Abstract

Little is known about the physiological role of alkylglycerol monooxygenase (AGMO), the only enzyme capable of cleaving the 1-O-alkyl ether bond of ether lipids. Expression and enzymatic activity of this enzyme can be detected in a variety of tissues including adipose tissue. This labile lipolytic membrane-bound protein uses tetrahydrobiopterin as a cofactor, and mice with reduced tetrahydrobiopterin levels have alterations in body fat distribution and blood lipid concentrations. In addition, manipulation of AGMO in macrophages led to significant changes in the cellular lipidome, and alkylglycerolipids, the preferred substrates of AGMO, were shown to accumulate in mature adipocytes. Here, we investigated the roles of AGMO in lipid metabolism by studying 3T3-L1 adipogenesis. AGMO activity was induced over 11 days using an adipocyte differentiation protocol. We show that RNA interference-mediated knockdown of AGMO did not interfere with adipocyte differentiation or affect lipid droplet formation. Furthermore, lipidomics revealed that plasmalogen phospholipids were preferentially accumulated upon Agmo knockdown, and a significant shift toward longer and more polyunsaturated acyl side chains of diacylglycerols and triacylglycerols could be detected by mass spectrometry. Our results indicate that alkylglycerol catabolism has an influence not only on ether-linked species but also on the degree of unsaturation in the massive amounts of triacylglycerols formed during in vitro 3T3-L1 adipocyte differentiation.

Supplementary key wordsAbbreviations: Adipoq (adiponectin), Agmo (alkylglycerol monooxygenase), Agps (alkylglycerone phosphate synthase), CE (cholesteryl ester), DEX (dexamethasone), DG (diacylglycerol), Elovl3 (elongation of very long chain fatty acids protein 3), Fabp4 (fatty acid-binding protein 4), Far1 (fatty acyl-CoA reductase 1), Fasn (fatty acidsynthase), Gnpat (glyceronephosphate O-acyltransferase), IBMX (3-isobutyl-1-methylxanthine), Lep (leptin), (L)PC[O]/[P] (alkyl-/alkenyl-(lyso)phosphatidylcholine), (L)PE[O]/[P] (alkyl-/alkenyl-(lyso)phosphatidylethanolamine), Lpl (lipoprotein lipase), Mgll (monoacylglycerol lipase), PC (phosphatidylcholine), PE (phosphatidylethanolamine), Pnpla2 (patatin-like phospholipase domaincontaining 2/adipose triglyceride lipase), Pparg (peroxisome proliferator-activated receptor gamma), RGZ (rosiglitazone), shRNA (short hairpin RNA), TG (triacylglycerol), TG[O/P] (alkyl-/alkenyl-diacylglycerol)Ether-linked lipid species such as plasmanyl-glycerophospholipids (alkyl-linked lipids) or plasmenyl-glycerophospholipids (alkenyl-linked lipids or plasmalogens) are known to be important integral membrane constituents in several organs including the brain (1Gorgas K. Teigler A. Komljenovic D. Just W.W. The ether lipid-deficient mouse: tracking down plasmalogen functions.). They carry an exceptionally high amount of polyunsaturated fatty acids at their sn-2 position (2Magnusson C.D. Haraldsson G.G. ), are essential for proper eye development, and play an important role in male fertility (1Gorgas K. Teigler A. Komljenovic D. Just W.W. The ether lipid-deficient mouse: tracking down plasmalogen functions.). Ether lipids interfere with kinase signaling pathways such as protein kinase C (3Warne T.R. Buchanan F.G. Robinson M. Growth-dependent accumulation of monoalkylglycerol in Madin-Darby canine kidney cells. Evidence for a role in the regulation of protein kinase C., 4Daniel L.W. Small G.W. Schmitt J.D. Marasco C.J. Ishaq K. Piantadosi C. Alkyl-linked diglycerides inhibit protein kinase C activation by diacylglycerols., 5McNeely T.B. Rosen G. Londner M.V. Turco S.J. Inhibitory effects on protein kinase C activity by lipophosphoglycan fragments and glycosylphosphatidylinositol antigens of the protozoan parasite Leishmania.) or protein kinase B (6Kozikowski A.P. Sun H. Brognard J. Dennis P.A. Novel PI analogues selectively block activation of the pro-survival serine/threonine kinase Akt.). Also platelet-activating factor, an important lipid biomediator, belongs to this lipid class (7Zimmerman G.A. McIntyre T.M. Prescott S.M. Stafforini D.M. The platelet-activating factor signaling system and its regulators in syndromes of inflammation and thrombosis., 8Rangholia N. Leisner T.M. Holly S.P. Bioactive ether lipids: primordial modulators of cellular signaling.). An important subcellular hub for ether lipid synthesis are peroxisomes, which also play a pivotal role in other fatty acid processes including very long-chain and branched-chain fatty acid degradation (9Lodhi I.J. Semenkovich C.F. Peroxisomes: a nexus for lipid metabolism and cellular signaling.).The only known enzyme able to degrade plasmanyl ether species is alkylglycerol monooxygenase (AGMO) (10Watschinger K. Werner E.R. Alkylglycerol monooxygenase.), a highly hydrophobic integral membrane protein (11Watschinger K. Fuchs J.E. Yarov-Yarovoy V. Keller M.A. Golderer G. Hermetter A. et al.Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase.). Because AGMO enzymatic activity is quickly lost during standard biochemical procedures, protein purification attempts have not succeeded so far (12[Studies on catabolism of ether lipids. -Solubilization of alkylglycerol monooxygenase from rat liver microsome (author’s transl)]., 13Solubilization and partial characterization of alkylglycerol monooxygenase from rat liver microsomes., 14Affinity purification of alkylglycerol monooxygenase from rat liver microsomes by chimyl alcohol-Sepharose 4B column chromatography.). In 2010, we were able to identify Tmem195 as the Agmo gene (15Watschinger K. Keller M.A. Golderer G. Hermann M. Maglione M. Sarg B. et al.Identification of the gene encoding alkylglycerol monooxygenase defines a third class of tetrahydrobiopterin-dependent enzymes.), which now enables us to examine its physiological role in detail. AGMO is differentially regulated in mouse macrophage polarization (16Tokuoka S.M. Kita Y. Shindou H. Shimizu T. Alkylglycerol monooxygenase as a potential modulator for PAF synthesis in macrophages., 17Watschinger K. Keller M.A. McNeill E. Alam M.T. Lai S. Sailer S. et al.Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome.), and activity manipulation in a murine macrophage cell line impacts on the composition of the cellular lipidome (17Watschinger K. Keller M.A. McNeill E. Alam M.T. Lai S. Sailer S. et al.Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome.). In the same study, modulations of AGMO activity in murine macrophages led to substantial accumulation of ether-linked phospholipids (plasmanyl and plasmenyl) and alkylglycerols. In earlier analyses, AGMO was also suggested to play a role in platelet-activating factor degradation (16Tokuoka S.M. Kita Y. Shindou H. Shimizu T. Alkylglycerol monooxygenase as a potential modulator for PAF synthesis in macrophages.). In the model organism Caenorhabditis elegans, mutants deficient for AGMO showed a more fragile cuticle and an altered sensitivity to bacterial infection (18Loer C.M. Calvo A.C. Watschinger K. Werner-Felmayer G. O'Rourke D. Stroud D. et al.Cuticle integrity and biogenic amine synthesis in Caenorhabditis elegans require the cofactor tetrahydrobiopterin (BH4).). Analysis of the cuticle lipid profile revealed alterations of ester lipids, glucosylceramides, a lower abundance of negatively charged lipid headgroups, and accumulation of higher molecular weight lipids with longer side chains (19Juarez J.F.B. Bada Juarez J.F. O'Rourke D. Judge P.J. Liu L.C. Hodgkin J. et al.Lipodisqs for eukaryote lipidomics with retention of viability: sensitivity and resistance to Leucobacter infection linked to C. elegans cuticle composition.). Furthermore, it was recently shown that ether-linked phosphatidylcholines and sphingolipids exert an inverse function in bidirectional endoplasmic reticulum trafficking of glycosylphosphatidylinositol anchors (20Jiménez-Rojo N. Leonetti M.D. Zoni V. Colom A. Feng S. Iyengar N.R. et al.Conserved functions of ether lipids and sphingolipids in the early secretory pathway.).Still, the exact physiological role of AGMO is not well understood. From genome-wide association studies in humans and from experimental evidence in model organisms, associations between the AGMO locus, and biologically relevant traits like energy homeostasis and infections were found (21Sailer S. Keller M.A. Werner E.R. Watschinger K. The emerging physiological role of AGMO 10 years after its gene identification.). Single nucleotide polymorphisms adjacent to or in the human AGMO gene were correlated with fasting glucose levels (22Dupuis J. Langenberg C. Prokopenko I. Saxena R. Soranzo N. Jackson A.U. et al.New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk.) and with recurrent leishmaniasis (23Marquet S. Bucheton B. Reymond C. Argiro L. El-Safi S.H. Kheir M.M. et al.Exome sequencing identifies two variants of the alkylglycerol monooxygenase gene as a cause of relapses in visceral leishmaniasis in children, in Sudan.), respectively. Manipulation of tetrahydrobiopterin levels, a crucial redox partner of AGMO, in mouse models showed that a complete cofactor deficiency leads to embryonic lethality (24Douglas G. Hale A.B. Crabtree M.J. Ryan B.J. Hansler A. Watschinger K. et al.A requirement for Gch1 and tetrahydrobiopterin in embryonic development.). If, however, modest tetrahydrobiopterin levels are maintained in mice during pregnancy, pups are born normally but have more body fat and altered fat distribution, as well as elevated blood glucose and cholesterol levels (25Korner G. Scherer T. Adamsen D. Rebuffat A. Crabtree M. Rassi A. et al.Mildly compromised tetrahydrobiopterin cofactor biosynthesis due to Pts variants leads to unusual body fat distribution and abdominal obesity in mice.). Recently, we succeeded in generation of the first Agmo knockout mouse model to study the physiological relevance of ether lipid degradation by AGMO in more detail (26Sailer S. Coassin S. Lackner K. Fischer C. McNeill E. Streiter G. et al.When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping.).Agmo is abundantly expressed and active in many tissues of rats and mice, including liver, gastrointestinal tract, and different fat tissues (15Watschinger K. Keller M.A. Golderer G. Hermann M. Maglione M. Sarg B. et al.Identification of the gene encoding alkylglycerol monooxygenase defines a third class of tetrahydrobiopterin-dependent enzymes., 27Werner E.R. Hermetter A. Prast H. Golderer G. Werner-Felmayer G. Widespread occurrence of glyceryl ether monooxygenase activity in rat tissues detected by a novel assay.). There are reports on ether lipids in adipocytes and adipogenesis claiming that incorporation of ether-linked lipid species, such as ethanolamine plasmalogens, helps adipocytes to maintain their membrane rigidity (28Pietiläinen K.H. Róg T. Seppänen-Laakso T. Virtue S. Gopalacharyulu P. Tang J. et al.Association of lipidome remodeling in the adipocyte membrane with acquired obesity in humans.). Another clue that ether lipids are relevant for adipose tissue came from lipidomic analyses of human plasma and adipose tissue samples, which revealed that levels of alkyl-linked and alkenyl-linked phospholipids are changed in obese compared with lean individuals (29Donovan E.L. Pettine S.M. Hickey M.S. Hamilton K.L. Miller B.F. Lipidomic analysis of human plasma reveals ether-linked lipids that are elevated in morbidly obese humans compared to lean., 30Kotronen A. Seppänen-Laakso T. Westerbacka J. Kiviluoto T. Arola J. Ruskeepää A.-L. et al.Comparison of lipid and fatty acid composition of the liver, subcutaneous and intra-abdominal adipose tissue, and serum., 31Pietiläinen K.H. Sysi-Aho M. Rissanen A. Seppänen-Laakso T. Yki-Järvinen H. Kaprio J. et al.Acquired obesity is associated with changes in the serum lipidomic profile independent of genetic effects–a monozygotic twin study.). As compared with ether-linked phospholipids, the general role of neutral ether lipids like 1-O-alkyl-2,3-diacylglycerols (DGs)—the ether analogues of triacylglycerols (TGs)—in physiology, however, is only marginally understood. These neutral ether lipids were shown to be upregulated in vitro in cell models of adipocyte differentiation (32Liaw L. Prudovsky I. Koza R.A. Anunciado-Koza R.V. Siviski M.E. Lindner V. et al.Lipid profiling of in vitro cell models of adipogenic differentiation: relationships with mouse adipose tissues.), to exert a proadipogenic stimulus in 3T3-L1 adipogenesis (33Homan E.A. Kim Y.-G. Cardia J.P. Saghatelian A. Monoalkylglycerol ether lipids promote adipogenesis.) and to be able to rescue peroxin 16 deficiency-mediated inhibition of adipocyte development (34Hofer D.C. Pessentheiner A.R. Pelzmann H.J. Schlager S. Madreiter-Sokolowski C.T. Kolb D. et al.Critical role of the peroxisomal protein PEX16 in white adipocyte development and lipid homeostasis., 35Structural and functional roles of ether lipids.). In adipocytes, peroxisomes, the crucial organelles for the initial steps of ether lipid biosynthesis, and lipid droplets get in close proximity (36Blanchette-Mackie E.J. Dwyer N.K. Barber T. Coxey R.A. Takeda T. Rondinone C.M. et al.Perilipin is located on the surface layer of intracellular lipid droplets in adipocytes.) and are essential for bidirectional lipid trafficking of ether-linked triradylglycerols (TG, DG[O], and DG[P]) to lipid droplets (37Bartz R. Li W.-H. Venables B. Zehmer J.K. Roth M.R. Welti R. et al.Lipidomics reveals that adiposomes store ether lipids and mediate phospholipid traffic.).

In light of these few scattered reports about the proadipogenic effect of alkylglycerols, as well as the putative connection of AGMO and obesity including its comorbidities, we studied Agmo expression and activity in 3T3-L1 adipocyte differentiation and knocked down its expression by RNA interference in 3T3-L1 preadipocytes, monitored consequences on differentiation, and found quite unexpected impacts of decreased AGMO activity on the global cellular lipidome.

Materials and methodsCell lines and cell culture

The 3T3-L1 preadipocyte cell line (American Type Culture Collection, Manassas, VA) was grown in sterile 75 cm2 polystyrene, cell+ growth surface flasks equipped with ventilated screw caps (Sarstedt, Nümbrecht, Germany) in basal medium (DMEM/GlutaMAX high glucose plus sodium pyruvate [Fisher Scientific, Vienna, Austria]) supplemented with 10% fetal bovine serum (Fisher Scientific) and 1% penicillin/streptomycin (Sigma, Vienna, Austria). Cells were split when reaching about 90% confluence with 1× trypsin-EDTA solution (Sigma) and were transferred to collagen-coated 6-well or 96-well plates, which were coated with collagen type I (rat tail; Fisher Scientific, 80 μg/ml working solution in 20 mM acetic acid) overnight at 4°C and afterward washed once with 1× PBS.

For adipocyte differentiation, 5-day postconfluent 3T3-L1 cells were exposed for 3 days to the differentiation medium 1 consisting of basal medium supplemented with 34.4 nM insulin (Sigma), 0.25 μM dexamethasone (DEX) (Sigma), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma), and 2 μM rosiglitazone (RGZ) (Cayman, Tallinn, Estonia). On day 4, the medium was changed to differentiation medium 2 (basal medium supplemented with 34.4 nM insulin only) for the rest of the differentiation protocol until day 11.

Lipid droplet staining of mature adipocytesTo quantify the amount of lipid droplets and cell nuclei, 3T3-L1 adipocytes were stained with Bodipy™ 493/503 (Fisher Scientific, InvitrogenTM, and Molecular ProbesTM) and Hoechst 33342 (Sigma). For this, cells were washed once with 1× PBS and fixed for 10 min in 4% paraformaldehyde (Merck, Darmstadt, Germany). After fixation, cells were washed twice with 1× PBS and then incubated for 15 min in the dark with a staining solution consisting of 2 μM Bodipy, 2 μg/ml Hoechst, and 1× PBS. Thereafter, cells were again washed twice and then stored in 1× PBS. Images were recorded on a Leica DM IL LED inverted fluorescence microscope (Leica, Wetzlar, Germany). All images were evaluated using the CellProfiler™ cell image analysis software (38Kamentsky L. Jones T.R. Fraser A. Bray M.A. Logan D.J. Madden K.L. et al.Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software.). Alternatively, lipid droplets of 3T3-L1 adipocytes were stained with the neutral lipid dye Oil Red O (Sigma). For this, fixed cells were washed twice with 1× PBS and once with 60% triethylphosphate (Sigma) solution in aqua destillata. A 0.5% Oil Red O solution was prepared in 60% triethylphosphate solution and added to the cells for 10 min. Afterward, the Oil Red O staining solution was aspirated and 1× PBS was added for 2 min and changed for fresh 1× PBS.RNA isolation and quantitative PCRTotal RNA from 3T3-L1 cells was prepared using the RNeasy Plus Mini Kit according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Transcription into complementary DNA was performed using the M-MLV reverse transcriptase (RNase H Minus, Point Mutant; Promega, Mannheim, Germany) and random hexamer primers (Microsynth, Balgach, Switzerland). For quantitative PCR (qPCR), the TaqMan assay technology using Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies, Vienna, Austria) and the Mx3005P qPCR system (Agilent) were used. TaqMan probes were labeled with fluorescein (FAM) (5′) and tetramethylrhodamine (TAMRA) (3′). Primer and TaqMan probe sequences are listed in supplemental Table S1.Western blot

Cell pellets were collected in 0.1 M Tris/0.25 M sucrose at day 0 and day 11 of adipocyte differentiation and assessed for protein content by Bradford assay using BSA as standard. The rest of the sample was mixed with 5× SDS sample buffer, and homogenates were sonicated and boiled for 5 min at 95°C. Twenty micrograms of samples were separated on a Novex™ WedgeWell™ 4–20% Tris-Glycine Gel (Fisher Scientific, Invitrogen), blotted onto PVDF membrane (Bio-Rad Laboratories, Inc, Hercules, CA), blocked with 5% skim milk (Sigma), and stained with either mouse anti-fatty acid binding protein 4 (FABP4) (1:1,000 dilution; Santa Cruz, Heidelberg, Germany) or mouse anti-PPARγ (1:500 dilution; Santa Cruz). For the loading control β-actin, mouse anti-actin (1:2,500 dilution; Millipore) was used. As secondary antibody, HRP-linked anti-mouse IgG (Promega) was applied. Blots were incubated with Westar Supernova ECL reagent (LabConsulting, Cyanagen, Bologna, Italy), and signals were recorded with the MicroChemi 4.2 chemiluminescent station (DNR, Neve Yamin, Israel). Western blot band pixel count was quantified with ImageQuant TL software (GE Healthcare Life Sciences, Vienna, Austria), and signals were normalized to the β-actin reference.

AGMO activity assayEnzyme activity was measured as described in a previous work (27Werner E.R. Hermetter A. Prast H. Golderer G. Werner-Felmayer G. Widespread occurrence of glyceryl ether monooxygenase activity in rat tissues detected by a novel assay.) with some modifications: Homogenates of 3T3-L1 cells were not centrifuged, and a protein concentration of >1 mg/ml was used to measure the enzymatic activity. These optimization steps were necessary to minimize quenching of accumulated lipids during adipocyte differentiation and to robustly detect the AGMO activity of in vitro differentiated adipocytes. Furthermore, fatty aldehyde dehydrogenase, essential for full conversion of the fatty aldehyde to the fatty acid, was added in its recombinant form to the assay mixture (39Keller M.A. Watschinger K. Golderer G. Maglione M. Sarg B. Lindner H.H. et al.Monitoring of fatty aldehyde dehydrogenase by formation of pyrenedecanoic acid from pyrenedecanal.). We carefully analyzed samples and controls of each replicate in parallel to exclude artifacts by day-to-day variability of the assay.Manipulation of AGMO activity by lentiviral constructsStable knockdown of Agmo gene expression using shRNAs was performed as already described in a previous work (17Watschinger K. Keller M.A. McNeill E. Alam M.T. Lai S. Sailer S. et al.Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome.). In brief, the pHR-SFFV-DEST-ires-Puro transfer plasmid containing the shRNA-encoding oligonucleotides of murine Agmo 1699–1717 for shAgmo1699 (GeneBank accession no.: NM_178767.5) was added to human embryonic kidney 293T cells together with the packaging plasmid (pSPAX2) and the pseudotyping vector pVSV-G for generation of infectious lentiviral particles. The infectious supernatant was harvested at 48 and 72 h after transfection, 0.45 μm filtered, and added to the target cells for 24 h. The shLuc control cell line expressing shRNA against luciferase (155–173 from pGL3 Luciferase; Promega) was generated in parallel. Afterward, cells were selected for puromycin resistance (3.5 μg/ml).Cellular lipidomics analysisFor lipidomics analysis, shLuc and shAgmo cells were harvested at day 0 and 11 of adipocyte differentiation. Cells were first washed once with PBS containing 0.5% fatty acid-free BSA (Sigma) and afterward washed with PBS only and trypsinized. Dry cell pellets were snap-frozen in liquid nitrogen and shipped to Amsterdam UMC (the Netherlands) for lipidomic analysis performed in the Core Facility Metabolomics and processing done with an in-house developed pipeline written in R (40Chambers M.C. Maclean B. Burke R. Amodei D. Ruderman D.L. Neumann S. et al.A cross-platform toolkit for mass spectrometry and proteomics., 41Patti G.J. Tautenhahn R. Siuzdak G. Meta-analysis of untargeted metabolomic data from multiple profiling experiments., 42Herzog K. Pras-Raves M.L. Vervaart M.A.T. Luyf A.C.M. van Kampen A.H.C. Wanders R.J.A. et al.Lipidomic analysis of fibroblasts from Zellweger spectrum disorder patients identifies disease-specific phospholipid ratios.). Internal standards for (phospho)lipid classes were added at known conc