Environmental Toxin Acrolein Alters Levels of Endogenous Lipids, Including TRP Agonists: A Potential Mechanism for Headache Driven by TRPA1 Activation.

Exposure to airborne toxins can trigger headaches, but the mechanisms are not well understood. Some environmental toxins, such as acrolein, activate transient receptor potential ankyrin 1 (TRPA1), a receptor involved in pain sensation that is highly expressed in the trigeminovascular system. It has been shown in rat models that repeated exposure to acrolein induces trigeminovascular sensitization to both TRPA1 and TRP vanilloid 1 (TRPV1) agonists, a phenomenon linked to headache. In this study, we test the hypothesis that the sensitization of trigeminovascular responses in rats after acrolein exposure via inhalation is associated with changes in levels of endogenous lipids, including TRPV1 agonists, in the trigeminal ganglia, trigeminal nucleus, and cerebellum. Lipidomics analysis of 80 lipids was performed on each tissue after acute acrolein, chronic acrolein, or room air control. Both acute and chronic acrolein exposure drove widespread alterations in lipid levels. After chronic acrolein exposure, levels of all 6 N-acyl ethanolamines in the screening library, including the endogenous cannabinoid and TRPV1 agonist, N-arachidonoyl ethanolamine, were elevated in trigeminal tissue and in the cerebellum. This increase in TRPV1 ligands by acrolein exposure may indicate further downstream signaling, in that we also show here that a combination of these TRPV1 endogenous agonists increases the potency of the individual ligands in TRPV1-HEK cells. In addition to these TRPV1 agonists, 3 TRPV3 antagonists, 4 TRPV4 agonists, and 25 orphan lipids were up and down regulated after acrolein exposure. These data support the hypothesis that lipid signaling may represent a mechanism by which repeated exposure to the TRPA1 agonist and environmental toxin, acrolein, drives trigeminovascular sensitization.

Novel endogenous N-acyl amides activate TRPV1-4 receptors, BV-2 microglia, and are regulated in brain in an acute model of inflammation.

A family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (Anandamide), and called N-acyl amides have emerged as a family of biologically active compounds at TRP receptors. N-acyl amides are constructed from an acyl group and an amine via an amide bond. This same structure can be modified by changing either the fatty acid or the amide to form potentially hundreds of lipids. More than 70 N-acyl amides have been identified in nature. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, using a unique in-house library of over 70 N-acyl amides we tested the following three hypotheses: (1) Additional N-acyl amides will have activity at TRPV1-4, (2) Acute peripheral injury will drive changes in CNS levels of N-acyl amides, and (3) N-acyl amides will regulate calcium in CNS-derived microglia. Through these studies, we have identified 20 novel N-acyl amides that collectively activate (stimulating or inhibiting) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we showed that levels of at least 10 of these N-acyl amides that activate TRPVs are regulated in brain after intraplantar carrageenan injection. We then screened the BV2 microglial cell line for activity with this N-acyl amide library and found overlap with TRPV receptor activity as well as additional activators of calcium mobilization from these lipids. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at ion channels, in microglia, and in the brain in the context of inflammation.

Brain levels of prostaglandins, endocannabinoids, and related lipids are affected by mating strategies.

Background. Endogenous cannabinoids (eCBs) are involved in the development and regulation of reproductive behaviors. Likewise, prostaglandins (PGs) drive sexual differentiation and initiation of ovulation. Here, we use lipidomics strategies to test the hypotheses that mating immediately activates the biosynthesis and/or metabolism of eCBs and PGs and that specific mating strategies differentially regulate these lipids in the brain. Methods. Lipid extractions and tandem mass spectrometric analysis were performed on brains from proestrous rats that had experienced one of two mating strategies (paced or standard mating) and two nonmated groups (chamber exposed and home cage controls). Levels of PGs (PGE2 and PGF2alpha), eCBs (AEA and 2-AG, N-arachidonoyl glycine), and 4 related lipids (4 N-acylethanolamides) were measured in olfactory bulb, hypothalamus, hippocampus, thalamus, striatum, midbrain, cerebellum, and brainstem. Results. Overall, levels of these lipids were significantly lower among paced compared to standard mated rats with the most dramatic decreases observed in brainstem, hippocampus, midbrain, and striatum. However, chamber exposed rats had significantly higher levels of these lipids compared to home cage controls and paced mated wherein the hippocampus showed the largest increases. Conclusions. These data demonstrate that mating strategies and exposure to mating arenas influence lipid signaling in the brain.

Targeted lipidomics in Drosophila melanogaster identifies novel 2-monoacylglycerols and N-acyl amides.

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling.

Oleoyl serine, an endogenous N-acyl amide, modulates bone remodeling and mass.

Bone mass is determined by a continuous remodeling process, whereby the mineralized matrix is being removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. Here we report the presence of endogenous amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) in mouse bone. Of these compounds, N-oleoyl-l-serine (OS) had the highest activity in an osteoblast proliferation assay. In these cells, OS triggers a Gi-protein-coupled receptor and Erk1/2. It also mitigates osteoclast number by promoting osteoclast apoptosis through the inhibition of Erk1/2 phosphorylation and receptor activator of nuclear-κB ligand (RANKL) expression in bone marrow stromal cells and osteoblasts. In intact mice, OS moderately increases bone volume density mainly by inhibiting bone resorption. However, in a mouse ovariectomy (OVX) model for osteoporosis, OS effectively rescues bone loss by increasing bone formation and markedly restraining bone resorption. The differential effect of exogenous OS in the OVX vs. intact animals is apparently a result of an OVX-induced decrease in skeletal OS levels. These data show that OS is a previously unexplored lipid regulator of bone remodeling. It represents a lead to antiosteoporotic drug discovery, advantageous to currently available therapies, which are essentially either proformative or antiresorptive.

The endocannabinoid anandamide is a precursor for the signaling lipid N-arachidonoyl glycine by two distinct pathways.

BACKGROUND: N-arachidonoyl glycine (NAGly) is an endogenous signaling lipid with a wide variety of biological activity whose biosynthesis is poorly understood. Two primary biosynthetic pathways have been proposed. One suggests that NAGly is formed via an enzymatically regulated conjugation of arachidonic acid (AA) and glycine. The other suggests that NAGly is an oxidative metabolite of the endogenous cannabinoid, anandamide (AEA), through an alcohol dehydrogenase. Here using both in vitro and in vivo assays measuring metabolites with LC/MS/MS we test the hypothesis that both pathways are present in mammalian cells. RESULTS: The metabolic products of deuterium-labeled AEA, D4AEA (deuterium on ethanolamine), indicated that NAGly is formed by the oxidation of the ethanolamine creating a D2NAGly product in both RAW 264.7 and C6 glioma cells. Significantly, D4AEA produced a D0NAGly product only in C6 glioma cells suggesting that the hydrolysis of AEA yielded AA that was used preferentially in a conjugation reaction. Addition of the fatty acid amide (FAAH) inhibitor URB 597 blocked the production of D0NAGly in these cells. Incubation with D8AA in C6 glioma cells likewise produced D8NAGly; however, with significantly less efficacy leading to the hypothesis that FAAH-initiated AEA-released AA conjugation with glycine predominates in these cells. Furthermore, the levels of AEA in the brain were significantly increased, whereas those of NAGly were significantly decreased after systemic injection of URB 597 in rats and in FAAH KO mice further supporting a role for FAAH in endogenous NAGly biosynthesis. Incubations of NAGly and recombinant FAAH demonstrated that NAGly is a significantly less efficacious substrate for FAAH with only ~50% hydrolysis at 30 minutes compared to 100% hydrolysis of AEA. Co-incubations of AEA and glycine with recombinant FAAH did not, however, produce NAGly. CONCLUSION: These data support the hypothesis that the signaling lipid NAGly is a metabolic product of AEA by both oxidative metabolism of the AEA ethanolamine moiety and through the conjugation of glycine to AA that is released during AEA hydrolysis by FAAH.