C]NAPE was observed only with the ɛ-expressing cells. Secondly, when the cells co-expressing ɛ and one of the other isoforms were analyzed, the increase in [14C]N-acyl-lysophosphatidylethanolamine (lysoNAPE) and [14C]NAE was seen with the combination of ɛ and γ isoforms. Furthermore, the purified cPLA2γ hydrolyzed not only NAPE to lysoNAPE, but also lysoNAPE to glycerophospho-N-acylethanolamine (GP-NAE). Thus, the produced GP-NAE was further hydrolyzed to NAE by glycerophosphodiesterase 1. These results suggested that cPLA2γ is involved in the biosynthesis of NAE by its phospholipase A1/A2 and lysophospholipase activities.

Phosphatidylserine-stimulated production of N-acyl-phosphatidylethanolamines by Ca2+-dependent N-acyltransferase.

N-acyl-phosphatidylethanolamine (NAPE) is known to be a precursor for various bioactive N-acylethanolamines including the endocannabinoid anandamide. NAPE is produced in mammals through the transfer of an acyl chain from certain glycerophospholipids to phosphatidylethanolamine (PE) by Ca2+-dependent or -independent N-acyltransferases. The ε isoform of mouse cytosolic phospholipase A2 (cPLA2ε) was recently identified as a Ca2+-dependent N-acyltransferase (Ca-NAT). In the present study, we first showed that two isoforms of human cPLA2ε function as Ca-NAT. We next purified both mouse recombinant cPLA2ε and its two human orthologues to examine their catalytic properties. The enzyme absolutely required Ca2+ for its activity and the activity was enhanced by phosphatidylserine (PS). PS enhanced the activity 25-fold in the presence of 1 mM CaCl2 and lowered the EC50 value of Ca2+ >8-fold. Using a PS probe, we showed that cPLA2ε largely co-localizes with PS in plasma membrane and organelles involved in the endocytic pathway, further supporting the interaction of cPLA2ε with PS in living cells. Finally, we found that the Ca2+-ionophore ionomycin increased [14C]NAPE levels >10-fold in [14C]ethanolamine-labeled cPLA2ε-expressing cells while phospholipase A/acyltransferase-1, acting as a Ca2+-independent N-acyltransferase, was insensitive to ionomycin for full activity. In conclusion, PS potently stimulated the Ca2+-dependent activity and human cPLA2ε isoforms also functioned as Ca-NAT.

A quantitative study on splice variants of N-acylethanolamine acid amidase in human prostate cancer cells and other cells.

N-Acylethanolamine acid amidase (NAAA) is a lysosomal enzyme, hydrolyzing various bioactive N-acylethanolamines with a preference for palmitoylethanolamide. Human NAAA mRNA was previously reported to consist of multiple 3'-end splice variants. However, their tissue distributions and roles have not been examined yet. In the present study, we first identified four major splice variants (tentatively referred to as a1, a2, b2, and c2) in a human prostate cancer cell line LNCaP, which were composed of exons 1-11, exons 1-10 and 12, exons 1-9 and 12, and exons 1-8 and 12, respectively. We next developed quantitative polymerase chain reaction methods to individually quantify these NAAA variants as well as collectively measure all the variants. Among various human prostate cancer cells, the total levels of NAAA mRNAs in androgen-sensitive cells like LNCaP were higher than those in androgen-insensitive cells. In all of these prostate cells and other human cells, variants a1 and b2 showed the highest and lowest expression levels, respectively, among the four variants. Interestingly, ratios of the four variants were different by cell type. Variants a1 and a2 encoded the same full-length NAAA protein, which was catalytically active, while b2 and c2 were translated to C-terminally truncated proteins. As expressed in HEK293 cells these truncated forms were detected as catalytically inactive precursor proteins, but not as mature forms. These results revealed wide distribution of multiple variants of NAAA mRNA in various human cells and suggested that the proteins from some variants are catalytically inactive.

Calcium-dependent generation of N-acylethanolamines and lysophosphatidic acids by glycerophosphodiesterase GDE7.

N-Acylethanolamines form a class of lipid mediators and include an endocannabinoid arachidonoylethanolamide (anandamide), analgesic and anti-inflammatory palmitoylethanolamide, and appetite-suppressing oleoylethanolamide. In animal tissues, N-acylethanolamines are synthesized from N-acylated ethanolamine phospholipids directly by N-acylphosphatidylethanolamine-hydrolyzing phospholipase D or through multi-step pathways via N-acylethanolamine lysophospholipids. We previously reported that glycerophosphodiesterase (GDE) 4, a member of the GDE family, has lysophospholipase D (lysoPLD) activity hydrolyzing N-acylethanolamine lysophospholipids to N-acylethanolamines. Recently, GDE7 was shown to have lysoPLD activity toward lysophosphatidylcholine to produce lysophosphatidic acid (LPA). Here, we examined the reactivity of GDE7 with N-acylethanolamine lysophospholipids as well as the requirement of divalent cations for its catalytic activity. When overexpressed in HEK293 cells, recombinant GDE7 proteins of human and mouse showed lysoPLD activity toward N-palmitoyl, N-oleoyl, and N-arachidonoyl-lysophosphatidylethanolamines and N-palmitoyl-lysoplasmenylethanolamine to generate their corresponding N-acylethanolamines and LPAs. However, GDE7 hardly hydrolyzed glycerophospho-N-palmitoylethanolamine. Overexpression of GDE7 in HEK293 cells increased endogenous levels of N-acylethanolamines and LPAs. Interestingly, GDE7 was stimulated by micromolar concentrations of Ca2+ but not by millimolar concentrations of Mg2+, while GDE4 was stimulated by Mg2+ but was insensitive to Ca2+. GDE7 was widely distributed in various tissues of humans and mice with the highest levels in their kidney tissues. These results suggested that GDE7 is a novel Ca2+-dependent lysoPLD, which is involved in the generation of both N-acylethanolamines and LPAs.

Mammalian enzymes responsible for the biosynthesis of N-acylethanolamines.

Bioactive N-acylethanolamines (NAEs) are ethanolamides of long-chain fatty acids, including palmitoylethanolamide, oleoylethanolamide and anandamide. In animal tissues, NAEs are biosynthesized from membrane phospholipids. The classical "transacylation-phosphodiesterase" pathway proceeds via N-acyl-phosphatidylethanolamine (NAPE), which involves the actions of two enzymes, NAPE-generating Ca2+-dependent N-acyltransferase (Ca-NAT) and NAPE-hydrolyzing phospholipase D (NAPE-PLD). Recent identification of Ca-NAT as Ɛ isoform of cytosolic phospholipase A2 enabled the further molecular biological approaches toward this enzyme. In addition, Ca2+-independent NAPE formation was shown to occur by N-acyltransferase activity of a group of proteins named phospholipase A/acyltransferases (PLAAT)-1-5. The analysis of NAPE-PLD-deficient mice confirmed that NAEs can be produced through multi-step pathways bypassing NAPE-PLD. The NAPE-PLD-independent pathways involved three members of the glycerophosphodiesterase (GDE) family (GDE1, GDE4 and GDE7) as well as α/ß-hydrolase domain-containing protein (ABHD)4. In this review article, we will focus on recent progress made and latest insights in the enzymes involved in NAE synthesis and their further characterization.

Comparative analyses of isoforms of the calcium-independent phosphatidylethanolamine N-acyltransferase PLAAT-1 in humans and mice.

N-Acylphosphatidylethanolamines (NAPEs) are a class of glycerophospholipids, which are known as precursors for different bioactive N-acylethanolamines. We previously reported that phospholipase A/acyltransferase-1 (PLAAT-1), which was originally found in mammals as a tumor suppressor, catalyzes N-acylation of phosphatidylethanolamines to form NAPEs. However, recent online database suggested the presence of an uncharacterized isoform of PLAAT-1 with an extra sequence at the N terminus. In the present study, we examined the occurrence, intracellular localization, and catalytic properties of this longer isoform, as well as the original shorter isoform from humans and mice. Our results showed that human tissues express the longer isoform but not the short isoform at all. In contrast, mice expressed both isoforms with different tissue distribution. Unlike the cytoplasmic localization of the shorter isoform, the long isoform was found in both cytoplasm and nucleus, inferring that the extra sequence harbors a nuclear localization signal. As assayed with purified proteins, neither isoform required calcium for full activity. Moreover, the overexpression of each isoform remarkably increased cellular NAPE levels. These results conclude that the new long isoform of PLAAT-1 is a calcium-independent N-acyltransferase existing in both cytoplasm and nucleus and suggest a possible formation of NAPEs in various membrane structures including nuclear membrane. J. Lipid Res 2016. 57: 2051-2060.

Interaction of Phospholipase A/Acyltransferase-3 with Pex19p: A POSSIBLE INVOLVEMENT IN THE DOWN-REGULATION OF PEROXISOMES.

Phospholipase A/acyltransferase (PLA/AT)-3 (also known as H-rev107 or AdPLA) was originally isolated as a tumor suppressor and was later shown to have phospholipase A1/A2 activity. We have also found that the overexpression of PLA/AT-3 in mammalian cells results in specific disappearance of peroxisomes. However, its molecular mechanism remained unclear. In the present study, we first established a HEK293 cell line, which stably expresses a fluorescent peroxisome marker protein (DsRed2-Peroxi) and expresses PLA/AT-3 in a tetracycline-dependent manner. The treatment with tetracycline, as expected, caused disappearance of peroxisomes within 24 h, as revealed by diffuse signals of DsRed2-Peroxi and a remarkable decrease in a peroxisomal membrane protein, PMP70. A time-dependent decrease in ether-type lipid levels was also seen. Because the activation of LC3, a marker of autophagy, was not observed, the involvement of autophagy was unlikely. Among various peroxins responsible for peroxisome biogenesis, Pex19p functions as a chaperone protein for the transportation of peroxisomal membrane proteins. Immunoprecipitation analysis showed that PLA/AT-3 binds to Pex19p through its N-terminal proline-rich and C-terminal hydrophobic domains. The protein level and enzyme activity of PLA/AT-3 were increased by its coexpression with Pex19p. Moreover, PLA/AT-3 inhibited the binding of Pex19 to peroxisomal membrane proteins, such as Pex3p and Pex11ßp. A catalytically inactive point mutant of PLA/AT-3 could bind to Pex19p but did not inhibit the chaperone activity of Pex19p. Altogether, these results suggest a novel regulatory mechanism for peroxisome biogenesis through the interaction between Pex19p and PLA/AT-3.

Glycerophosphodiesterase GDE4 as a novel lysophospholipase D: a possible involvement in bioactive N-acylethanolamine biosynthesis.

Bioactive N-acylethanolamines include anti-inflammatory palmitoylethanolamide, anorexic oleoylethanolamide, and an endocannabinoid arachidonoylethanolamide (anandamide). In animal tissues, these molecules are biosynthesized from N-acylethanolamine phospholipids directly by phospholipase D-type enzyme or through multi-step routes via N-acylethanolamine lysophospholipids. We previously found that mouse brain has a lysophospholipase D (lysoPLD) activity hydrolyzing N-acylethanolamine lysophospholipids to N-acylethanolamines and that this activity could be partially attributed to glycerophosphodiesterase (GDE) 1. In the present study, we examined catalytic properties of GDE4, another member of the GDE family. When overexpressed in HEK293 cells, murine GDE4 mostly resided in the membrane fraction. Purified GDE4 showed lysoPLD activity toward various lysophospholipids, including N-acylethanolamine lysophospholipids as well as lysophosphatidylethanolamine and lysophosphatidylcholine. When HEK293 cells were metabolically labeled with N-[(14)C]palmitoylethanolamine lysophospholipid, the transient expression of GDE4 increased the [(14)C]palmitoylethanolamide level, while the knockdown of endogenous GDE4 decreased this level. These results suggested that GDE4 functions as an N-acylethanolamine-generating lysoPLD in living cells. Moreover, the expression of GDE4 increased most species of lysophosphatidic acid (LPA), which can be produced from various lysophospholipids by the lysoPLD activity of GDE4. GDE4 mRNA was widely distributed among mouse tissues including brain, stomach, ileum, colon, and testis. In conclusion, GDE4 may act as a lysoPLD, which is involved in the generation of N-acylethanolamines and LPA.

Involvement of phospholipase A/acyltransferase-1 in N-acylphosphatidylethanolamine generation.

Anandamide and other bioactive N-acylethanolamines (NAEs) are a class of lipid mediators and are produced from glycerophospholipids via N-acylphosphatidylethanolamines (NAPEs). Although the generation of NAPE by N-acylation of phosphatidylethanolamine is thought to be the rate-limiting step of NAE biosynthesis, the enzyme responsible, N-acyltransferase, remains poorly characterized. Recently, we found that five members of the HRAS-like suppressor (HRASLS) family, which were originally discovered as tumor suppressors, possess phospholipid-metabolizing activities including NAPE-forming N-acyltransferase activity, and proposed to call HRASLS1-5 phospholipase A/acyltransferase (PLA/AT)-1-5, respectively. Among the five members, PLA/AT-1 attracts attention because of its relatively high N-acyltransferase activity and predominant expression in testis, skeletal muscle, brain and heart of human, mouse and rat. Here, we examined the formation of NAPE by PLA/AT-1 in living cells. As analyzed by metabolic labeling with [(14)C]ethanolamine or [(14)C]palmitic acid, the transient expression of human, mouse and rat PLA/AT-1s in COS-7 cells as well as the stable expression of human PLA/AT-1 in HEK293 cells significantly increased the generation of NAPE and NAE. Liquid chromatography-tandem mass spectrometry also exhibited that the stable expression of PLA/AT-1 enhanced endogenous levels of NAPE, N-acylplasmenylethanolamine, NAE and glycerophospho-NAE. Furthermore, the knockdown of endogenous PLA/AT-1 in mouse ATDC5 cells lowered NAPE levels. Interestingly, the dysfunction of peroxisomes, which was caused by PLA/AT-2 and -3, was not observed in the PLA/AT-1-expressing HEK293 cells. Altogether, these results suggest that PLA/AT-1 is at least partly responsible for the generation of NAPE in mammalian cells.

New players in the fatty acyl ethanolamide metabolism.

Fatty acyl ethanolamides represent a class of endogenous bioactive lipid molecules and are generally referred to as N-acylethanolamines (NAEs). NAEs include palmitoylethanolamide (anti-inflammatory and analgesic substance), oleoylethanolamide (anorexic substance), and anandamide (endocannabinoid). The endogenous levels of NAEs are mainly regulated by enzymes responsible for their biosynthesis and degradation. In mammalian tissues, the major biosynthetic pathway starts from glycerophospholipids and is composed of two enzyme reactions. The first step is N-acylation of ethanolamine phospholipids catalyzed by Ca(2+)-dependent N-acyltransferase and the second step is the release of NAEs from N-acylated ethanolamine phospholipids by N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD). As for the degradation of NAEs, fatty acid amide hydrolase plays the central role. However, recent studies strongly suggest the involvement of other enzymes in the NAE metabolism. These enzymes include members of the HRAS-like suppressor family (also called phospholipase A/acyltransferase family), which were originally discovered as tumor suppressors but can function as Ca(2+)-independent NAPE-forming N-acyltransferases; multiple enzymes involved in the NAPE-PLD-independent multi-step pathways to generate NAE from NAPE, which came to light by the analysis of NAPE-PLD-deficient mice; and a lysosomal NAE-hydrolyzing acid amidase as a second NAE hydrolase. These newly recognized enzymes may become the targets for the development of new therapeutic drugs. Here, we focus on recent enzymological findings in this area.

Regulation of peroxisomal lipid metabolism by catalytic activity of tumor suppressor H-rev107.

H-rev107 is a mammalian protein belonging to the HRAS-like suppressor family. Although the protein was originally found as a tumor suppressor, currently it is receiving considerable attention as a regulator of adipocyte lipolysis. We recently revealed that purified recombinant H-rev107 has phospholipase A(1/2) activity, releasing free fatty acids from glycerophospholipids with a preference for esterolysis at the sn-1 position. In the present study, we constitutively expressed H-rev107 in cloned HEK293 cells to examine its biological function in living cells. Initially, the cells accumulated free fatty acids. We also found a remarkable decrease in the levels of ether-type lipids, including plasmalogen and ether-type triglyceride, with a concomitant increase in fatty alcohols, substrates for the biosynthesis of ether-type lipids. Considering that peroxisomes are involved in the ether-type lipid biosynthesis, we next focused on peroxisomes and found that the peroxisomal markers 70-kDa peroxisomal membrane protein and catalase were abnormally distributed in the transfected cells. These biochemical and morphological abnormalities were not seen in HEK293 cells stably expressing a catalytically inactive mutant of H-rev107. When H-rev107 or its fusion protein with enhanced green fluorescence protein was transiently expressed in mammalian cells, both proteins were associated with peroxisomes in some of the observed cells. These results suggest that H-rev107 interferes with the biosynthesis of ether-type lipids and is responsible for the dysfunction of peroxisomes in H-rev107-expressing cells.

Generation of N-acylphosphatidylethanolamine by members of the phospholipase A/acyltransferase (PLA/AT) family.

Bioactive N-acylethanolamines (NAEs), including N-palmitoylethanolamine, N-oleoylethanolamine, and N-arachidonoylethanolamine (anandamide), are formed from membrane glycerophospholipids in animal tissues. The pathway is initiated by N-acylation of phosphatidylethanolamine to form N-acylphosphatidylethanolamine (NAPE). Despite the physiological importance of this reaction, the enzyme responsible, N-acyltransferase, remains molecularly uncharacterized. We recently demonstrated that all five members of the HRAS-like suppressor tumor family are phospholipid-metabolizing enzymes with N-acyltransferase activity and are renamed HRASLS1-5 as phospholipase A/acyltransferase (PLA/AT)-1-5. However, it was poorly understood whether these proteins were involved in the formation of NAPE in living cells. In the present studies, we first show that COS-7 cells transiently expressing recombinant PLA/AT-1, -2, -4, or -5, and HEK293 cells stably expressing PLA/AT-2 generated significant amounts of [(14)C]NAPE and [(14)C]NAE when cells were metabolically labeled with [(14)C]ethanolamine. Second, as analyzed by liquid chromatography-tandem mass spectrometry, the stable expression of PLA/AT-2 in cells remarkably increased endogenous levels of NAPEs and NAEs with various N-acyl species. Third, when NAPE-hydrolyzing phospholipase D was additionally expressed in PLA/AT-2-expressing cells, accumulating NAPE was efficiently converted to NAE. We also found that PLA/AT-2 was partly responsible for NAPE formation in HeLa cells that endogenously express PLA/AT-2. These results suggest that PLA/AT family proteins may produce NAPEs serving as precursors of bioactive NAEs in vivo.

Assay of NAT Activity.

In animal tissues, N-acyltransferase (NAT) catalyzes the first reaction in the biosynthetic pathway of bioactive N-acylethanolamines, in which an acyl chain is transferred from the sn-1 position of the donor phospholipid, such as phosphatidylcholine, to the amino group of phosphatidylethanolamine, resulting in the formation of N-acylphosphatidylethanolamine. NAT has long been known to be stimulated by Ca2+ and hence referred to as Ca2+-dependent NAT. Later, this enzyme was identified as cPLA2ε (also referred to as PLA2G4E). On the other hand, members of the phospholipase A/acyltransferase (PLAAT) family (also known as HRAS-like suppressor family) show Ca2+-independent NAT activity. In this chapter, we describe (1) partial purification of Ca2+-dependent NAT from rat brain, (2) purification of recombinant cPLA2ε and PLAAT-2, and (3) NAT assay using radiolabeled substrate.

ß-1,4-Galactan suppresses lipid synthesis in sebaceous gland cells via TLR4.

Sebum is a lipid mixture secreted from sebaceous glands of the skin. The excessive secretion of sebum causes acne vulgaris and seborrheic dermatitis, while its deficiency causes xerosis. Therefore, the appropriate control of sebum secretion is crucially important to keep the skin healthy. In the present study, we evaluated the effects of naturally occurring polysaccharides on lipid biosynthesis in hamster sebaceous gland cells. Among the tested polysaccharides, ß-1,4-galactan, the main chain of type I arabinogalactan, most potently suppressed lipid synthesis in the sebaceous gland cells as analysed by oil red O staining. Toll-like receptor (TLR)4 inhibitors counteracted this suppressive effect and lipopolysaccharide, a TLR4 ligand, mimicked this effect, suggesting the involvement of the TLR4 signalling pathway. In the cells ß-1,4-galactan significantly decreased mRNA levels of lipogenesis-related transcription factors (peroxisomeGraphical Abstract$\includegraphics{\bwartpath }$ proliferator-activated receptor γ and sterol regulatory element-binding protein 1) and enzymes (acetyl-CoA carboxylase and fatty acid synthase) as well as the glucose transporter GLUT4. Furthermore, ß-1,4-galactan increased the production of lactic acid serving as a natural moisturizing factor and enhanced the proliferation of sebaceous gland cells. These results suggest potential of ß-1,4-galactan as a material with therapeutic and cosmetic values for the skin.

PLAAT1 expression triggers fragmentation of mitochondria in an enzyme activity-dependent manner.

The phospholipase A and acyltransferase (PLAAT) family is a protein family consisting of five members (PLAAT1-5), which acts as phospholipid-metabolizing enzymes with phospholipase A1/A2 and N-acyltransferase activities. Since we previously reported that the overexpression of PLAAT3 in mammalian cells causes the specific disappearance of peroxisomes, in the present study we examined a possible effect of PLAAT1 on organelles. We prepared HEK293 cells expressing mouse PLAAT1 in a doxycycline-dependent manner and found that the overexpression of PLAAT1 resulted in the transformation of mitochondria from the original long rod shape to a round shape, as well as their fragmentation. In contrast, the overexpression of a catalytically inactive point mutant of PLAAT1 did not generate any morphological change in mitochondria, suggesting the involvement of catalytic activity. PLAAT1 expression also caused the reduction of peroxisomes, while the levels of the marker proteins for ER, Golgi apparatus and lysosomes were almost unchanged. In PLAAT1-expressing cells, the level of dynamin-related protein 1 responsible for mitochondrial fission was increased, whereas those of optic atrophy 1 and mitofusin 2, both of which are responsible for mitochondrial fusion, were reduced. These results suggest a novel role of PLAAT1 in the regulation of mitochondrial biogenesis.

Vasoactive intestinal peptide blockade suppresses tumor growth by regulating macrophage polarization and function in CT26 tumor-bearing mice.

Macrophages are a major population of immune cells in solid cancers, especially colorectal cancers. Tumor-associated macrophages (TAMs) are commonly divided into M1-like (tumor suppression) and M2-like (tumor promotion) phenotypes. Vasoactive intestinal peptide (VIP) is an immunoregulatory neuropeptide with a potent anti-inflammatory function. Inhibition of VIP signaling has been shown to increase CD8+ T cell proliferation and function in viral infection and lymphoma. However, the role of VIP in macrophage polarization and function in solid tumors remains unknown. Here, we demonstrated that conditioned medium from CT26 (CT26-CM) cells enhanced M2-related marker and VIP receptor (VPAC) gene expression in RAW264.7 macrophages. VIP hybrid, a VIP antagonist, enhanced M1-related genes but reduced Mrc1 gene expression and increased phagocytic ability in CT26-CM-treated RAW264.7 cells. In immunodeficient SCID mice, VIP antagonist alone or in combination with anti-PD-1 antibody attenuated CT26 tumor growth compared with the control. Analysis of tumor-infiltrating leukocytes found that VIP antagonist increased M1/M2 ratios and macrophage phagocytosis of CT26-GFP cells. Furthermore, Vipr2 gene silencing or VPAC2 activation affected the polarization of CT26-CM-treated RAW264.7 cells. In conclusion, the inhibition of VIP signaling enhanced M1 macrophage polarization and macrophage phagocytic function, resulting in tumor regression in a CT26 colon cancer model.

Enzymatic formation of N-acylethanolamines from N-acylethanolamine plasmalogen through N-acylphosphatidylethanolamine-hydrolyzing phospholipase D-dependent and -independent pathways.

Bioactive N-acylethanolamines include anandamide (an endocannabinoid), N-palmitoylethanolamine (an anti-inflammatory), and N-oleoylethanolamine (an anorexic). In the brain, these molecules are formed from N-acylphosphatidylethanolamines (NAPEs) by a specific phospholipase D, called NAPE-PLD, or through NAPE-PLD-independent multi-step pathways, as illustrated in the current study employing NAPE-PLD-deficient mice. Although N-acylethanolamine plasmalogen (1-alkenyl-2-acyl-glycero-3-phospho(N-acyl)ethanolamine, pNAPE) is presumably a major class of N-acylethanolamine phospholipids in the brain, its enzymatic conversion to N-acylethanolamines is poorly understood. In the present study, we focused on the formation of N-acylethanolamines from pNAPEs. While recombinant NAPE-PLD catalyzed direct release of N-palmitoylethanolamine from N-palmitoylethanolamine plasmalogen, the same reaction occurred in the brain homogenate of NAPE-PLD-deficient mice, suggesting that this reaction occurs through both the NAPE-PLD-dependent and -independent pathways. Liquid chromatography-mass spectrometry revealed a remarkable accumulation of 1-alkenyl-2-hydroxy-glycero-3-phospho(N-acyl)ethanolamines (lyso pNAPEs) in the brain of NAPE-PLD-deficient mice. We also found that brain homogenate formed N-palmitoylethanolamine, N-oleoylethanolamine, and anandamide from their corresponding lyso pNAPEs by a Mg(2+)-dependent "lysophospholipase D". Moreover, the brain levels of alkenyl-type lysophosphatidic acids, the other products from lyso pNAPEs by lysophospholipase D, also increased in NAPE-PLD-deficient mice. Glycerophosphodiesterase GDE1 can hydrolyze glycerophospho-N-acylethanolamines to N-acylethanolamines in the brain. In addition, we discovered that recombinant GDE1 has a weak activity to generate N-palmitoylethanolamine from its corresponding lyso pNAPE, suggesting that this enzyme is at least in part responsible for the lysophospholipase D activity. These results strongly suggest that brain tissue N-acylethanolamines, including anandamide, can be formed from N-acylated plasmalogen through an NAPE-PLD-independent pathway as well as by their direct release via NAPE-PLD.

Heparanase-enhanced shedding of syndecan-1 by myeloma cells promotes endothelial invasion and angiogenesis.

Heparanase enhances shedding of syndecan-1 (CD138), and high levels of heparanase and shed syndecan-1 in the tumor microenvironment are associated with elevated angiogenesis and poor prognosis in myeloma and other cancers. To explore how the heparanase/syndecan-1 axis regulates angiogenesis, we used myeloma cells expressing either high or low levels of heparanase and examined their impact on endothelial cell invasion and angiogenesis. Medium conditioned by heparanase-high cells significantly stimulated endothelial invasion in vitro compared with medium from heparanase-low cells. The stimulatory activity was traced to elevated levels of vascular endothelial growth factor (VEGF) and syndecan-1 in the medium. We discovered that the heparan sulfate chains of syndecan-1 captured VEGF and also attached the syndecan-1/VEGF complex to the extracellular matrix where it then stimulated endothelial invasion. In addition to its heparan sulfate chains, the core protein of syndecan-1 was also required because endothelial invasion was blocked by addition of synstatin, a peptide mimic of the integrin activating region present on the syndecan-1 core protein. These results reveal a novel mechanistic pathway driven by heparanase expression in myeloma cells whereby elevated levels of VEGF and shed syndecan-1 form matrix-anchored complexes that together activate integrin and VEGF receptors on adjacent endothelial cells thereby stimulating tumor angiogenesis.