biomedicines

Article Release of Anti-Inflammatory Palmitoleic Acid and Its Positional Isomers by Mouse Peritoneal Macrophages

Alma M. Astudillo 1,2, Clara Meana 1,2, Miguel A. Bermúdez 1,2 , Alfonso Pérez-Encabo 3, María A. Balboa 1,2 and Jesús Balsinde 1,2,*

1 Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Valladolid, 47003 Valladolid, Spain; [email protected] (A.M.A.); [email protected] (C.M.); [email protected] (M.A.B.); [email protected] (M.A.B.) 2 Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 28029 Madrid, Spain 3 Instituto CINQUIMA, Departamento de Química Orgánica, Universidad de Valladolid, 47011 Valladolid, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-983-423-062



Received: 15 October 2020; Accepted: 2 November 2020; Published: 6 November 2020


Abstract: Positional isomers of hexadecenoic acid are considered as fatty acids with anti-inflammatory properties. The best known of them, palmitoleic acid (cis-9-hexadecenoic acid, 16:1n-7), has been identified as a lipokine with important beneficial actions in metabolic diseases. Hypogeic acid (cis-7-hexadecenoic acid, 16:1n-9) has been regarded as a possible biomarker of foamy cell formation during atherosclerosis. Notwithstanding the importance of these isomers as possible regulators of inflammatory responses, very little is known about the regulation of their levels and distribution and mobilization among the different lipid pools within the cell. In this work, we describe that the bulk of hexadecenoic fatty acids found in mouse peritoneal macrophages is esterified in a unique phosphatidylcholine species, which contains palmitic acid at the sn-1 position, and hexadecenoic acid at the sn-2 position. This species markedly decreases when the macrophages are activated with inflammatory stimuli, in parallel with net mobilization of free hexadecenoic acid. Using pharmacological inhibitors and specific gene-silencing approaches, we demonstrate that hexadecenoic acids are selectively released by calcium-independent group VIA phospholipase A2 under activation conditions. While most of the released hexadecenoic acid accumulates in free fatty acid form, a significant part is also transferred to other phospholipids to form hexadecenoate-containing inositol phospholipids, which are known to possess growth-factor-like-properties, and are also used to form fatty acid esters of hydroxy fatty acids, compounds with known anti-diabetic and anti-inflammatory properties. Collectively, these data unveil new pathways and mechanisms for the utilization of palmitoleic acid and its isomers during inflammatory conditions, and raise the intriguing possibility that part of the anti-inflammatory activity of these fatty acids may be due to conversion to other lipid mediators.

Keywords: palmitoleic acid; phospholipid hydrolysis; phospholipase A2; lipid mediators; fatty acid esters of hydroxy fatty acids; inflammation; monocytes/macrophages

1. Introduction Accumulating evidence suggests a key role for the cis-unsaturated fatty acid palmitoleic acid (cis-9-hexadecenoic acid; 16:1n-7) in protecting against hepatic steatosis and improving overall insulin sensitivity in metabolic tissues [1,2]. In murine models, strong evidence is available to support a role for palmitoleic acid as an anti-inflammatory mediator and a positive correlate of insulin sensitivity.

Biomedicines 2020, 8, 480; doi:10.3390/biomedicines8110480 www.mdpi.com/journal/biomedicines Biomedicines 2020, 8, 480 2 of 21

In humans, however, opposing results have been reported. Some studies have indicated that circulating levels of palmitoleate positively correlate with insulin sensitivity and decreased inflammatory markers, while others showed that the circulating levels of palmitoleate correlate with the degree of hepatic steatosis and adiposity, which promotes fat deposition in hepatocytes. Clearly, more research in humans is needed to validate the promising therapeutic potential of palmitoleic acid suggested by murine and in vitro studies [1–4]. While the direct actions of 16:1n-7 on cells of innate immunity remain largely unexplored, it appears clear that this fatty acid manifests marked anti-inflammatory effects [5,6]. Notably, recent work has demonstrated that innate immune cells also contain significant levels of a positional isomer of palmitoleic acid, cis-7-hexadecenoic acid (16:1n-9; hypogeic acid), which also manifests strong anti-inflammatory properties on monocytes and macrophages [7–9]. Furthermore, 16:1n-9 is synthesized by human phagocytic cells via β-oxidation of oleic acid and its levels are elevated in lipid droplet-laden monocytes, suggesting that it may constitute a biomarker for foamy cell formation [8]. When added exogenously, 16:1n-9 exhibits potent anti-inflammatory actions in vitro and in vivo, which are distinguishable from those of 16:1n-7 and comparable in magnitude to those exerted by omega-3 fatty acids [8,9]. Monocytes and macrophages also contain significant quantities of a third 16:1 positional isomer, cis-6-hexadecenoic acid (16:1n-10; sapienic acid). 16:1n-10 also exhibits anti-inflammatory activity, although generally at higher concentrations than those observed for 16:1n-7 or 16:1n-9 [9]. It is well established that fatty acyl chains of membrane lipids play a variety of biological functions including signaling; thus, the enzymes regulating phospholipid fatty acid recycling constitute a key step for the fine regulation of lipid mediator production during cell activation. Phospholipase A2 (PLA2) enzymes, which cleave the sn-2 position of glycerophospholipids, are key regulators of these processes [10–12]. Multiple PLA2 enzymes co-exist in a single cell, each exhibiting potentially different headgroup and/or fatty acid preferences. PLA2s are classified in 16 groups, each containing several sub-groups [13,14]. However, based on biochemical features, these enzymes are frequently grouped 2+ into six major families: secreted PLA2s, Ca -independent PLA2s, cytosolic PLA2s, platelet activating factor acetyl hydrolases (also known as lipoprotein-associated PLA2), lysosomal PLA2, and the adipose PLA2 [13,14]. Of these families, two of them have been shown to play key roles in regulating fatty acid mobilization reactions in innate immune cells, namely the cytosolic group IVA phospholipase 2+ A2 (cPLA2α) and the Ca -independent group VIA phospholipase A2 (iPLA2-VIA). cPLA2α is the critical enzyme regulating stimulus-induced arachidonic acid release in cells under a wide variety of conditions [15,16]. iPLA2-VIA is a multifaceted enzyme with multiple roles in cell physiology and pathophysiology, being of special relevance in regulating bone formation, apoptosis, insulin secretion, and sperm development [17,18]. Unlike cPLA2α, iPLA2-VIA does not manifest specificity with regard to the fatty acid released in in vitro assays [10,13]. However, studies carried out in murine macrophages have shown that cPLA2α and iPLA2-VIA act on different cellular phospholipid pools [19,20]. While the former regulates arachidonic acid release and eicosanoid production, the latter appears to act on phospholipids that do not contain arachidonate [19,20]. In this regard, we show in this paper, for the first time, that macrophages do release 16:1 fatty acids when challenged by activating stimuli, and that the process appears to be exquisitely regulated by iPLA2-VIA. Moreover, we also show that part of the 16:1 fatty acids initially present in choline-containing glycerophospholipids (PC) are transferred to phosphatidylinositol (PI) and/or used to produce 16:1-containing branched fatty acid esters of hydroxy fatty acids (FAHFAs), a family of compounds that displays significant anti-inflammatory activity in vivo [21].

2. Materials and Methods

2.1. Reagents Cell culture medium was from Corning (Glendale, AZ, USA). Organic solvents were from Fisher Scientific (Madrid, Spain). Lipid standards were from Avanti Polar Lipids (Alabaster, AL, USA) Biomedicines 2020, 8, 480 3 of 21

or Larodan Fine Chemicals (Malmö, Sweden). PLA2 inhibitors were from Cayman (Ann Arbor, MI, USA). The Coenzyme A-independent transacylase inhibitor SKF98625 [22,23] was synthesized in our laboratory. Deuterated 9-cis-hexadecenoic acid (16:1n-7) was from Cayman, and deuterated cis-7-hexadecenoic acid (16:1n-9) was synthesized in our laboratory. The detailed procedure is described in AppendixA. All other reagents were from Sigma-Aldrich (Madrid, Spain).

2.2. Cell Culture Resident peritoneal macrophages from Swiss male mice (University of Valladolid Animal House, 10–12 weeks old) were obtained by peritoneal lavage using 5 mL cold phosphate-buffered saline, as described elsewhere [24]. The cells were plated at 2 106 per well (6-well plates) in 2 mL Roswell Park × Memorial Institute 1640 medium with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, and allowed to adhere for 20 h in a humidified atmosphere of 5% CO2 at 37 ◦C. Wells were extensively washed to remove nonadherent cells. Adherent macrophages were then used for experimentation. All procedures involving animals were carried out under the supervision of the Institutional Committee of Animal Care and Usage of the University of Valladolid (approval number 7406000; date: 04/19/2016; renewed: 10/08/2019), and are in accordance with the guidelines established by the Spanish Ministry of Agriculture, Food, and Environment and the European Union. For the antisense inhibition experiments, RAW264.7 macrophage-like cells were used. These cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine at 37 ◦C in a humidified atmosphere of 5% CO2 at 37 ◦C, as previously described [25,26]. For experiments, the cells were treated in serum-free medium for 1 h before addition of stimulants for the indicated concentrations and periods of time. When inhibitors were used, they were added to the media 30 min before stimulating the cells. Zymosan was prepared as described [27]. Only zymosan preparations that showed no endogenous PLA2 activity, as measured by enzyme assay [28–31], were used in this study. Protein was measured using a commercial kit (BioRad, Hercules, CA, USA).

2.3. Gas Chromatography/Mass Spectrometry (GC/MS) Analyses Total lipids from approximately 107 cells were extracted according to Bligh and Dyer [32], and internal standards were added. Phospholipids were separated from neutral lipids by thin-layer chromatography, using n-hexane/diethyl ether/acetic acid (70:30:1, v/v/v) as the mobile phase [33]. Phospholipid classes were separated twice with chloroform/methanol/28% (w/w) ammonium hydroxide (60:37.5:4, v/v/v) as the mobile phase, using plates impregnated with boric acid [34]. The bands corresponding to the different lipid classes were scraped from the plate, and fatty acid methyl esters were obtained from the various lipid fractions by transmethylation with 0.5 M KOH in methanol for 60 min at 37 ◦C. Fatty acid methyl esters were analyzed by GC/MS as previously described [35,36], using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass-selective detector operated in electron impact mode (EI, 70 eV), equipped with an Agilent 7693 autosampler, and an Agilent DB23 column (60 m length 0.25 mm internal diameter 0.15 µm film thickness) (Agilent × × Technologies, Santa Clara, CA, USA). Data analysis was carried out with the Agilent G1701EA MSD Productivity Chemstation software, revision E.02.00. Derivatization of fatty acid methyl esters to dimethyl disulfide (DMDS) adducts was carried out according to the procedure described by Sansone et al. [37], as modified by Astudillo et al. [12]. Identification of 16:1 isomers was carried out by elution time of the different DMDS adducts (corresponding to the position of the double bond), and by specific fragments of the mass spectra generated by cleavage of the C-C bond that originally contained the double bond (ω-fragment, ∆-fragment and ∆-fragment with the loss of methanol). Biomedicines 2020, 8, 480 4 of 21

2.4. Liquid Chromatography/Mass Spectrometry (LC/MS) Analyses of Phospholipids Lipids corresponding to 50 µg of cell homogenate protein were extracted according Bligh and Dyer [32]. The samples were re-dissolved in hexanes/2-propanol/water (42:56:2, v/v/v) and injected into a Thermo Fisher Scientific Dionex Ultimate 3000 high-performance liquid chromatograph equipped with an Ultimate HPG-3400SD standard binary pump and an Ultimate ACC-3000 autosampler column compartment (Thermo Fisher Scientific, Waltham, MA, USA). Separation was carried using a FORTIS HILIC column (150 mm 3 mm, 3 µm particle size) (Fortis Technologies, Neston, UK). × The mobile phase consisted of a gradient of solvent A (hexanes/isopropanol 30:40, v/v) and solvent B (hexanes/isopropanol/20 mM ammonium acetate in water, 30:40:7, v/v/v). The gradient started at 75% A from 0 to 5 min, then decreased from 75% A to 40% A at 15 min, from 40% A to 5% A at 20 min, was held at 5% until 40 min, and then increased to 75% at 41 min. Then, the column was re-equilibrated holding 75% A for an additional 14 min period before the next sample injection [38]. The flow rate through the column was fixed at 0.4 mL/min. The liquid chromatography system was coupled online to an Sciex QTRAP 4500 mass spectrometer equipped with a Turbo V Ion Source and a TurbolonSpray probe for electrospray ionization (AB Sciex, Framingham, MA, USA). Source parameters were set as follows: ion spray voltage, 4500 V; curtain gas, 30 psi; nebulizer gas, 50 psi; desolvation gas, 60 psi; − temperature, 425 ◦C. Phospholipid species were analyzed in scheduled multiple reaction monitoring mode with negative ionization, detecting in Q3 the m/z of 253.2, corresponding to 16:1 fatty acids, as [M-H]−. The chromatographic peaks of each species were integrated and normalized to the peak area of the internal standard corresponding to each phospholipid class.

2.5. FAHFA Analysis by LC/MS FAHFA determination by LC/MS was carried out according to Zhang et al. [39], as modified by Rodríguez et al. [40]. Analyses were performed using a high-performance liquid chromatograph Thermo Fisher Scientific Dionex Ultimate 3000 equipped with an Ultimate HPG-3400SD standard binary pump and an Ultimate ACC-3000 Autosampler (Thermo Fisher Scientific, Waltham, MA, USA), coupled to a Sciex QTRAP 4500 Mass Spectrometer operated in negative ion mode (AB Sciex, Framingham, MA, USA). For the quantitative measurement of 16:1-derived FAHFA species, the instrument was set to multiple reaction monitoring mode, selecting three transitions for each FAHFA: parent ion hydroxy fatty acid, parent ion hydroxy fatty acid minus water loss and parent ion → → → m/z of esterified fatty acid as carboxylate ion.

2.6. iPLA2-VIA Antisense Inhibition Studies

The iPLA2-VIA antisense oligonucleotide utilized in this work has been described in previous studies [41–46]. The iPLA2-VIA antisense sequence (50-CTC CTT CAC CCG GAA TGG GT) corresponds to nucleotides 59–78 in the murine iPLA2-VIA sequence. As a control, the sense complement was used (50-ACC CAT TCC GGG TGA AGG AG). Both sense and antisense oligonucleotides contained phosphorothioate linkages to limit degradation. The oligonucleotides were mixed with Lipofectamine RNAiMAX (Thermo Fisher Scientific, Walthman, MA, USA), following the manufacturer’s instructions. Oligonucleotide treatment and culture conditions were not toxic for the cells as assessed by the trypan blue dye exclusion assay and by quantitating adherent cellular protein. iPLA2 activity was measured exactly as described by Balboa et al. [29].

2.7. Data Analysis The results are shown as means standard error of the mean and were analyzed for statistical ± significance by t-test (two groups) or by ANOVA (more than two groups), followed by Tukey’s post hoc test, using GraphPad Prism software (version 9.0.0, GraphPad Software, Inc., San Diego, CA, USA). A value of p < 0.05 was considered statistically significant. Biomedicines 2020, 8, 480 5 of 21

Biomedicines 2020, 8, 480 5 of 21 3. Results 3. Results 3.1. Endogenous Distribution of 16:1 Fatty Acids among Macrophage Phospholipids 3.1. Endogenous Distribution of 16:1 Fatty Acids among Macrophage Phospholipids Given the biological relevance of 16:1 fatty acids as endogenous molecules with anti-inflammatory properties,Given we the began biological this work relevance by assessing of 16:1 the fatty cellular acids distribution as endogenous of these molecules fatty acids with in murineanti- residentinflammatory peritoneal properties, macrophages. we began Quantitative this work by analysisassessing of the the cellular distribution distribution of 16:1 of these fatty fatty acids acids within phospholipidin murine resident classes peritoneal was performed macrophages. by gas Quantitative chromatography analysis coupled of the distribution with mass of spectrometry 16:1 fatty (GCacids/MS). within Strikingly, phospholipid more than classes 90% of was total performed cellular 16:1 by gas fatty chromatography acid was found coupled in choline-containing with mass glycerophospholipidsspectrometry (GC/MS). (PC). Strikingly, Other major more than phospholipid 90% of total classes cellular such 16:1 as fatty ethanolamine-containing acid was found in phospholipidscholine-containing (PE), phosphatidylinositol glycerophospholipids (PI) (PC). or phosphatidylserine Other major phospholipid (PS) contained classes only low such amounts as ofethanolamine this fatty acid-containing (Figure1A). phospholipids (PE), phosphatidylinositol (PI) or phosphatidylserine (PS) containedThis phospholipid only low amounts 16:1 of fatty this fatty acid acid distribution (Figure 1A). is striking and prompted us to determine This phospholipid 16:1 fatty acid distribution is striking and prompted us to determine the the distribution profile of 16:1-containing molecular species by using high-performance liquid distribution profile of 16:1-containing molecular species by using high-performance liquid chromatography coupled to a QTRAP 4500 triple quadrupole in multiple reaction mode (LC/MS). chromatography coupled to a QTRAP 4500 triple quadrupole in multiple reaction mode (LC/MS). The 16:1-containing species were unequivocally identified by detecting the production of an m/z The 16:1-containing species were unequivocally identified by detecting the production of an m/z fragment of 253.2, corresponding to the [M-H] ion of 16:1. Following nomenclature recommendations, fragment of 253.2, corresponding to the− [M-H]− ion of 16:1. Following nomenclature fattyrecommendations, chains within phospholipids fatty chains within are designated phospholipids by their are number designated of carbons:double by their number bonds. of A designationcarbons:double of O- bonds. before A the designation first fatty chainof O- before indicates the first that fatty the sn-1 chain position indicates is ether-linked,that the sn-1 position whereas a P-is designation ether-linked, indicates whereas a a plasmalogen P- designation form indicates (sn-1 vinyla plasmalogen ether linkage) form (sn [47-].1 vinyl A total ether of 14 linkage) major [47]. species wereA total detected, of 14 major comprising species ~90%were detected, of total cellular comprising 16:1 (Figure~90% of1 totalB). Strikingly cellular 16:1 again, (Figure ~75% 1B). of Strikingly total 16:1 in PCagain, was found~75% of in total a single 16:1 phospholipidin PC was found species, in a single namely phospholipid PC(16:0/16:1). species, namely PC(16:0/16:1).

FigureFigure 1. 1.The The 16:1-containing16:1-containing phospholipid species species in in resident resident peritoneal peritoneal macrophages. macrophages. (A) ( A 16:1) 16:1 contentcontent in in phospholipids as as shown shown by class. by class. (B) Distribution (B) Distribution profile of profile 16:1-containing of 16:1-containing choline- choline-containingcontaining glycerophospholipids glycerophospholipids (PC) (red), (PC) ethanolamine (red), ethanolamine-containing-containing phospholipids phospholipids (PE) (green), (PE) (green),phosphatidylinositol phosphatidylinositol (PI) (yellow), (PI) (yellow), and phosphatidylserine and phosphatidylserine (PS) (pink) (PS) (pink)species species in macrophages, in macrophages, as asdetermined determined by by LC/MS. LC/MS. The The results results are are shown shown as means as means ± standardstandard error error (n = 6). (n = 6). ± 3.2.3.2. Decreased Decreased Levels Levels of of 16:1-Containing 16:1-Containing Phospholipid Species Species in in Stimulated Stimulated Macrophages Macrophages TheThe unexpected unexpected high high amounts amounts ofof 16:116:1 withinwithin oneone single sp speciesecies make make it it reasonable reasonable to to speculate speculate a rolea role for thisfor this species species in stimulus-response in stimulus-response coupling. coupling. Consistent Consistent with with this this view, view, previous previous lipidomic lipidomic work fromwork our from laboratory our laboratory characterizing characterizing phospholipid phospholipid changes in changes resting versusin resting activated versus macrophages, activated identifiedmacrophages, PC (32:1) identified as a species PC (32:1) whose as levels a species decreased whose after levels activation decreased [19 after]. This activation species was [19]. found This to containspecies 16:1 was at found the sn-2to contain position, 16:1 asat the assessed sn-2 position, by fragmentation as assessed by experiments fragmentation using experiments an ion-trap using mass an ion-trap mass spectrometer [19]. To further extend these initial findings, we set out to measure the Biomedicines 2020, 8, 480 6 of 21

Biomedicines 2020, 8, 480 6 of 21 spectrometer [19]. To further extend these initial findings, we set out to measure the changes in the levelschanges of all 16:1-containing in the levels of all PC 16:1 species-containing after PC activation. species after For activation. these experiments, For these experiments, we used yeast-derived we used zymosanyeast- asderived a macrophage zymosan stimulus. as a macrophage Cellular stimulus. stimulation Cellular significantly stimulation reduced significantly the levels reduced of all the major 16:1-containinglevels of all PC major species 16:1-containing in a time-dependent PC species manner, in a time with-dependent PC (16:0 manner,/16:1) experiencing with PC (16:0/16:1) the most pronouncedexperiencing decreases the most (Figure pronounced2A,B). As fordecreases the rest (Figure of 16:1-containing 2A,B). As for phospholipid the rest of species16:1-containing not of the PC class,phospholipid apart from species being not present of the PC at class, very apart low levelsfrom being in comparison present at very with low PC levels species in comparison (Figure1), no significantwith PC changes species were(Figure appreciated 1), no significant upon changes zymosan were stimulation. appreciated upon zymosan stimulation.

3 15 A B *** 12 *** PC(16:0/16:1) *** 2 9

6 * PC(18:0/16:1) * 1 3 * PC(18:1/16:1) *

Relative peak area (a.u.) Relative area peak

16:1 in PC (nmol/mg protein) in PC (nmol/mg 16:1 PC(O-16:0/16:1) 0 0 0 20 40 60 80 100 120 Time (min)

Control

Zymosan

3 16 3 C D E * ** 12 ** 2 2

8 * 1 1 4 **

Relative peak area (a.u.) Relative area peak

16:1 in PC (nmol/mg protein) in PC (nmol/mg 16:1 protein) in PC (nmol/mg 16:1

0 0 0

LPS

Control Control

Man+Lam

PC(16:0/16:1) PC(18:1/16:1) PC(18:0/16:1)

PC(O-16:0/16:1) FigureFigure 2. E ff2.ect Effect of macrophageof macrophage activation activation on on the the cellularcellular levels of of 16:1 16:1-containing-containing PC. PC. (A () AThe) The cells cells werewere either either untreated untreated (control) (control) or or stimulated stimulated with with 1 1 mgmg/mL/mL zymosan for for 1 1h. h. Afterward, Afterward, the the content content of 16:1of 16:1 fatty fatty acid acid in PCin PC was was determined. determined. These results results are are shown shown as asmeans means ± standardstandard error error(n = 6). ( n***= 6). ± *** p

BiomedicinesIt is worth2020, 8 ,noting 480 that zymosan preparations are yeast cell wall homogenates, and thus they7 may of 21 contain undefined amounts of 16:1 fatty acids. To prevent the lipid component of zymosan influencing the responses measured, we assayed a combination of the two major carbohydrate It is worth noting that zymosan preparations are yeast cell wall homogenates, and thus they may polymers present in zymosan, mannan (α-[1-6]-mannose) and laminarin (β-[1-3]-glucan), which are contain undefined amounts of 16:1 fatty acids. To prevent the lipid component of zymosan influencing the responsible for activating phospholipid turnover pathways in macrophages via engagement of a the responses measured, we assayed a combination of the two major carbohydrate polymers present in variety of C-type lectin receptors [48]. The results, shown in Figure 2C,D, indicate that the zymosan, mannan (α-[1-6]-mannose) and laminarin (β-[1-3]-glucan), which are the responsible for combination of mannan and laminarin produced almost identical results to those obtained with activating phospholipid turnover pathways in macrophages via engagement of a variety of C-type zymosan, thus ruling out an effect of endogenous zymosan lipids on macrophage 16:1 fatty acid lectin receptors [48]. The results, shown in Figure2C,D, indicate that the combination of mannan and mobilization. Given that macrophages respond to a wide variety of stimuli, it was interesting to laminarin produced almost identical results to those obtained with zymosan, thus ruling out an effect investigate next whether other stimuli of the innate immune response were able to also induce the of endogenous zymosan lipids on macrophage 16:1 fatty acid mobilization. Given that macrophages hydrolysis of 16:1-containing PC phospholipids. Figure 2E shows show that stimulation of respond to a wide variety of stimuli, it was interesting to investigate next whether other stimuli of the macrophages with bacterial lipopolysaccharide (LPS), a Toll-like receptor 4 agonist, did induce PC innate immune response were able to also induce the hydrolysis of 16:1-containing PC phospholipids. hydrolysis to an extent that was comparable to that found for zymosan or mannan plus laminarin. Figure2E shows show that stimulation of macrophages with bacterial lipopolysaccharide (LPS), These data suggest that the reduction in 16:1-containing PC levels may constitute a hallmark of a Toll-like receptor 4 agonist, did induce PC hydrolysis to an extent that was comparable to that found macrophage activation by innate stimuli. for zymosan or mannan plus laminarin. These data suggest that the reduction in 16:1-containing PC levels may constitute a hallmark of macrophage activation by innate stimuli. 3.3. Fatty Acid Isomers in the Response 3.3. FattyIn previous Acid Isomers work in from the Response our laboratory, we showed that at least three 16:1 fatty acid positional isomersIn previous co-exist at work significant from our levels laboratory, in macrophages we showed and that other at innate least three immune 16:1 cells, fatty namely acid positional 16:1n-7 isomers(cis-9-hexadecenoic co-exist at significant acid; palmitoleic levels in acid), macrophages 16:1n-9 and (cis- other7-hexadecenoic innate immune acid; cells, hypogeic namely acid), 16:1n-7 and (cis-9-hexadecenoic16:1n-10 (cis-6-hexadecenoic acid; palmitoleic acid; sapienic acid), acid), 16:1n-9 and that (cis-7-hexadecenoic the three of them acid; exhibit hypogeic anti-inflammatory acid), and 16:1n-10activity to (cis-6-hexadecenoic different degrees [8,9]. acid; It sapienic was therefore acid), andimportant that the to three assess of whether them exhibit or not anti-inflammatory the three isomers activityare differentially to different released degrees upon [8,9]. Itzymosan was therefore activation important of the to cells. assess Figure whether 3A orshows not the that three stimulated isomers aremacrophages differentially exhibited released decreased upon zymosan amounts activation of all three of isomers, the cells. suggesting Figure3A that shows whatever that stimulated may be the macrophagespathway for exhibited 16:1 mobilization, decreased it amounts does not of all distinguish three isomers, the position suggesting of that the whatever double bond. may be In the an pathwayanalogous for manner 16:1 mobilization, to arachidonic it does-acid not containing distinguish phospholipids, the position ofthe the decreases double bond.of which In an after analogous cellular mannerstimulation to arachidonic-acid are the result of containing increased phospholipids, free arachidonic the decreasesacid release of which[49–51], after we cellular reasoned stimulation that the aredecrease the result in cellular of increased 16:1- freecontaining arachidonic phospholipids acid release is [49 due–51 ], to we the reasoned release that of 16:1 the decrease fatty acids in cellularduring 16:1-containingactivation. As shown phospholipids in Figure is 3B, due this to thewas release found ofto 16:1be the fatty case, acids and during a time activation.-dependent As increase shown inin Figurefree 16:13B, fattythis was acid found formation to bethe could case, be and detected a time-dependent after activation increase of inthe free macrophages. 16:1 fatty acid At formation 1 h, 16:1 couldrelease be had detected increased after two activation-fold over of the unstimulated macrophages. levels, At 1which h, 16:1 represents release had 10 increased–15% of total two-fold fatty overacid unstimulatedinitially present levels, in the which cells.represents 10–15% of total fatty acid initially present in the cells.

Figure 3. 3.Release Release of of 16:1 16:1 fatty fatty acids acids in activated in activated macrophages. macrophages. (A) 16:1 (A fatty) 16:1 acid fatty isomers acid were isom analyzeders were byanalyzed GC/MS by after GC/MS transmethylation after transmethylation and transformation and transformation into dimethyl into disulfide dimethyl (DMDS) disulfide derivatives. (DMDS) Thederivatives. content ofThe each content isomer of each was analyzedisomer was in analyzed control unstimulated in control unstimulated cells (open bars)cells (open or cells bars) stimulated or cells withstimulated mannan with plus mannan laminarin plus (0.5laminarin mg/mL (0.5 each) mg/mL for 1 eac h.h) (B )for Time-course 1 h. (B) Time of- thecourse release of the of release 16:1 fatty of acid16:1 fromfatty mannanacid from plus mannan laminarin-stimulated plus laminarin- macrophages,stimulated macrophages, as assessed byas GCassessed/MS after by GC/MS derivatizing after thederivatizing free fatty the acids free to fatty methyl acids esters. to methyl Results esters. are shownResults as are means shown asstandard means error± standard (n = 6). error * p (

Biomedicines 2020, 8, 480 8 of 21 3.4. PLA2 Involvement in 16:1 Release 3.4. PLA2 Involvement in 16:1 Release Since our previous lipidomic studies indicated that 16:1 fatty acids are primarily located at the sn-2 positionSince of phospholipids our previous inlipidomic resident studies peritoneal indicated macrophages that 16:1 [fatty19], itacids would are beprimarily logical tolocated expect at thatthe a sn-2 position of phospholipids in resident peritoneal macrophages [19], it would be logical to expect PLA2 mediates 16:1 fatty acid mobilization from the activated cells. In order to verify this point, we first that a PLA2 mediates 16:1 fatty acid mobilization from the activated cells. In order to verify this point, tested the effect of selective inhibitors of the two major intracellular PLA2s potentially responsible for we first tested the effect of selective inhibitors of the two major intracellular PLA2s potentially fatty acid release in activated macrophages, i.e., cPLA2α and iPLA2-VIA [19,20]. To inhibit cPLA2α, responsible for fatty acid release in activated macrophages, i.e., cPLA2α and iPLA2-VIA [19,20]. To we used pyrrophenone. To inhibit iPLA2-VIA, we used the fluoroketone FKGK18 and the structurally inhibit cPLA2α, we used pyrrophenone. To inhibit iPLA2-VIA, we used the fluoroketone FKGK18 and unrelated compound bromoenol lactone (BEL). These inhibitors are the most potent and selective the structurally unrelated compound bromoenol lactone (BEL). These inhibitors are the most potent compounds currently available to block cPLA2α and iPLA2-VIA in cells [13,52]. In addition, we also and selective compounds currently available to block cPLA2α and iPLA2-VIA in cells [13,52]. In tested the effect of SKF98625, an inhibitor of coenzyme A-independent transacylase, which acts in addition, we also tested the effect of SKF98625, an inhibitor of coenzyme A-independent transacylase, concert with cPLA α to remodel phospholipids with polyunsaturated fatty acids [53]. Figure4A which acts in concert2 with cPLA2α to remodel phospholipids with polyunsaturated fatty acids [53]. shows that 16:1 fatty acid release was almost completely inhibited by the two iPLA -VIA inhibitors, Figure 4A shows that 16:1 fatty acid release was almost completely inhibited by the 2two iPLA2-VIA whileinhibitors, pyrrophenone while pyrrophenone and SKF98625 and had SKF98625 no effect. had Under no effect. the conditions Under the utilized, conditions none utilized, of the inhibitorsnone of exertedthe inhibitors any detectable exerted e anyffect detectable on cell viability effect on (Figure cell viability S1). (Figure S1).

0.5 A 100 B 0.4 80 0.3 60 ** 0.2 40 ** 0.1 ** 20

Relative mRNA Level mRNA Relative

16:1 Release (nmol/mg protein) (nmol/mg Release 16:1 0.0 0 Control Sense Antisense

Pyrr

BEL

Unstim

FKGK18

No Inhib. No

SKF98625 120 0.4 C D 100 0.3 80

60 ** 0.2 40

Activity (% Hydrolysis) Activity 0.1 **

2 20

iPLA

0 protein) (nmol/mg Release 16:1 0.0 Control Sense Antisense Control Sense Antisense

FigureFigure 4. Release 4. Release of 16:1 of fatty 16:1 acids fatty by acids stimulated by stimulated macrophages. macrophages. (A) The cells (A) were The either cells were unstimulated either or stimulatedunstimulated by or mannan stimulated plus by laminarin mannan plus (0.5 laminarin mg/mL each) (0.5 mg/mL for 1 h each) (colored for 1 bars) h (colored in the bars) absence in the (no inhibition)absence or (no presence inhibition) of the or following presence inhibitors:of the following 10 µM inhibitors: FKGK18, 5 10µ M µM bromoenol FKGK18, lactone 5 µM bromoenol (BEL), 2 µM pyrrophenonelactone (BEL), (pyrr), 2 µMor pyrrophenone 10 µM SKF98625. (pyrr), Afterward, or 10 µM SKF98625. total content Afterward, of 16:1 total fatty content acids was of 16:1 measured fatty by GCacids/MS. was The measured fatty acid by releaseGC/MS. wasThe calculatedfatty acid release by subtracting was calculated the amount by subtracting of phospholipid-bound the amount of 16:1phospholipid in stimulated-bound cells from16:1 in that stimulated in unstimulated cells from cells. that Results in unstimulated are shown cells. as mean Results values are shownstandard as ± errorme ofan thevalues mean. ± standard (n = 4). error ** p

To confirm the results obtained with inhibitors, we next examined the effect of an iPLA2-VIA antisense oligonucleotide, which we and others have previously used with success to reduce the expression of this enzyme in a variety of cells [41–46]. Since we were unable to find reliable antibodies against murine iPLA2-VIA, the efficiency of antisense inhibition was analyzed by determining mRNA levels by qPCR, and also by assaying the BEL-inhibitable iPLA2 activity of cell homogenates after the different treatments. Using these techniques, we observed an mRNA decrease of 50–60% (Figure4B), and an enzyme activity decrease of 60–70% (Figure4C). Notably, even with this incomplete inhibition, the stimulus-induced mobilization of 16:1 fatty acids from the macrophages was almost completely abrogated (Figure4D). Collectively, these data strongly suggest that iPLA 2-VIA is the key enzyme regulating 16:1 fatty acid mobilization in activated macrophages.

3.5. Studies with Deuterated Fatty Acids An important question that arises from the results showing that activated macrophages release 16:1 is whether, after being released, the fatty acid stays in free fatty acid form or is utilized for the formation of new 16:1-containing lipids, which might also convey biological activity. It has been shown that some fatty acids released by activated macrophages, i.e., arachidonate and other polyunsaturates, are also transferred between various phospholipid species via remodeling reactions that are necessary to maintain the function and activity of the macrophages [53]. By analogy, we first set out to analyze whether the 16:1 released from PC is used for phospholipid fatty acid remodeling reactions, resulting in the enrichment of particular phospholipid species not of the PC class. To analyze the metabolic fate of each 16:1 isomer separately and also to improve detection by LC/MS, we utilized deuterated fatty acids for these experiments. Figure5 shows the characteristic profile of natural 16:1, deuterated 16:1n-7 (16:1n-7d) and deuterated 16:1n-9 (16:1n-9d). Deuterated 16:1n-10 was not available at the time of carrying out these experiments. 16:1n-7d was obtained from commercial sources and contained 14 deuterium atoms; thus, it produced a bell-shaped set of peaks due to the presence of various isotopomers with a maximum at m/z 267, corresponding to the m/z of the native molecule plus 14 deuteriums. 16:1n-9d was synthesized in our laboratory and contained 27 deuterium atoms (all hydrogen atoms bound to saturated carbons); thus, it produced a bell-shaped set of peaks with a maximum at m/z 277, that is, the m/z of native 16:1 plus 27. The signal produced by native 16:1 was very different, showing a decay from a maximum at m/z 253, allowing clear differentiation. For experiments, the cells were spiked with either 16:1n-7d or 16:1n-9d for 1 h before stimulation for an additional 1 h. Exogenous deuterated fatty acids incorporated and distributed into phospholipids in a manner that closely resembled that of endogenous fatty acid (Figure6). For both 16:1n-7d and 16:1n-9d, it was observed that cellular stimulation promoted the loss of 16:1 from PC, with similar amounts of both fatty acids being lost. This is analogous to that previously found for endogenous 16:1 and thus confirms the validity of our metabolipidomic approach and, in turn, provides further evidence that stimulated 16:1 release is not isomer-specific. Importantly, a small but clearly significant increase in the levels of the species PI(18:0/16:1), containing either 16:1n-7 or 16:1n-9, could be detected (Figure6). These results demonstrate that part of the 16:1 released from PC is transferred to PI during receptor-activation of the macrophages. Biomedicines 20202020,, 88,, 480480 10 of 21 Biomedicines 2020, 8, 480 10 of 21

Figure 5.5. Detection of 16:1 isomers by massmass spectrometry.spectrometry. Naturally occurring 16:1 (A),), deuterateddeuterated Figure 5. Detection of 16:1 isomers by mass spectrometry. Naturally occurring 16:1 (A), deuterated 16:1n-716:1n-7 ((BB),), andand deuterated 16:1n-916:1n-9 (C)) were injected directly into the mass spectrometer and their m/z 16:1n-7 (B), and deuterated 16:1n-9 (C) were injected directly into the mass spectrometer and their m/z spectra were determineddetermined.. The m/z of the most abundant ions are highlightedhighlighted inin red.red. spectra were determined. The m/z of the most abundant ions are highlighted in red.

100 14 10080 A 1412 * B * 10 80 A 12 * B 60 * 108 6040 86 4 4020 * 6 * * 42 * 200 20 0 0

PC(16:0/16:1n-7d) PC(18:2/16:1n-7d) PC(18:1/16:1n-7d) PC(18:0/16:1n-7d)

PC(16:0/16:1n-9d) PC(18:2/16:1n-9d) PC(18:1/16:1n-9d) PC(18:0/16:1n-9d)

PC(O-16:0/16:1n-7d)

PC(O-16:0/16:1n-9d)

PC(16:0/16:1n-7d) PC(18:2/16:1n-7d) PC(18:1/16:1n-7d) PC(18:0/16:1n-7d)

PC(16:0/16:1n-9d) PC(18:2/16:1n-9d) PC(18:1/16:1n-9d) PC(18:0/16:1n-9d)

PC(O-16:0/16:1n-7d)

20 2.5 PC(O-16:0/16:1n-9d) 20 C 2.5 D 15 2.0 C D 15 2.01.5 10 1.51.0 10 5 1.00.5 5 0 0.50.0 0 0.0

PE(16:0/16:1n-9d) PE(18:2/16:1n-9d) PE(18:1/16:1n-9d) PE(18:0/16:1n-9d)

PE(16:0/16:1n-7d) PE(18:2/16:1n-7d) PE(18:1/16:1n-7d) PE(18:0/16:1n-7d)

R e l a t a k i v e P a (a. u.) R e A r e e

PE(P-16:0/16:1n-9d)

PE(P-16:0/16:1n-7d)

PE(16:0/16:1n-9d) PE(18:2/16:1n-9d) PE(18:1/16:1n-9d) PE(18:0/16:1n-9d)

PE(16:0/16:1n-7d) PE(18:2/16:1n-7d) PE(18:1/16:1n-7d) PE(18:0/16:1n-7d)

R e l a t a k i v e P a (a. u.) R e A r e e

PE(P-16:0/16:1n-9d) 25 PE(P-16:0/16:1n-7d) 6 2520 * E 65 * F 4 2015 * E 5 * F 43 1510 32 10 5 21 50 10 0 0

PI(18:1/16:1n-9d) PI(18:0/16:1n-9d) PI(20:4/16:1n-9d)

PI(16:0/16:1n-7d) PI(18:1/16:1n-7d) PI(18:0/16:1n-7d) PI(20:4/16:1n-7d)

PI(18:1/16:1n-9d) PI(18:0/16:1n-9d) PI(20:4/16:1n-9d) PI(16:0/16:1n-7d) PI(18:1/16:1n-7d) PI(18:0/16:1n-7d) PI(20:4/16:1n-7d) Figure 6. 6. Analysis of phospholipid species containing deuterated 16:1 fatty fatty acids. acids. The cells were Figure 6. Analysis of phospholipid species containing deuterated 16:1 fatty acids. The cells were incubated with either deuterated deuterated 16:1n-716:1n-7 (left column, panels A,,C,E) or or deuterated deuterated 16:1n-9 16:1n-9 (right (right incubated with either deuteratedµ 16:1n-7 (left column, panels A,C,E) or deuterated 16:1n-9 (right column, panels panelsB ,BD,,DF),F (10) (10M, µM, 1 h). 1 After h). extensivelyAfter extensively washing washing the cells tothe remove cells tothe remove non-incorporated the non- column, panels B,D,F) (10 µM, 1 h). After extensively washing the cells to remove the non- fattyincorporated acid, the cells fatty were acid, either the cells untreated were (open either bars) untreated or treated (open with bars) mannan or treated plus laminarin with mannan (0.5 mg plus/mL incorporated fatty acid, the cells were either untreated (open bars) or treated with mannan plus each,laminarin 1 h). The(0.5 variousmg/mL deuteratedeach, 1 h). speciesThe various were determineddeuterated byspecies LC/MS. were The determined results are shownby LC/MS. as means The laminarinstandard (0.5 error mg/mL (n = 6). each, * p < 10.05, h). The significantly various deuterated different from species untreated were determined cells. by LC/MS. The ±results are shown as means ± standard error (n = 6). * p < 0.05, significantly different from untreated results are shown as means ± standard error (n = 6). * p < 0.05, significantly different from untreated cells. cells. BiomedicinesBiomedicines2020 2020, 8, ,8 480, 480 1111 of of 21 21

3.6. Synthesis of 16:1-Containing FAHFA 3.6. Synthesis of 16:1-Containing FAHFA Recent work has described in mammalian cells the existence of a novel family of lipids consisting Recent work has described in mammalian cells the existence of a novel family of lipids consisting of a fatty acid esterified to the hydroxyl group of a hydroxy fatty acid (fatty acid esters of hydroxy of a fatty acid esterified to the hydroxyl group of a hydroxy fatty acid (fatty acid esters of hydroxy fatty fatty acids, FAHFA), with anti-diabetic and anti-inflammatory properties [21]. Since 16:1 has been acids, FAHFA), with anti-diabetic and anti-inflammatory properties [21]. Since 16:1 has been shown to shown to be one of the fatty acid constituents of FAHFA, we sought for the possible presence of 16:1- be one of the fatty acid constituents of FAHFA, we sought for the possible presence of 16:1-containing containing FAHFA in the activated macrophages. Three such species were detected in resting cells, FAHFA in the activated macrophages. Three such species were detected in resting cells, namely the namely the hexadecenoic acid esters of hydroxyhexadecenoic (16:1/OH-16:1), hydroxypalmitic, hexadecenoic acid esters of hydroxyhexadecenoic (16:1/OH-16:1), hydroxypalmitic, (16:1/OH-16:0) (16:1/OH-16:0) and hydroxystearic acids (16:1/OH-18:0) (Figure 7). Importantly, the latter clearly and hydroxystearic acids (16:1/OH-18:0) (Figure7). Importantly, the latter clearly increased upon increased upon macrophage activation, indicating that part of the 16:1 released during the activation macrophage activation, indicating that part of the 16:1 released during the activation process is used to process is used to form compounds known to possess anti-inflammatory activity. form compounds known to possess anti-inflammatory activity.

3.00 * 2.75 *

16:1/OH-16:1 2.50 16:1/OH-16:0 16:1/OH-18:0 2.25

FAHFA (a.u.) FAHFA 1.00 0.50

0 2 4 6 Time (h) Figure 7. Identification of 16:1-derived branched fatty acid esters of hydroxy fatty acids (FAHFAs). Figure 7. Identification of 16:1-derived branched fatty acid esters of hydroxy fatty acids (FAHFAs). The cells were stimulated with mannan plus laminarin (0.5 mg/mL each) for the indicated times. Then, The cells were stimulated with mannan plus laminarin (0.5 mg/mL each) for the indicated times. Then, 16:1-containing FAHFAs were determined by LC/MS. Three species were detected: hydroxyhexadecenoic 16:1-containing FAHFAs were determined by LC/MS. Three species were detected: acid (16:1/OH-16:1) (cyan), hydroxypalmitic acid (16:1/OH-16:0) (blue) and hydroxystearic acid hydroxyhexadecenoic acid (16:1/OH-16:1) (cyan), hydroxypalmitic acid (16:1/OH-16:0) (blue) and (16:1/OH-18:0) (dark blue). A representative experiment is shown, and the data are expressed as mean hydroxystearic acid (16:1/OH-18:0) (dark blue). A representative experiment is shown, and the data values of three individual replicates. * p < 0.05, significantly different from unstimulated cells. are expressed± as mean values ± of three individual replicates. * p < 0.05, significantly different from 4. Discussionunstimulated cells.

4. DiscussionIn the last few years, 16:1 fatty acids have been under the spotlight in bioactive lipid research as promising nutritional and metabolic biomarkers [54]. We have shown in this work that most of the 16:1In the present last few in macrophages years, 16:1 fatty accumulates acids have inbeen just under one phospholipid the spotlight in molecular bioactive species, lipid research namely as PC(16:0promising/16:1), nutritional from which and itmetabolic is released biomarkers under cell [54]. activation We have conditions. shown in Thisthis work is, to that the bestmost of of our the knowledge,16:1 present the in first macrophages study addressing accumulates the metabolism in just one and phospholipid dynamics of molecular 16:1 fatty acids species, in cells namely in aPC(16:0/16:1), comprehensive from manner. which Whileit is released not focusing under cell specifically activation on conditions. 16:1 fatty acids,This is, recent to the studies best of that our includedknowledge, lipidomic the first profiling study addressing of macrophages the metabolism also identified and dynamics the species of PC(32:1),16:1 fatty likelyacids in composed cells in a ofcomprehensive 16:0 and 16:1, although manner. other While compositions not focusing are specifically also possible on (e.g., 16:1 fatty14:0 and acids, 18:1, recent or 18:0 studies and 14:1). that Montenegro-Burkeincluded lipidomic et profiling al. [55] analyzedof macrophages the lipid also composition identified ofthe polarized species PC(32:1), human monocyte-derivedlikely composed of macrophages,16:0 and 16:1, findingalthough six other species compositions that significantly are also di ffpossibleered among (e.g., the14:0 various and 18:1, states—one or 18:0 and of these14:1). wasMontenegro PC(32:1).-Burke Another et study,al. [55] also analyzed analyzing the lipid the glycerophospholipid composition of polarized profile human of human monocyte macrophages,-derived reportedmacrophages, a decrease finding in monounsaturated six species that signifi phospholipidscantly differed such as among PC(32:1) the during various polarized states— activationone of these of thewas cells PC(32:1). to either Another M1 or study, M2 phenotypes also analyzing [56]. the In contrast, glycerophospholipid studies with murineprofile of macrophages human macrophages, reported increasedreported levelsa decrease of PC(32:1) in monounsaturated following polarization phospholipids with granulocyte such as PC(32:1)/macrophage during polarized colony-stimulating activation factorof the and cells bacterial to either lipopolysaccharide M1 or M2 phenotypes [57,58]. [56]. In contrast, studies with murine macrophages reported increased levels of PC(32:1) following polarization with granulocyte/macrophage colony- stimulating factor and bacterial lipopolysaccharide [57,58]. Biomedicines 2020, 8, 480 12 of 21

The use of specific inhibitors together with antisense oligonucleotide approaches have allowed us to uncover the involvement of iPLA2-VIA in 16:1 mobilization. The other major intracellular PLA2 present in macrophages, cPLA2α, does not seem to participate in the process, as the specific inhibitor pyrrophenone had no discernible effect. The blockade of coenzyme A-independent transacylation also had no effect, indicating that 16:1 is not directly transferred between phospholipid species by mechanisms similar to those involving omega-6 fatty acids [53]. These results confirm and extend further our previous work on the role of iPLA2-VIA in mediating phospholipid fatty acid release responses in macrophages that do not involve arachidonic acid [19,20]. Moreover, the finding that the major substrate for iPLA2-VIA-mediated 16:1 release is PC(16:0/16:1) is also relevant in view of previous data showing that, in activated cells, iPLA2-VIA manifests selectivity for PC molecules containing palmitic acid at the sn-1 position, and that some of the major species hydrolyzed by the enzyme contain 16:1 at the sn-2 position [19,59]. Collectively, these results suggest that iPLA2-VIA is the key enzyme for 16:1 mobilization, and that the substrate selectivity of the enzyme in a whole cell context may be more stringent than what in vitro assays have suggested [60], probably due to cellular compartmentalization. Due to the anti-inflammatory character of the 16:1 fatty acids, it is conceivable that the regulated release of such fatty acids constitutes an important step for the mitigation or resolution of an inflammatory reaction. This study also addresses the involvement of part of the 16:1 liberated from PC in phospholipid fatty acid remodeling reactions. This is an important aspect in lipid signaling, as fatty acid remodeling reactions may increase phospholipid diversity and generate novel 16:1-containing phospholipid signatures that could be identified as fingerprints of specific activation states of the macrophages. More importantly, transfer of 16:1 moieties between phospholipids could lead to the generation of novel 16:1-containing species, or to increase the levels of pre-existing ones, which endow the cell with novel or improved functions. To explore this challenging possibility, we took advantage of the use of deuterated fatty acids. This strategy improves sensitivity and allows the characterization of the various isomers separately. Importantly, our approach identified a significant increase in the levels of a particular PI species, namely PI(18:0/16:1). This phenomenon was observed with the two deuterated fatty acids analyzed, and thus implies that the enzymes involved in the transfer do not distinguish positional isomers. Note, in this regard, that iPLA2-VIA also hydrolyzes all 16:1 isomers from PC equally well. It should be noted that the amount of 16:1 gained by the PI species is rather low, which suggests that 16:1 remodeling between phospholipids utilizes only small amounts of the fatty acid mobilized by iPLA2-VIA. Based on the relative peak areas of the samples and internal standards in LC/MS experiments, we estimate that as much as 5–10% of the 16:1 initially present in PC is transferred to PI. Notwithstanding, the fact that the cells actively make 16:1-containing PI strongly suggests that this lipid may have some signaling function. As a matter of fact, work by Koeberle et al. [61] has recently established that 16:1-containing PI possesses growth factor-like properties when added to fibroblast-like cell cultures. Clearly further work will be needed to explore a signaling role and possible cellular functions of 16:1-containing PI in macrophages. Our studies also unveil a second potentially important metabolic fate for the 16:1 mobilized from phospholipids in activated macrophages. The data show that part of the 16:1 hydrolyzed by iPLA2-VIA is diverted to the formation of FAHFA, a family of lipids with anti-inflammatory properties [21,62]. It is interesting that, of the three 16:1-containing FAHFA identified in resting macrophages, only one of them, 16:1/OH-18:0, decidedly increases in the activated cells. This suggests some sort of specificity in the formation of such species, which would be compatible with it playing a specific biological role. Similar to the situation discussed above regarding phospholipid 16:1 remodeling, formation of 16:1-containing FAHFA also appears to utilize small amounts of the fatty acid, accounting for as much 1% of total 16:1 mobilized from phospholipids. However, comparisons of the relative peak areas corresponding to the 16:1-containing FAHFA identified in our LC/MS experiments with that of a similar standard (palmitic acid ester of hydroxystearic acid) indicate that FAHFA levels increase Biomedicines 2020, 8, 480 13 of 21 inBiomedicines activated 2020 cells, 8, 480 up to approximately 4 nM, a value that is in the same range as that reported13 of for 21 FAHFA levels in mice serum (0.4–2.5 nM), and also comparable to the range of signaling lipids such as levels in mice serum (0.4–2.5 nM), and also comparable to the range of signaling lipids such as eicosanoids and endocannabinoids [21]. Thus, it is possible that even at the levels identified in this eicosanoids and endocannabinoids [21]. Thus, it is possible that even at the levels identified in this study, 16:1-containing FAHFA may be of pathophysiological relevance (Scheme1). Experimentation is study, 16:1-containing FAHFA may be of pathophysiological relevance (Scheme 1). Experimentation currently underway to validate this intriguing possibility. is currently underway to validate this intriguing possibility.

SchemeScheme 1.1. DynamicsDynamics ofof 16:116:1 fattyfatty acidacid utilizationutilization byby macrophages.macrophages. DottedDotted arrowsarrows indicateindicate thethe routeroute maymay involveinvolve severalseveral undefinedundefined steps.steps. 5. Conclusions 5. Conclusions Our study constitutes the first report addressing the dynamics of 16:1 utilization by macrophages. Our study constitutes the first report addressing the dynamics of 16:1 utilization by A key issue in this area of research is to determine whether the 16:1 fatty acid isomers exert their macrophages. A key issue in this area of research is to determine whether the 16:1 fatty acid isomers actions as free fatty acids per se or, instead, they are converted/incorporated into another lipid structure exert their actions as free fatty acids per se or, instead, they are converted/incorporated into another conveying the biological activity. We demonstrate that 16:1 fatty acids are released from phospholipids lipid structure conveying the biological activity. We demonstrate that 16:1 fatty acids are released via an iPLA2-VIA-regulated pathway. Moreover, while most of the released fatty acids remain in from phospholipids via an iPLA2-VIA-regulated pathway. Moreover, while most of the released fatty free fatty acid form, small parts are utilized in phospholipid fatty acid remodeling reactions to enrich acids remain in free fatty acid form, small parts are utilized in phospholipid fatty acid remodeling PI with 16:1, and also to form 16:1-containing FAHFA (Scheme1). As both 16:1-containing PI and reactions to enrich PI with 16:1, and also to form 16:1-containing FAHFA (Scheme 1). As both 16:1- FAHFA are known to possess biological activity, our results would be compatible with the challenging containing PI and FAHFA are known to possess biological activity, our results would be compatible possibility that (a) highly-regulated metabolite(s) of 16:1 mediate(s) at least part of the effects attributed with the challenging possibility that (a) highly-regulated metabolite(s) of 16:1 mediate(s) at least part to the fatty acid. Thus, our work provides new concepts and ideas that may help to explain the of the effects attributed to the fatty acid. Thus, our work provides new concepts and ideas that may mechanisms through which the 16:1 fatty acids exert their anti-inflammatory actions. help to explain the mechanisms through which the 16:1 fatty acids exert their anti-inflammatory Supplementaryactions. Materials: The following are available online at http://www.mdpi.com/2227-9059/8/11/480/s1, Figure S1: PLA2 inhibitors do not affect macrophage viability. AuthorSupplementary Contributions: Materials:Formal The analysis, following A.M.A., are available C.M., M.A.B.online at (Mar www.mdpi.com/2227ía A. Balboa) and J.B.;-9059/8/11/480/s1 funding acquisition,, Figure M.A.B.S1: PLA (Mar2 inhibitorsía A. Balboa) do not affect and J.B.; macrophage investigation, viability. A.M.A., C.M., M.A.B. (Miguel A. Bermúdez) and A.P.-E.; methodology, A.M.A., C.M., M.A.B. (Miguel A. Bermúdez) and A.P.-E.; project administration, M.A.B. (María A.Author Balboa) Contributions: and J.B.; supervision, Formal analysis, M.A.B. A.M.A., (María A. C.M., Balboa) M.A.B. and (María J.B.; writing—original A. Balboa) and J.B.; draft, funding A.M.A. acquisition, and J.B.; writing—reviewM.A.B. (María A. and Balboa) editing, and A.M.A., J.B.; A.P.-E.,investigation, M.A.B. A.M.A., (María A. C.M., Balboa) M.A.B. and J.B. (Miguel All authors A. Bermúdez) have read and and A.P. agreed-E.; tomethodology, the published A.M.A., version C.M., of the M.A.B. manuscript. (Miguel A. Bermúdez) and A.P.-E.; project administration, M.A.B. (María Funding:A. Balboa)This and researchJ.B.; supervision, was funded M.A.B. by (María the Spanish A. Balboa) Ministry and J.B.; of Science writing and—original Innovation, draft, A.M.A. grant numbers and J.B.; SAF2016-80883-Rwriting—review an andd editing,PID2019-105989RB-I00. A.M.A., A.P.-E., M.A.B. M.A.B. (Miguel (María A. Berm Balboa)údez) and was J.B. supported All authors by have a predoctoral read and fellowshipagreed to the from published the Spanish version Ministry of the of manuscript. Science and Innovation. CIBERDEM is an initiative of Instituto de Salud Carlos III. Funding: This research was funded by the Spanish Ministry of Science and Innovation, grant numbers SAF2016- Acknowledgments: 80883-R and PID2019We-105989RB thank Montse-I00. M.A.B. Duque (Miguel for excellent A. Bermúdez) technical assistance.was supported by a predoctoral fellowship Conflictsfrom the Spanish of Interest: MinistryThe authors of Science declare and Innovation. no conflict CIBERDEM of interest. Theis an funders initiative had of Instituto no role in de the Salud design Carlos of the III. study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publishAcknowledgments: the results. We thank Montse Duque for excellent technical assistance. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Biomedicines 2020, 8, 480 14 of 21

Abbreviations

GC/MS Gas chromatography coupled to mass spectrometry Biomedicines 2020, 8, 480 14 of 21 LC/MSBiomedicines 2020Liquid, 8, 480 chromatography coupled to mass spectrometry 14 of 21 FAHFA Branched fatty acid esters of hydroxy fatty acids PC AbbreviationsAbbreviationsCholine -containing glycerophospholipid PE Ethanolamine-containing glycerophospholipid GCGC/MS/MS GasGas chromatography chromatography coupled coupled to massto mass spectrometry spectrometry PI LCLC/MS/MS Phosphatidylinositol LiquidLiquid chromatography chromatography coupled coupled to massto mass spectrometry spectrometry PS FAHFAFAHFA Phosphatidylserine BranchedBranched fatty fatty acid acid esters esters of hydroxyof hydroxy fatty fatty acids acids PC Choline-containing glycerophospholipid PLAPC2 PhospholipaseCholine-containing A2 glycerophospholipid PE Ethanolamine-containing glycerophospholipid cPLAPE2α CytosolicEthanolamine group- containingIVA phospholipase glycerophospholipid A2 PI Phosphatidylinositol iPLAPI2- VIA CalciumPhosphatidylinositol-independent group VIA phospholipase A2 PS Phosphatidylserine PS Phosphatidylserine PLA2 Phospholipase A2 PLA2 Phospholipase A2 AppendixcPLA2α A Cytosolic group IVA phospholipase A2 cPLA2α Cytosolic group IVA phospholipase A2 iPLA2-VIA Calcium-independent group VIA phospholipase A2 AppendixiPLA A.1.2-VIA Synthesis Calcium of -Deuteratedindependent Fattygroup AcidsVIA phospholipase A2 Appendix A Appendix1H NMR A (400 MHz) and 13C NMR (100.6 MHz) spectra were recorded in CDCl3 as solvent. ChemicalAppendix shifts A.1. for Synthesis protons of Deuterated are reported Fatty Acidsin ppm from TMS with the residual CHCl3 resonance as Appendix A.1. Synthesis of Deuterated Fatty Acids internal reference. Chemical shifts for carbons are reported in ppm from TMS and are reference to 1 13 H NMR (400 MHz) and C NMR (100.6 MHz) spectra were recorded in CDCl3 as solvent. the carbon1H resonance NMR (400 of MHz) solvent. and Data13C NMR are reported (100.6 MHz) as follows: spectra chemical were recorded shift, inmultiplicity CDCl3 as solvent. (s = singlet, Chemical shifts for protons are reported in ppm from TMS with the residual CHCl3 resonance as d = Chemical doublet, tshifts = triplet, for protons q = quartet, are reported m = in multiplet, ppm from br TMS = broad) with the coupling residual constants CHCl3 resonance in Hertz, as and internal reference. Chemical shifts for carbons are reported in ppm from TMS and are reference to internal reference. Chemical shifts for carbons are reported in ppm from TMS and are reference to integration.the carbon The resonance high-resolution of solvent. Data mass are spectrometer reported as follows: used chemical was Agilent shift, multiplicity 5973, ESI (s-QTOF.= singlet, Flash the carbon resonance of solvent. Data are reported as follows: chemical shift, multiplicity (s = singlet, chromatographyd = doublet, t was= triplet, carried q = outquartet, using m silica= multiplet, gel (230 br–=240broad) mesh). coupling Thin-layer constants chromatography in Hertz, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad) coupling constants in Hertz, and analysesand integration. were performed The high-resolution using glass-backed mass spectrometer plates coated used with was silica Agilent gel 60 5973, and ESI-QTOF. F254 indicator Flash and integration. The high-resolution mass spectrometer used was Agilent 5973, ESI-QTOF. Flash visualizedchromatography by either was ultraviolet carried out irradiation using silica or gel by (230–240 staining mesh). with phosphomolybdic Thin-layer chromatography acid solution. analyses chromatography was carried out using silica gel (230–240 mesh). Thin-layer chromatography were performed using glass-backed plates coated with silica gel 60 and F254 indicator and visualized analysesThe deuterated were performed fatty acids using were glass synthesized-backed plates according coated with to Darwish silica gel et60 al.and [63] F254 with indicator modifications, and by either ultraviolet irradiation or by staining with phosphomolybdic acid solution. as describedvisualized below. by either The ultraviolet synthetic irradiation procedure or usesby staining two fragments with phosphomolybdic that, through acid a Wittig solution. reaction and The deuterated fatty acids were synthesized according to Darwish et al. [63] with modifications, final saponification,The deuterated leads fatty to acids deuterated were synthesized fatty acids according (Schemes to DarwishA1–A3). et al. [63] with modifications, as described below. The synthetic procedure uses two fragments that, through a Wittig reaction and as described below. The synthetic procedure uses two fragments that, through a Wittig reaction and final saponification, leads to deuterated fatty acids (Schemes A1–A3). final saponification, leads to deuterated fatty acids (Schemes A1–A3).

Scheme A1. Synthesis of the first fragment for the Wittig reaction, deuterated heptanal as a phosphoniumSchemeScheme A1. A1. bromideSynthesis Synthesis of salt the of (Wittig first the fragment first salt) fragment for (7a,b). the Wittigfor Pimelic the reaction, Wittig acid deuterated reaction, (1a,b) was heptanal deuterated used as as a heptanal phosphoniumstarting asmaterial. a Experimentalbromidephosphonium salt conditions: (Wittig bromide salt) (salta (7a,b).) Pd(C) (Wittig Pimelic 10%, salt) D(7a,b). acid2O, (1a,b) NaOD Pimelic was (40%), acid used (1a,b) 260 as starting °C, was 30 used material. bar; as (b starting) AC Experimental2O, material. 150 °C; (c) conditions: (a) Pd(C) 10%, D O, NaOD (40%), 260 C, 30 bar; (b) AC O, 150 C; (c) CH OH, 70 C; CH3ExperimentalOH, 70 °C; (d conditions:) BH3THF; ((ae)) 2 Pd(C)CBr4 Ph 10%,3P; D(f2)O, Ph NaOD3P,◦ CH (40%),3CN, 100 260 °C. °C, 2 30 bar; ◦ (b) AC2O,3 150 °C; ◦ (c) (CHd) BH3OH,3THF; 70 °C; (e) ( CBrd) BH4 Ph3THF;3P; (f(e)) Ph CBr3P,4 CHPh33P;CN, (f) 100Ph3P,◦C. CH3CN, 100 °C.

Scheme A2. Synthesis of the second fragment for the Wittig reaction, deuterated nonanal (11a,b). SchemeScheme A2. A2. Synthesis Synthesis of ofthe the second second fragmentfragment for thethe Wittig Wittig reaction, reaction, deuterated deuterated nonanal nonanal (11a,b). (11a,b). Nonanoic acid (8a,b) was used as starting material. Experimental conditions: (a) Pd(C) 10%, D2O, NonanoicNonanoic acid acid (8a,b) (8a,b) was was used used as as starting starting material. ExperimentalExperimental conditions: conditions: (a) (Pd(Ca) Pd(C) 10%,) 10%, D2O, D2O, NaOD (40%), 260 ◦C, 30 bar; (b) LiAlH4/THF; (c) PCC/DCM. NaODNaOD (40%), (40%), 260 260 °C, °C, 30 30 bar; bar; (b ()b LiAlH) LiAlH44/THF;/THF; (c) PCC/DCM.PCC/DCM. Biomedicines 2020, 8, 480 15 of 21 Biomedicines 2020, 8, 480 15 of 21

Scheme A3. Synthesis of deuterated fatty acids. Coupling [D17]nonanal and [D13]heptanal with the Scheme A3. Synthesis of deuterated fatty acids. Coupling [D17]nonanal and [D13]heptanal with the phosphonium bromide achieved by heating in acetonitrile and cesium carbonate. The cis-specific Wittig phosphoniumreaction leadsbromide to the achieved formation of by the heating cis-double in bond acetonitrile under mild and conditions. cesium Experimental carbonate. conditions: The cis-specific

Wittig reaction(a) 7a,b, Cs leads2CO3 ,to CH the3CN; formation (b) LiOH, Hof2O, the CH cis3OH.-double bond under mild conditions. Experimental conditions: (a) 7a,b, Cs2CO3, CH3CN; (b) LiOH, H2O, CH3OH. Appendix A.2. Deuteration of Pimelic Acid (General Procedure)

Appendix A.2.A mixtureDeuteration of pimelic of Pimelic acid (7 Acid g, 43.7 (General mmol), Procedure) Pd(C) (10%) (1.1 g) and 40% w/w NaOD (8 g, 19.7 mmol) in D2O (70 mL) was loaded into the Parr pressure reactor. The contents of the reactor were A mixturdegassede byof purgingpimelic with acid hydrogen (7 g, 43.7 gas mmol), and then sealedPd(C) and (10%) heated (1.1 to g) 260 and◦C (30 40% bar), w with/w NaOD constant (8 g, 19.7 mmol) instirring D2O for(70 six mL) days. was The loaded reactor wasinto cooled the Parr to room pressure temperature, reactor. and The the contents contents were of filtered the reactor off. were The catalyst was washed with water (4 25 mL). The aqueous filtrate was acidified to pH 2 using 3 M degassed by purging with hydrogen gas× and then sealed and heated to 260 °C (30 bar), with constant HCl. The white solid that was formed upon acidification was extracted from the aqueous solution stirring usingfor six ethyl days. acetate The (5 reactor100 mL) was and cooled washed to with room saturated temperature, NaCl. The and organic the layers contents were combinedwere filtered off. × The catalystand dried was with washed anhydrous with MgSO water4, and (4 × the 25 solvent mL). wasThe removed aqueous under filtrate vacuum was to giveacidified white solidto pH (5.4 2 using 3 1 M HCl. g,The 72%). white [D10 ]pimelicsolid that acid wa (94%s formed D). H NMR upon (400 acidification MHz, CDCl3 ):wasδH 1.36extracted (residual from CH2 ),the 1.60 aqueous (residual solution 13 using ethyl2 CH acetate2), 2.31 (residual(5 × 100 2mL)CH 2and). Cwashed NMR (100.6 with MHz, saturated CDCl3): NaCl.δC 179.85 The (2 organicCO2H). [Dlayers14]azelaic were acid combined × 1 × × (92% D): H NMR (400 MHz, CDCl3): δH 1.30 (residual 3 CH2), 1.55 (residual 2 CH2), 2.28 (residual 4 and dried with 13anhydrous MgSO , and the solvent was× removed under vacuum× to give white solid 2 CH2). C NMR (100.6 MHz, CDCl3): δC 178.80 (2 CO2H). (5.4 g, 72%).× [D10]pimelic acid (94% D). 1H NMR × (400 MHz, CDCl3): δH 1.36 (residual CH2), 1.60 (residualAppendix 2×CH A.3.2), 2.31 Deuteration (residual of Nonanoic 2×CH Acid2). and13C Heptanoic NMR Acid (100.6 MHz, CDCl3): δC 179.85 (2×CO2H). 1 [D14]azelaic [D acid17]nonanoic (92% D): and H [D 13 NMR]heptanoic (400 acids MHz, were CDCl3): prepared δH according 1.30 (residual to the general 3×CH2), procedure. 1.55 (residual 13 1 2×CH2),[D 2.2817]nonanoic (residual acid 2×CH (77%,2 95%). C D): NMRH NMR (100.6 (400 MHz, MHz, CDCl3):CDCl3):δ HδC0.81 178.80 (residual (2×CO CH3),2H). 1.22 (residual 5 CH ), 1.57 (residual CH ), 2.29 (residual CH ). 13C NMR (100.6 MHz, CDCl ): δ 13.5 (CD ), 21.2 × 2 2 2 3 C 3 Appendix(CD A.3.2), 23.9Deuteration (CD2), 28.6 of (3 NonanoicCD2), 31.1 Acid (CD 2and), 33.1 Heptanoic (CD2), 179.85 Acid (CO 2H). [D13]heptanoic acid (78%, 93% 1 × D): H NMR (400 MHz, CDCl3): δH 0.80 (residual CH3), 1.19 (residual 3 CH2), 1.59 (residual CH2), 13 × [D172.31]nonanoic (residual CH2). and [DC13 NMR]heptanoic (100.6 MHz, acids CDCl3): were δC prepared14.0 (CD3 ), according 22.5 (CD2), 24.4 to the(CD 2 general), 27.9 (CD procedure.2), [D17]nonanoic30.7 (CD acid2), 32.7 (77%, (CD2 ),95% 177.90 D): (CO 1H2 H).NMR (400 MHz, CDCl3): δH 0.81 (residual CH3), 1.22 (residual 13 5×CH2), Appendix1.57 (residual A.4. Synthesis CH2), of Anhydride2.29 (residual 3a,b CH2). C NMR (100.6 MHz, CDCl3): δC 13.5 (CD3), 21.2 (CD2), 23.9 (CD2), 28.6 (3×CD2), 31.1 (CD2), 33.1 (CD2), 179.85 (CO2H). [D13]heptanoic acid (78%, 93% A solution of [D10]pimelic acid 2a (8.16 g, 48 mmol) in acetic anhydride (170 mL) was refluxed for 1 D): H NMR4 h. The (400 solution MHz, was CDCl3): cooled to δ roomH 0.80 temperature (residual and CH the3), solvent1.19 (residual was removed 3×CH at reduced2), 1.59 pressure. (residual CH2), 2.31 (residualXylene (100CH2). mL) 13C was NMR added, (100.6 and the MHz, mix wasCDCl3): stirred δ forC 14.0 10 min (CD at3), room 22.5 temperature. (CD2), 24.4 The (CD solvent2), 27.9 (CD2), 1 30.7 (CDwas2), 32.7 removed (CD at2), reduced177.90 (CO pressure2H). to yield a white solid. (7.0 g, 97%): H NMR (400 MHz, CDCl3): δ 1.40 (residual CH ), 1.65 (residual 2 CH ), 2.42 (residual 2 CH ). 13C NMR (100.6 MHz, CDCl ): H 2 × 2 × 2 3 δ 19.9 (CD ), 22.3 (2 CD ), 36.4 (2 CD ), 168.68 (CO ). [D ]azelaic anhydride (95%): 1H NMR AppendixC A.4. Synthesis2 of Anhydride× 2 3a,b× 2 2− 14 (400 MHz, CDCl ): δ 1.37 (residual 3 CH ), 1.61 (residual 2 CH ), 2.38 (residual 2 CH ). 13C NMR 3 H × 2 × 2 × 2 A solution(100.6 MHz, of [D CDCl3):10]pimelicδC 19.9 acid (CD 2a2), (8.16 21.2 (2 g, CD48 2mmol)), 27.5 (2 inCD acetic2), 32.7 anhydride (2 CD2), 167.53(170 mL) (2 CO was2−). refluxed for 4 × × × × h. The solutionAppendix was A.5. cooled Synthesis to ofroom Compounds temperature 4a,b and the solvent was removed at reduced pressure. Xylene (100 mL) was added, and the mix was stirred for 10 min at room temperature. The solvent was removed A solution of [D10]pimelic anhydride 3a (11.25 g, 74.0 mmol) in anhydrous methanol (110 mL) at reduced pressure to yield a white solid. (7.0 g, 97%): 1H NMR (400 MHz, CDCl3): δH 1.40 (residual CH2), was refluxed under argon for 3 h. The solvent was removed in vacuo. The resulting liquid (13.29 g, 1.65 (residual 2×CH2), 2.42 (residual 2×CH2). 13C 1NMR (100.6 MHz, CDCl3): δC 19.9 (CD2), 22.3 (2×CD2), 72.22 mmol, 98%) was used in the next step. H NMR (400 MHz, CDCl3): δH 1.27 (residual CH2), 36.4 (2×CD1.542), (residual 168.68 2(COCH2−).), 2.29[D14 (residual]azelaic 2anhydrideCH ), 3.58 (95%): (s, 3H, CH1H ONMR). 13 C(400 NMR MHz, (100.6 CDCl MHz,3 CDCl): δH 1.37): δ (residual × 2 × 2 3 − 3 C 3×CH2), 24.11.61 (CD (residual2), 28.1 (22×CHCD22),), 34.4 2.38 (2 (residualCD2), 51.29 2×CH (CH32O). −13),C 174.21 NMR (CO (100.62−), 179.54 MHz, (CO CDCl3):2H). Compound δC 19.9 4b(CD2), 21.2 1 × × (97%): H NMR (400 MHz, CDCl3): δ 1.24 (residual− 3 CH2), 1.60 (residual 2 CH ), 2.29 (residual (2×CD2), 27.5 (2×CD2), 32.7 (2×CD2), 167.53H (2×CO2 ). × × 2

Appendix A.5. Synthesis of Compounds 4a,b

A solution of [D10]pimelic anhydride 3a (11.25 g, 74.0 mmol) in anhydrous methanol (110 mL) was refluxed under argon for 3 h. The solvent was removed in vacuo. The resulting liquid (13.29 g, 72.22 mmol, 98%) was used in the next step. 1H NMR (400 MHz, CDCl3): δH 1.27 (residual CH2), 1.54 (residual 2×CH2), 2.29 (residual 2×CH2), 3.58 (s, 3H, CH3O−). 13C NMR (100.6 MHz, CDCl3): δC 24.1 (CD2), 28.1 (2×CD2), 34.4 (2×CD2), 51.29 (CH3O−), 174.21 (CO2−), 179.54 (CO2H). Compound 4b (97%): 1H NMR (400 MHz, CDCl3): δH 1.24 (residual 3×CH2), 1.60 (residual 2×CH2), 2.29 (residual 2×CH2), 3.56 (s, 3H, CH3O−) 13C NMR (100.6 MHz, CDCl3): 20.1 (CD2), 22.8 (2×CD2), 28.1 (2×CD2), 32.3 (2×CD2), δC 51.09 (CH3O−), 173.71 (CO2−), 179.74 (CO2H). Biomedicines 2020, 8, 480 16 of 21

2 CH ), 3.56 (s, 3H, CH O ) 13C NMR (100.6 MHz, CDCl ): 20.1 (CD ), 22.8 (2 CD ), 28.1 (2 CD ), × 2 3 − 3 2 × 2 × 2 32.3 (2 CD ), δ 51.09 (CH O ), 173.71 (CO ), 179.74 (CO H). × 2 C 3 − 2− 2 Appendix A.6. Synthesis of Compounds 5a,b Borane-methyl complex (41 mL, 81.9 mmol) was added dropwise to a stirred solution of 4a (12.15 g, 66.03 mmol) in anhydrous THF (82 mL) at 0 ◦C under an inert atmosphere. The solution was maintained at 0 ◦C for 2 h and then allowed to warm up gradually to room temperature overnight. MeOH (10 mL) was added dropwise and the mixture was stirred for 2 h. The solvent was removed in vacuo and water and ethyl acetate were added to the residue, and the aqueous layer was extracted twice more with ethyl acetate. The combined organic layer was washed with NaHCO3, brine and 1 H2O, dried over MgSO4 and the solvent removed in vacuo to yield a clear liquid. (7.8 g, 70%). H NMR (400 MHz, CDCl3): δH 1.24 (residual 2 CH2), 1.50 (residual CH2), 2.18 (residual CH2), 2.39 × 13 (residual CH2), 3.48 (br s, 2H, CH2OH), 3.56 (s, 3H, CH3O−). C NMR (100.6 MHz, CDCl3): δC 23.3 (3 CD ), 29.1 (CD ), 31.9 (CD ), 51.29 (CH O ), 62.33 (CH OH), 174.21 (CO ). Compound 5a: 1H × 2 2 2 3 − 2 2− NMR (400 MHz, CDCl ): δ 1.22 (residual 4 CH ), 1.53 (residual 2 CH ), 2.13 (residual CH ), 3.45 (s, 3 H × 2 × 2 2 2H, CH OH), 3.58 (s, 3H, CH O ). 13C NMR (100.6 MHz, CDCl ): δ 19.8 (4 CD ), 27.4 (CD ), 34.2 2 3 − 3 C × 2 2 (CD2), 51.09 (CH3O−), 62.54 (CH2OH), 174.45 (CO2−).

Appendix A.7. Synthesis of Compounds 6a,b

Triphenylphosphine (13.6 g, 51.8 mmol) was added to a solution of ethyl [D10]7-hydroxyheptanoate (7.8 g, 46.2 mmol) and CBr4 (16.6 g, 50.0 mmol) in dichloromethane (50 mL) at 0 ◦C under an inert atmosphere. The mixture was stirred overnight. The solvent was removed in vacuo and the residue was triturated with heptane (70 mL). The solid was removed by filtration and the solvent was passed through a Celite pad. The solvent was removed in vacuo to yield a clear liquid (9.75 g, 91%). 1H NMR (400 MHz, CDCl3): δH 1.29 (residual CH2), 1.42 (residual CH2), 1.59 (residual CH2), 1.81 (residual 13 CH2), 2.26 (residual CH2), 3.37 (br s, 2H, CH2), 3.65 (s, 3H, CH3O−). C NMR (100.6 MHz, CDCl3): δC 1 22.3 (CD2), 27.4 (CD2), 33.58 (CH2Br), 51.30 (CH3O−), 174.21 (CO2−). Compound 6b (90%): H NMR (400 MHz, CDCl ): δ 1.34 (residual 3 CH ), 1.40 (residual CH ), 1.53 (residual CH ), 1.83 (residual 3 H × 2 2 2 CH2), 2.28 (residual CH2), 3.35 (s, 2H, CH2), 3.61 (s, 3H, CH3O−) 13C NMR (100.6 MHz, CDCl3): δC 18.3 (2 CD ), 22.0 (2 CD ), 28.1 (2 CD ), 33.48 (CH Br), 51.38 (CH O ), 174.44 (CO ). × 2 × 2 × 2 2 3 − 2− Appendix A.8. Synthesis of Compounds 7a,b (Wittig Salt) Anhydrous triphenylphosphine (27.4 g, 104.5 mmol) was added to a solution of ethyl [D10]7-bromoheptanoate (9.7 g, 41.8 mmol) and K2CO3 (1 g, 7.2 mmol) in acetonitrile (170 mL) at 0 ◦C under an inert atmosphere. The mixture was refluxed overnight. The solvent was removed in vacuo and the residue was triturated with methanol (60 mL). The Wittig salt was purified by flash chromatography (silica gel, eluent: DCM/methanol 20:1) to yield a clear oil. (7.0 g, 34%). 1H NMR (400 MHz, CDCl3): δH 1.25 (residual CH2), 1.48 (residual CH2), 1.54 (residual CH2), 1.77 (residual 13 CH2), 2.22 (residual CH2), 3.60 (s, 3H, CH3O−), 3.74 (m, 2H, CH2P), 7.59–7.92 (m, 15H). C NMR (100.6 MHz, CDCl3): δC 22.3 (CD2), 22.53 (CH2P), 25.9 (CD2), 28.8 (CD2), 25.1 (CD2), 33.7 (CD2), 51.68 (CH3O−), 118.38 (Ar), 119.2 (Ar), 130.42 (Ar), 131.3 (Ar), 133.65 (Ar), 134.92 (Ar), 137.5 (Ar), 174.21 (CO ). Compound 7b (41%): 1H NMR (400 MHz, CDCl ): δ 1.28 (residual 2 CH ), 1.47 (residual 2− 3 H × 2 2 CH2), 1.53 (residual CH2), 1.79 (residual CH2), 2.24 (residual CH2), 3.64 (s, 3H, CH3O−), 3.72 (m, 2H, × 13 CH2P), 7.53–7.5 (m, 15H). C NMR (100.6 MHz, CDCl3): δC 21.9 (CD2), 22.43 (CH2P), 25.4 (CD2), 28.6 (3 CD ), 25.5 (CD ), 33.9 (CD ), 51.88 (CH O ), 118.58 (Ar), 119.3 (Ar), 130.7 (Ar), 131.2 (Ar), 133.65 × 2 2 2 3 − (Ar), 134.82 (Ar), 138.3 (Ar), 174.31 (CO2−).

Appendix A.9. Synthesis of Compounds 10a,b

A solution of 7a (28.5 g, 162.4 mmol) in THF (260 mL) was added dropwise at 0 ◦C to a suspension of LiAlH4 (12.3 g, 324.8 mmol) in anhydrous THF (350 mL). The mixture was refluxed for 3 h with Biomedicines 2020, 8, 480 17 of 21 vigorous stirring. The reaction was quenched by addition of NaOH (12.3 mL, 2 M), water (12.3 mL) and NaOH (36.9 mL, 2 M). The white solid was filtrated off. The solvent was removed in vacuo to 1 yield a clear liquid. (23.8 g, 90%). H NMR (400 MHz, CDCl3): δH 0.82 (residual CH3), 1.22 (residual 6 CH ), 1.48 (residual CH ), 3.58 (s, 2H, CH OH). 13C NMR (100.6 MHz, CDCl ): δ 13.8 (CD ), 22.1 × 2 2 2 3 C 3 (CD ), 25.2 (CD ), 29.3 (3 CD ), 30.1 (CD ), 32.2 (CD ), 62.73 (CH OH). Compound 10b (88%): 1H 2 2 × 2 2 2 2 NMR (400 MHz, CDCl3): δH 0.80 (residual CH3), 1.25 (residual 7 CH2), 1.48 (residual 2 CH2), 3.55 (s, 13 × × 2H, CH2OH). C NMR (100.6 MHz, CDCl3): δC 14.0 (CD3), 22.5 (CD2), 25.7 (CD2), 29.6 (CD2), 31.7 (CD2), 33.6 (CD2), 62.53 (CH2OH).

Appendix A.10. Synthesis of Compounds 11a,b

A solution of [D17]1-nonanol (5 g, 31 mmol) was added to a suspension of pyridinium chlorochromate (1.0 g, 46.6 mmol) and silica gel (10 g) in DCM (62 mL). Once the reaction was completed, diethyl ether (200 mL) was added and filtered over silica gel. The solvent was removed in vacuo to yield a yellow liquid. The aldehyde was distilled under reduced pressure to obtain a clear 1 liquid. (110 ◦C, 15.5 torr, 2.4 g, 48%). H NMR (400 MHz, CDCl3): δH 0.80 (residual CH3), 1.22 (residual 5 CH ), 1.56 (residual CH ), 2.35 (residual CH ), 9.69 (s, 1H, CHO). 13C NMR (100.6 MHz, CDCl ): δ × 2 2 2 3 C 14.0 (CD ), 22.7 (2 CD ), 29.4 (4 CD ), 43.7 (CD ), 202.73 (CHO). Compound 11b (50%): 1H NMR (400 3 × 2 × 2 2 MHz, CDCl ): δ 0.83 (residual CH ), 1.25 (residual 3 CH ), 1.53 (residual CH ), 2.55 (residual CH ), 3 H 3 × 2 2 2 9.55 (s, 1H, CHO). 13C NMR (100.6 MHz, CDCl ): δ 13.9 (CD ), 22.5 (CD ), 28.4 (2 CD ), 31.8 (CD ), 3 C 3 2 × 2 2 43.5 (CD2), 0.53 (CHO).

Appendix A.11. Synthesis of Compounds 12a,b

[D17]nonanal (0.27 g, 1.7 mmol) was added at room temperature to a mixture of phosphonium salt 7a (0.84 g, 1.7 mmol) in dichloromethane (30 mL) and Cs2CO3 (2.25 g, 7 mmol) under inert atmosphere. The mixture was refluxed for 24 h with vigorous stirring. The progress of the reaction was monitored by thin-layer chromatography on silica gel using ethyl acetate-heptane 1:10 (Rf = 0.25). When the reaction was completed, it was cooled at room temperature, and the mixture was filtered through a Celite pad, which was then washed with DCM. The solvent was evaporated under reduced pressure and purified on a column of silica gel to obtain a methyl ester as a clear liquid. (0.4 g, 79%). 1H NMR (400 MHz, CDCl3): δH 0.73 (residual CH3), 1.21 (residual 8 CH2), 1.55 (residual CH2), 1.90 (residual × 13 CH2), 2.25 (residual CH2), 3.66 (s, 3H, CH3O−), 5.33 (m, 2H, HC=C). C NMR (100.6 MHz, CDCl3): δC 14.0 (CD3), 22.7 (CD2), 25.3 (CD2), 27.8 (3 CD2), 29.6 (4 CD2), 31.8 (CD2), 33.7 (CD2), 51.30 (CH3O−), × × 1 129.1 (HC=C), 129.9 (HC=), 174.60 (CO2−). Methyl [D27](Z)-9-hexadecenoic ester (69%). H NMR (400 MHz, CDCl3): δH 0.75 (residual CH3), 1.25 (residual 8 CH2), 1.57 (residual CH2), 1.89 (residual CH2), 13 × 2.27 (residual CH2), 3.63 (s, 3H, CH3O−). C NMR (100.6 MHz, CDCl3): δC 13.8 (CD3), 22.8 (CD2), 25.0 (CD ), 27.7 (2 CD ), 29.5 (5 CD ), 31.9 (CD ), 33.8 (CD ), 51.22 (CH O ), 129.2 (HC=C), 130.0 2 × 2 × 2 2 2 3 − (HC=), 174.50 (CO2−). A solution of lithium hydroxide (90 mL, 1.6 M) was added to the methyl ester (2.5 g, 8.8 mmol) in methanol (80 mL) with stirring. The hydrolysis reaction was monitored by thin-layer chromatography. The reaction reached completion after 24 h. The aqueous layer was washed with diethyl ether (3 75 × mL) and HCl (1 M) was added to the reaction mixture to acidify it to pH 1. The turbid aqueous solution was extracted three times with diethyl ether. The organic phase was washed once with brine and dried over magnesium sulfate. The solvent was evaporated under reduced pressure to give a pale-yellow oil. The oil was purified on a silica gel column (eluent heptane:ethyl acetate) to give a colorless oil 1 of deuterated acid. (2.1 g, 8.3 mmol, 94%). H NMR (400 MHz, CDCl3): δH 0.83 (residual CH3), 1.30 (residual 8 CH ), 1.65 (residual CH ), 1.92 (residual CH ), 2.31 (residual CH ), 5.30 (m, 2H, HC=C). × 2 2 2 2 13C NMR (100.6 MHz, CDCl ): δ 14.0 (CD ), 22.5 (CD ), 25.1 (CD ), 27.6 (3 CD ), 29.3 (4 CD ), 3 C 3 2 2 × 2 × 2 31.5 (CD2), 33.8 (CD2), 51.32 (CH3O−), 129.0 (HC=C), 129.8 (HC=), 174.63 (CO2−). Compound 12b 1 (69%). H NMR (400 MHz, CDCl3): δH 0.77 (residual CH3), 1.27 (residual 8 CH2), 1.55 (residual CH2), 13 × 1.87 (residual CH2), 2.29 (residual CH2), 3.60 (s, 3H, CH3O−). C NMR (100.6 MHz, CDCl3): δC 13.9 Biomedicines 2020, 8, 480 18 of 21

(CD ), 22.6 (CD ), 25.1 (CD ), 27.9 (2 CD ), 29.1 (5 CD ), 31.4 (CD ), 33.5 (CD ), 51.22 (CH O ), 129.3 3 2 2 × 2 × 2 2 2 3 − (HC=C), 130.0 (HC=), 174.55 (CO2−).

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