Although seemingly banal, the structure-function relationship of glycerophospholipid (GPL) is far more complex and insightful than first meets the eye. While the amphoteric nature of the molecule importantly allows for chemical partitioning, the individual functional groups serve numerous other specific purposes. The charged hydrophilic head group creates the possibility for lipids to be directed to particular subcellular compartments, whereby the lipophilic FAs can impart variable physicochemical properties to the molecule and initiate signaling (1Jackson Catherine L. Walch L. Verbavatz J.-M. Lipids and their trafficking: an integral part of cellular organization.). Arguably, two of the most important processes in dictating the modeling and remodeling of mammalian GPL structure are the Kennedy pathway and the Lands cycle. While the Kennedy pathway describes the de novo synthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from cytidine-diphosphate diacylglycerol (2The function of cytidine coenzymes in the biosynthesis of phospholipides.), the Lands cycle describes the process by which phospholipase and acyltransferase enzymes remodel GPL-FAs (3Stories about acylchains.). While these processes logically have implications in cellular energy production, whether de novo generated FAs or extracellular FAs (from uptake) have identical or varied roles is not well understood. Given that cancer cells are known to modulate and oscillate between de novo and uptake FA metabolism to opportunistically source energy for growth (4Koundouros N. Poulogiannis G. Reprogramming of fatty acid metabolism in cancer., 5Hosios Aaron M. Hecht Vivian C. Danai Laura V. Johnson Marc O. Rathmell Jeffrey C. Steinhauser Matthew L. et al.Amino acids rather than glucose account for the majority of cell mass in proliferating mammalian cells.), understanding if subfractions of FAs serve discrete purposes is of importance when elucidating the molecular mechanisms of cancer. For example, a cancer cell actively increasing the production of FAs only to oxidize these to meet energy demands is counterproductive, and thus, the cell might instead be compelled to source extracellular FAs. For this to be possible however, there must be compartmentalization and discrete metabolic treatment of de novo or uptake FAs—a sentiment that is contrary to the current idea that the cell has a common “pool” of FAs, used to meet metabolic demand (4Koundouros N. Poulogiannis G. Reprogramming of fatty acid metabolism in cancer., 6Nagarajan S.R. Butler L.M. Hoy A.J. The diversity and breadth of cancer cell fatty acid metabolism.).One of the more important enzymes within the Lands cycle is phospholipase A2 (PLA2), a lipase that is responsible for the selective cleavage of the FA located at the sn-2 position within GPLs. Notably, PLA2 has been shown to exhibit a high-level of selectivity toward GPLs containing PUFAs, such as arachidonic acid (AA; 20:4 n-6) and DHA (22:6 n-3) (7Diez E. Chilton F.H. Stroup G. Mayer R.J. Winkler J.D. Fonteh A.N. Fatty acid and phospholipid selectivity of different phospholipase A2 enzymes studied by using a mammalian membrane as substrate., 8Lipid nutrition: “In silico” studies and undeveloped experiments.). Amongst other functions, the release of AA and DHA in turn initiates proinflammatory or anti-inflammatory responses through the cyclooxygenase and lipoxygenase pathways (8Lipid nutrition: “In silico” studies and undeveloped experiments., 9Omega-3 fatty acids and inflammatory processes., 10Arachidonic acid in cell signaling.). Concurrent with the release of the sn-2 FA is the formation of a 1-acyl-2-lyso-GPL, or 2-lyso-GPL, with a hydroxyl group at the sn-2 position of the glycerol backbone. While these lysospecies are themselves signaling mediators (11Meyer Zu Heringdorf D. Jakobs K.H. Lysophospholipid receptors: signalling, pharmacology and regulation by lysophospholipid metabolism.), they also readily undergo re-esterification, reverting to diacyl-GPLs via acyltransferase enzymes, such as the lysophosphatidic acid acyltransferase or lysophosphatidylcholine acyltransferase (LPCAT) isoforms (3Stories about acylchains., 12Hishikawa D. Shindou H. Kobayashi S. Nakanishi H. Taguchi R. Shimizu T. Discovery of a lysophospholipid acyltransferase family essential for membrane asymmetry and diversity., 13Hishikawa D. Hashidate T. Shimizu T. Shindou H. Diversity and function of membrane glycerophospholipids generated by the remodeling pathway in mammalian cells.). The labile nature of lyso-GPLs also allows physicochemical properties such as pH and temperature to influence a behavior known as acyl chain migration, whereby a FA is able to migrate from one sn-position to a free hydroxyl group on the glycerol backbone. Unlike the sn-selectivity of PLA2, acyltransferase enzymes are rarely reported to exhibit selectivity or specificity for 1-lyso or 2-lyso acceptor lipids. Results from some studies however suggest that perhaps certain LPCAT isoforms (e.g., LPCAT1) are nonspecific to 1-lyso or 2-lyso lipids, whereas other isoforms (e.g., LPCAT3) and acyltransferase enzymes (e.g., LYCAT) exhibit a degree of selectivity depending on lysoacceptor and FA donor species (14Kawana H. Kano K. Shindou H. Inoue A. Shimizu T. Aoki J. An accurate and versatile method for determining the acyl group-introducing position of lysophospholipid acyltransferases.). Generally, it is accepted that acyl chain migration occurring prior to esterification will be influential in lipid molecular structure. Because Kennedy and Lands metabolic pathways affect different structural motifs of the GPL molecule, they are able to dictate both the transport of lipids around the cell and the initiation of signaling cascades through the release of biologically active FAs. This allows for independent organelle membrane modifications as well as the initiation of signaling cascades through the release of biologically active FAs. Although the various mechanisms and outcomes of lipid remodeling have been studied extensively, little is known about the impact that sn-isomeric lipid species have on remodeling and transport, or conversely, the impact these mechanisms have on lipid isomer populations. While it is now known that the GPLs of both prokaryotic and eukaryotic cells exhibit a preference for unsaturated FAs to be at the sn-2 position, prior to 2003, there was little-to-no indication that sn-positional isomers were present in eukaryotic cells (15Ekroos K. Ejsing C.S. Bahr U. Karas M. Simons K. Shevchenko A. Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation., 16Renooij W. Van Golde L.M. Zwaal R.F. Van Deenen L.L. Topological asymmetry of phospholipid metabolism in rat erythrocyte membranes. Evidence for flip-flop of lecithin.). Ekroos et al. (15Ekroos K. Ejsing C.S. Bahr U. Karas M. Simons K. Shevchenko A. Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation.) displayed that not only was there a quantifiable population of sn-1 unsaturated GPLs, but the relative abundance of these regioisomer populations can vary between mammalian GPL samples. This was exemplified in more recent studies into mammalian GPL regioisomers, which revealed that bovine liver (17Wozny K. Lehmann W.D. Wozny M. Akbulut B.S. Brügger B. A method for the quantitative determination of glycerophospholipid regioisomers by UPLC-ESI-MS/MS.), egg yolk, and sheep kidney (18Kozlowski R.L. Mitchell T.W. Blanksby S.J. A rapid ambient ionization-mass spectrometry approach to monitoring the relative abundance of isomeric glycerophospholipids.) exhibited a stronger preference for canonical sn-positions than synthetically prepared standards.Although an independent lipase enzyme, phospholipase A1, is known to catalyze the cleavage of FAs in the sn-1 position of specific GPL classes (19Aoki J. Inoue A. Makide K. Saiki N. Arai H. Structure and function of extracellular phospholipase A1 belonging to the pancreatic lipase gene family.), the preference for unsaturated FAs to exist in the sn-2 position is thought to be strongly linked with Lands cycle lipid remodeling and PLA2 functionality (7Diez E. Chilton F.H. Stroup G. Mayer R.J. Winkler J.D. Fonteh A.N. Fatty acid and phospholipid selectivity of different phospholipase A2 enzymes studied by using a mammalian membrane as substrate., 10Arachidonic acid in cell signaling., 20Hughes-Fulford M. Tjandrawinata R.R. Li C.-F. Sayyah S. Arachidonic acid, an omega-6 fatty acid, induces cytoplasmic phospholipase Ainf2/inf in prostate carcinoma cells.). This enhanced sn-positional specificity in biological systems implies that cellular intervention is required for maintenance. Studies into the peroxidation of unilamellar liposomes comprised of specific PC sn-isomers (containing palmitic acid [PA] and linoleic acid [LA]) revealed oxidation rates were sn-isomer dependent (21Mazari A. Iwamoto S. Yamauchi R. Effects of linoleic acid position in phosphatidylcholines and cholesterol addition on their rates of peroxidation in unilamellar liposomes.). Using radiolabeled FAs in mouse models, others showed that while the supplemented cis-FA (oleic acid) showed esterification specificity to the sn-2 position of GPLs, the trans-FA species (elaidic acid) was equally esterified to sn-1 and sn-2 positions. More recently, molecular dynamics simulations revealed that unsaturated FAs at the sn-1 position of GPLs create more ordered (and hence less fluid) membranes than their unsaturated sn-2 counterparts (22Martinez-Seara H. Róg T. Karttunen M. Vattulainen I. Reigada R. Why is the sn-2 chain of monounsaturated glycerophospholipids usually unsaturated whereas the sn-1 chain is saturated? studies of 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (SOPC) and 1-Oleoyl-2-stearoyl-sn-glycero-3-phosphatidylcholine (OSPC) membranes with and without cholesterol.). These functional differences may help explain variations in sn-isomer distribution that are observed to exist in recent tissue models. In a pivotal study, Paine et al. (23Paine M.R.L. Poad B.L.J. Eijkel G.B. Marshall D.L. Blanksby S.J. Heeren R.M.A. et al.Mass spectrometry imaging with isomeric resolution enabled by ozone-induced dissociation.) showed that lipid isomers were tightly regulated and highly organized within the gray and white matter structures of brain tissue and were subsequently disrupted in murine cancer tumors. Therefore, because lipid species containing saturated FAs (SFAs) at the sn-1 and unsaturated FAs at the sn-2 are the typical structures identified as the majority isomer in lipid canon, to aid with explanation throughout, these isomers will be referred to as “canonomers.” Conversely, lipids displaying atypical or apocryphal sn-isomeric structure (with the unsaturated FA contained at the sn-1 position) will be referred to “apocromers.”DiscussionWith exception to mitochondria, FA modification is predominantly undertaken in the endoplasmic reticulum (ER) and thus trafficking of FAs and lipids is necessary to reach the plasma and organelle membranes. Although there are exceptions, lipids mainly require active transport mechanisms to shuttle around the cell. The three conventionally accepted trafficking mechanisms involve membrane transport (e.g., budding from a donor membrane), carrier proteins (e.g., fatty acyl binding proteins [FABPs]), or transfer across membrane contact sites (e.g., ER/mitochondria lipid exchange) (44Blom T. Somerharju P. Ikonen E. Synthesis and biosynthetic trafficking of membrane lipids.). Alongside de novo lipogenesis, cells can also obtain extracellular FAs from uptake and incorporate these into lipid structures. From our work within, these differential trafficking mechanisms can be inferred from the variations in the PC (and other GPL) lipid species profile(s) (vide supra Fig. 1D; other GPLs, cf., supplemental Fig. S2). For example, tracing incorporation and modification(s) of labeled PAs and SAs reveals that each FA is either incorporated into more highly unsaturated GPLs or are themselves desaturated more frequently (Fig. 1D; up 13PA/up 13SA; e.g., PC 32:2, PC 34:2, PC 34:3, and PC 36:3). In contrast, GPLs solely incorporating FAs synthesized by cellular machinery are influenced by the supplemented FA such that unlabeled PC 34:0 and PC 36:6 species are discretely increased (Fig. 1D; dn 13PA/dn 13SA). These differences suggest that the cellular management of FAs is dependent on both FA source origin and FA speciation. Thus, we propose that there are distinct mechanisms for the trafficking of FAs originating from de novo lipogenesis and those the cell has acquired from uptake. Previous research has also revealed FA-dependent trafficking differences. One such example can be found in FABP5, a FA binding protein that delivers FAs from the cytosol to nuclear PPARβ/δ. Researchers were able to show that while FAs from uptake all complexed with FABP5, a SFA (PA) inhibited FABP5 activation of the PPARβ/δ pathway, whereas a PUFA (LA; FA 18:2 n-6) instead activated PPARβ/δ (45Levi L. Wang Z. Doud M.K. Hazen S.L. Noy N. Saturated fatty acids regulate retinoic acid signalling and suppress tumorigenesis by targeting fatty acid-binding protein 5.). This FA-driven modulation of PPARβ/δ has downstream effects in the activation of lipometabolic-centered genes such as pyruvate dehydrogenase kinase 4 (PDK4), angiopoietin 4 (ANGPTL4), perilipin 2 (PLIN2), and cluster of differentiation/FA translocase (CD36). Considering the changes that occur to FA metabolism in cancer, especially including FA uptake and energy production from FAs, the translation of these genes into proteins has widespread implications for oncometabolism (46Song X. Liu J. Kuang F. Chen X. Zeh III, H.J. Kang R. et al.PDK4 dictates metabolic resistance to ferroptosis by suppressing pyruvate oxidation and fatty acid synthesis., 47Baba K. Kitajima Y. Miyake S. Nakamura J. Wakiyama K. Sato H. et al.Hypoxia-induced ANGPTL4 sustains tumour growth and anoikis resistance through different mechanisms in scirrhous gastric cancer cell lines., 48Zhu P. Tan Ming J. Huang R.-L. Tan Chek K. Chong Han C. Pal M. et al.Angiopoietin-like 4 protein elevates the prosurvival intracellular O2−:H2O2 ratio and confers Anoikis resistance to tumors., 49McIntosh A.L. Senthivinayagam S. Moon K.C. Gupta S. Lwande J.S. Murphy C.C. et al.Direct interaction of Plin2 with lipids on the surface of lipid droplets: a live cell FRET analysis., 50Hao J.-W. Wang J. Guo H. Zhao Y.-Y. Sun H.-H. Li Y.-F. et al.CD36 facilitates fatty acid uptake by dynamic palmitoylation-regulated endocytosis., 51The enigmatic membrane fatty acid transporter CD36: new insights into fatty acid binding and their effects on uptake of oxidized LDL.). Furthermore, this FABP5/PPARβ/δ mechanism highlights how trafficking mechanisms can be entangled with signaling events, while this work provides the foundation for the discovery of the proteins involved in partitioning of dn- and up-FAs.Given the widespread impact FA trafficking can have on signaling for gene expression and protein translation, it is logical to assume that the cell has fine control over FA uptake, FA trafficking, and regulation of lipid remodeling. This is exemplified in our work by the DB or sn-position differences observed between labeled or unlabeled FAs and possibly relates to the known compartmentalization of desaturase enzymes. Within the pie charts of Fig. 2D, the percentage of n-10 (requiring FADS2 desaturation) is observed to be higher when the labeled PA is modified by desaturation or by combined desaturation and elongation, whereas n-7 and n-9 (requiring stearoyl-CoA desaturase 1 [SCD-1] desaturation) are seen to be higher within labeled lipids where the unlabeled FA is the unsaturated chain. This would suggest that a higher proportion of FAs from uptake are transported to the mitochondria (where FADS2 desaturation mainly occurs) (52Park H.G. Park W.J. Kothapalli K.S.D. Brenna J.T. The fatty acid desaturase 2 (FADS2) gene product catalyzes Δ4 desaturation to yield n-3 docosahexaenoic acid and n-6 docosapentaenoic acid in human cells.), whereas de novo synthesized FAs are more readily transported to the ER (where SCD-1 desaturation occurs) (53Role of stearoyl-coenzyme A desaturase in lipid metabolism.). Furthermore, this would indicate that FA speciation and origin (i.e., uptake or de novo) both determine subcellular destination as opposed to the conventional theory that all FAs form a common “pool.” One explanation for this compartmentalization of FA fractions would be for net-positive energy production. Catabolizing FAs that have been actively de novo synthesized, although sometimes required, is counterproductive to energy production. Instead, trafficking FAs from uptake to β-oxidation sites would provide externally produced energy (and carbon) to the cell. Interestingly, FA uptake influences PPARβ/δ activity and subsequent activation of PDK4, which regulates the conversion of pyruvate and glucose to acetyl-CoA and suppresses ferroptosis (46Song X. Liu J. Kuang F. Chen X. Zeh III, H.J. Kang R. et al.PDK4 dictates metabolic resistance to ferroptosis by suppressing pyruvate oxidation and fatty acid synthesis.). These functions directly implicate compartmentalized FA metabolism as mitochondria are responsible for cellular energy production via β-oxidation and FA synthesis, whereas the peroxisomes mediate an alternate FA β-oxidation pathway and are largely responsible for the formation of reactive oxidative species and ferroptosis.A related result that supports the hypothesis of specific FA species compartmentalization can be found in the unusual lipid DB profile displaying 16:1 n-12 and increased 16:1 n-9 (Fig. 2D) after a labeled SA has undergone partial β-oxidation and desaturation to FA 16:1ⱡ. As stated, within the cell, both the mitochondria and peroxisomes have the capacity for β-oxidation; however, peroxisome β-oxidation and stimulation has been shown to have a complex relationship with the genetic expression of FADS1 and FADS2 and hence FADS1 and FADS2 enzymes (54Hall D. Poussin C. Velagapudi V.R. Empsen C. Joffraud M. Beckmann J.S. et al.Peroxisomal and microsomal lipid pathways associated with resistance to hepatic steatosis and reduced pro-inflammatory state., 55Song He W. Nara T.Y. Nakamura M.T. Delayed induction of Δ-6 and Δ-5 desaturases by a peroxisome proliferator.). These enzymes catalyze Δ4, Δ5, Δ6, and Δ8 desaturation (37Young R.S.E. Bowman A.P. Williams E.D. Tousignant K.D. Bidgood C.L. Narreddula V.R. et al.Apocryphal FADS2 activity promotes fatty acid diversification in cancer., 52Park H.G. Park W.J. Kothapalli K.S.D. Brenna J.T. The fatty acid desaturase 2 (FADS2) gene product catalyzes Δ4 desaturation to yield n-3 docosahexaenoic acid and n-6 docosapentaenoic acid in human cells.) and in conjunction with β-oxidation would lead to the formation of the unusual 16:1 n-12 species observed within Fig. 2D (mid). This may suggest that the SA supplement is being split between two trafficking pathways: i) to the ER for SCD-1 desaturation and then membrane-contact transport to the peroxisomes for β-oxidation to yield 16:1 n-9 and stimulate the peroxisomal influence on FADS2 and ii) to the mitochondria for FADS2 desaturation and β-oxidation to yield 16:1 n-12.Another example of remodeling compartmentalization can be observed in Fig. 3 (left), where PC 16:0_16:1 n-9 and PC 16:0_16:1 n-10 from nonsupplemented LNCaP showed equal apocromer/canonomer distribution, whereas PC 16:0_16:1 n-7 markedly favored canonomeric incorporation. This is a remarkable result as both β-oxidation and FADS2 desaturation (leading to FA 16:1 n-9 and n-10, respectively) are mitochondrial processes (52Park H.G. Park W.J. Kothapalli K.S.D. Brenna J.T. The fatty acid desaturase 2 (FADS2) gene product catalyzes Δ4 desaturation to yield n-3 docosahexaenoic acid and n-6 docosapentaenoic acid in human cells., 56Park H.G. Kothapalli K.S.D. Park W.J. Deallie C. Liu L. Liang A. et al.Palmitic acid (16:0) competes with omega-6 linoleic and omega-3 ɑ-linolenic acids for FADS2 mediated Δ6-desaturation.), whereas the SCD-1 desaturation forming FA 16:1 n-7 would occur at the ER. It should also be noted that the desaturation of FA 16:0 by FADS2 is performed at a rate determined by the relative abundance of FA components in the substrate mixture (56Park H.G. Kothapalli K.S.D. Park W.J. Deallie C. Liu L. Liang A. et al.Palmitic acid (16:0) competes with omega-6 linoleic and omega-3 ɑ-linolenic acids for FADS2 mediated Δ6-desaturation.). Similarly, the PC 14:0_18:1 in Fig. 3 (right) shows that sn-isomer distribution of FA 18:1 n-7 and n-10 is quite similar, whereas n-9 is distinctly different. To form PC 14:0_18:1, both FA 16:1 n-7 and FA 16:1 n-10 would require elongation, whereas FA 18:1 n-9 would be directly desaturated from FA 18:0. Interestingly, subcellular organelles are observed to have discrete pH ranges with mitochondrial pH being 7.58 for the matrix and 6.88 for the intermembrane space (57Porcelli A.M. Ghelli A. Zanna C. Pinton P. Rizzuto R. Rugolo M. pH difference across the outer mitochondrial membrane measured with a green fluorescent protein mutant.), ER pH being 7.1 at a resting state (58Kim J.H. Johannes L. Goud B. Antony C. Lingwood C.A. Daneman R. et al.Noninvasive measurement of the pH of the endoplasmic reticulum at rest and during calcium release.) and peroxisome pH varying between 7.4 and 8.1 depending on metabolic activity and cell-type properties (59Determination of peroxisomal pH in living mammalian cells using pHRed.), Considering acyl chain migration (while lipids exist as lysospecies) is a pH-dependant equilibrium, the remodeling of lipids under the different pH conditions of subcellular compartments could indeed lead to differences in the sn-positional isomer distributions. Therefore, the sn-positional isomers may be a further indication of compartmentalized FA modification events. Because FA 16:1 n-9 has been recently identified as a mediator for an anti-inflammatory response (60Astudillo A.M. Meana C. Guijas C. Pereira L. Lebrero P. Balboa M.A. et al.Occurrence and biological activity of palmitoleic acid isomers in phagocytic cells., 61Guijas C. Meana C. Astudillo Alma M. Balboa María A. Balsinde J. Foamy monocytes are enriched in cis-7-Hexadecenoic fatty acid (16:1n-9), a possible biomarker for early detection of cardiovascular disease.), FA 16:1 n-7 is known as a lipokine that regulates SCD-1 expression (62Cao H. Gerhold K. Mayers J.R. Wiest M.M. Watkins S.M. Hotamisligil G.S. Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.) and FA 16:1 n-10 has antimicrobial properties (63Fischer C.L. Blanchette D.R. Brogden K.A. Dawson D.V. Drake D.R. Hill J.R. et al.The roles of cutaneous lipids in host defense.), it is paramount to observe and measure any changes to the sn-isomeric composition in order to better understand the enzymes responsible for their catalytic release in metabolic diseases such as cancer.FAs in their free form have the potential for signaling cellular processes and mechanisms (20Hughes-Fulford M. Tjandrawinata R.R. Li C.-F. Sayyah S. Arachidonic acid, an omega-6 fatty acid, induces cytoplasmic phospholipase Ainf2/inf in prostate carcinoma cells., 45Levi L. Wang Z. Doud M.K. Hazen S.L. Noy N. Saturated fatty acids regulate retinoic acid signalling and suppress tumorigenesis by targeting fatty acid-binding protein 5., 64