Molecular species selectivity of lipid transport creates a mitochondrial sink for di-unsaturated phospholipids.

Mitochondria depend on the import of phospholipid precursors for the biosynthesis of phosphatidylethanolamine (PE) and cardiolipin, yet the mechanism of their transport remains elusive. A dynamic lipidomics approach revealed that mitochondria preferentially import di-unsaturated phosphatidylserine (PS) for subsequent conversion to PE by the mitochondrial PS decarboxylase Psd1p. Several protein complexes tethering mitochondria to the endomembrane system have been implicated in lipid transport in yeast, including the endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES), ER-membrane complex (EMC), and the vacuole and mitochondria patch (vCLAMP). By limiting the availability of unsaturated phospholipids, we created conditions to investigate the mechanism of lipid transfer and the contributions of the tethering complexes in vivo. Under these conditions, inactivation of ERMES components or of the vCLAMP component Vps39p exacerbated accumulation of saturated lipid acyl chains, indicating that ERMES and Vps39p contribute to the mitochondrial sink for unsaturated acyl chains by mediating transfer of di-unsaturated phospholipids. These results support the concept that intermembrane lipid flow is rate-limited by molecular species-dependent lipid efflux from the donor membrane and driven by the lipid species' concentration gradient between donor and acceptor membrane.

Automated, parallel mass spectrometry imaging and structural identification of lipids.

We report a method that enables automated data-dependent acquisition of lipid tandem mass spectrometry data in parallel with a high-resolution mass spectrometry imaging experiment. The method does not increase the total image acquisition time and is combined with automatic structural assignments. This lipidome-per-pixel approach automatically identified and validated 104 unique molecular lipids and their spatial locations from rat cerebellar tissue.

Quantification of Cholesterol and Cholesteryl Ester by Direct Flow Injection High-Resolution Fourier Transform Mass Spectrometry Utilizing Species-Specific Response Factors.

The quantification of free cholesterol (FC) and cholesteryl ester (CE) in mammalian samples is of great interest for basic science and clinical lipidomics. Here, we evaluated the feasibility of direct flow injection analysis (FIA) coupled to electrospray ionization high-resolution mass spectrometry (ESI-HRMS) to quantify FC and CE in lipid extracts from human serum, cultured cells, and mouse liver. Despite poor ionization efficiency of FC, the limit of quantitation was sufficient for precise and accurate quantification of FC by multiplexed HRMS (MSX) analysis without using a derivatization step. However, it was demonstrated that, upon full scan Fourier transform MS (FTMS) quantification, CE species show substantial differences in their analytical responses depending on number of double bonds, length of the acyl chain, infused lipid concentration, and other lipid components. A major determinant for these response differences is their susceptibility to in-source fragmentation. In particular, introduction of double bonds lowers the degree of in-source fragmentation. Therefore, CE species-specific response factors need to be applied for CE quantification by FTMS to achieve accurate concentrations. Method validation demonstrated that FIA-ESI-HRMS (MSX and FTMS) is applicable for quantification of FC and CE in samples used in basic science as well as clinical studies such as cultured cells, tissue homogenates, and serum.

The PNPLA-family phospholipases involved in glycerophospholipid homeostasis of HeLa cells.

Mammalian cells maintain the glycerophospholipid (GPL) compositions of their membranes nearly constant. To achieve this, GPL synthesis and degradation must be coordinated. There is strong evidence that A-type phospholipases (PLAs) are key players in homeostatic degradation of GPLs, but the identities of the PLAs involved have not been established. However, some members of the Patatin-like phospholipase domain-containing proteins (PNPLAs) have been implicated. Accordingly, we knocked down all the PNPLAs significantly expressed in human HeLa cells using RNA interference and then determined whether the turnover of the major glycerophospholipids is affected by using mass spectrometry and metabolic labeling with stable isotope-labeled precursors. Knockdown of PNPLA9, PNPLA6 or PNPLA4 significantly (30-50%) reduced the turnover of phosphatidylcholine, -ethanolamine and -serine. In a notable contrast, turnover of phosphatidylinositol was not significantly affected by the knockdown of any PNPLA. Depletion of PNPLA9 and PNPLA4 also inhibited G0/G1 to S cell cycle progression, which could thus be regulated by GPL turnover. These results strongly suggest that PNPLA9, -6 and -4 play a key role in GPL turnover and homeostasis in human cells. A hypothetical model suggesting how these enzymes could recognize the relative concentration of the different GPLs is proposed.

Reporting of lipidomics data should be standardized.

This article highlights, to our opinion, some of the most pertinent issues related to producing high quality lipidomics data. These issues include pitfalls related to sample collection and storage, lipid extraction, the use of shotgun and LC-MS-based lipidomics approaches, and the identification, annotation and quantification of lipid species. We hope that highlighting these issues will help stimulate efforts to implement reporting standards for dissemination of lipidomics data. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.

Decreased lipogenesis in white adipose tissue contributes to the resistance to high fat diet-induced obesity in phosphatidylethanolamine N-methyltransferase-deficient mice.

Mice lacking phosphatidylethanolamine N-methyltransferase (PEMT, Pemt(-/-) mice) are resistant to high-fat diet (HFD)-induced obesity (DIO) but develop non-alcoholic steatohepatitis. PEMT expression is strongly induced during differentiation of 3T3-L1 adipocytes. Hence, we hypothesized that white adipose tissue (WAT) might be a key player in the protection against DIO in Pemt(-/-) mice. We fed Pemt(-/-) and Pemt(+/+) mice the HFD for 2 weeks, after which we examined adipocyte differentiation, adipogenesis and lipolysis in WAT. Pemt(-/-) mice gained less body weight, had reduced WAT mass and had smaller adipocytes than Pemt(+/+) mice. The protein levels of adipose differentiation markers FABP4, PPARγ and C/EBPß were not altered by genotype, but acetyl-CoA carboxylase expression and activation was reduced in the Pemt(-/-) mice. The in vivo conversion of [¹4C]acetate to [¹4C]TG in WAT was also lower in Pemt(-/-) mice. The release of glycerol from WAT explants was comparable between Pemt(+/+) and Pemt(-/-) mice under basal condition and in the presence of isoproterenol, indicating unaffected lipolytic capacity. Furthermore, the amounts of leptin, cytokines and chemokines in WAT were not altered by genotype in mice fed the HFD for 2 weeks. However, after 10 weeks of HFD, WAT from Pemt(-/-) mice had dramatically lower leptin, inflammatory cytokines (IL-1 and TNF-α) and chemokines (MCP-1 and RANTES), and significantly higher anti-inflammatory cytokine IL-10 than Pemt(+/+) mice. Together, our data show that PEMT deficiency did not affect the capability for differentiation and lipolysis in WAT. Decreased lipogenesis in WAT may contribute to the resistance to DIO in Pemt(-/-) mice.

Sphingomyelin synthase-related protein SMSr is a suppressor of ceramide-induced mitochondrial apoptosis.

Cells synthesize ceramides in the endoplasmic reticulum (ER) as precursors for sphingolipids to form an impermeable plasma membrane. As ceramides are engaged in apoptotic pathways, cells would need to monitor their levels closely to avoid killing themselves during sphingolipid biosynthesis. How this is accomplished remains to be established. Here we identify SMSr (SAMD8), an ER-resident ceramide phosphoethanolamine (CPE) synthase, as a suppressor of ceramide-mediated cell death. Disruption of SMSr catalytic activity causes a rise in ER ceramides and their mislocalization to mitochondria, triggering a mitochondrial pathway of apoptosis. Blocking de novo ceramide synthesis, stimulating ceramide export from the ER or targeting a bacterial ceramidase to mitochondria rescues SMSr-deficient cells from apoptosis. We also show that SMSr-catalyzed CPE production, although essential, is not sufficient to suppress ceramide-induced cell death and that SMSr-mediated ceramide homeostasis requires the N-terminal sterile α-motif, or SAM domain, of the enzyme. These results define ER ceramides as bona fide transducers of mitochondrial apoptosis and indicate a primary role of SMSr in monitoring ER ceramide levels to prevent inappropriate cell death during sphingolipid biosynthesis.

Intact sphingomyelin biosynthetic pathway is essential for intracellular transport of influenza virus glycoproteins.

Cells genetically deficient in sphingomyelin synthase-1 (SGMS1) or blocked in their synthesis pharmacologically through exposure to a serine palmitoyltransferase inhibitor (myriocin) show strongly reduced surface display of influenza virus glycoproteins hemagglutinin (HA) and neuraminidase (NA). The transport of HA to the cell surface was assessed by accessibility of HA on intact cells to exogenously added trypsin and to HA-specific antibodies. Rates of de novo synthesis of viral proteins in wild-type and SGMS1-deficient cells were equivalent, and HA negotiated the intracellular trafficking pathway through the Golgi normally. We engineered a strain of influenza virus to allow site-specific labeling of HA and NA using sortase. Accessibility of both HA and NA to sortase was blocked in SGMS1-deficient cells and in cells exposed to myriocin, with a corresponding inhibition of the release of virus particles from infected cells. Generation of influenza virus particles thus critically relies on a functional sphingomyelin biosynthetic pathway, required to drive influenza viral glycoproteins into lipid domains of a composition compatible with virus budding and release.

Phosphatidylcholine metabolism and choline kinase in human osteoblasts.

There is a paucity of information about phosphatidylcholine (PC) biosynthesis in bone formation. Thus, we characterized PC metabolism in both primary human osteoblasts (HOB) and human osteosarcoma MG-63 cells. Our results show that the CDP-choline pathway is the only de novo route for PC biosynthesis in both HOB and MG-63 cells. Both CK activity and CKα expression in MG-63 cells were significantly higher than those in HOB cells. Silencing of CKα in MG-63 cells had no significant effect on PC concentration but decreased the amount of phosphocholine by approximately 80%. The silencing of CKα also reduced cell proliferation. Moreover, pharmacological inhibition of CK activity impaired the mineralization capacity of MG-63 cells. Our data suggest that CK and its product phosphocholine are required for the normal growth and mineralization of MG-63 cells.

Choline kinase beta is required for normal endochondral bone formation.

BACKGROUND: Choline kinase has three isoforms encoded by the genes Chka and Chkb. Inactivation of Chka in mice results in embryonic lethality, whereas Chkb(-/-) mice display neonatal forelimb bone deformations. METHODS: To understand the mechanisms underlying the bone deformations, we compared the biology and biochemistry of bone formation from embryonic to young adult wild-type (WT) and Chkb(-/-) mice. RESULTS: The deformations are specific to the radius and ulna during the late embryonic stage. The radius and ulna of Chkb(-/-) mice display expanded hypertrophic zones, unorganized proliferative columns in their growth plates, and delayed formation of primary ossification centers. The differentiation of chondrocytes of Chkb(-/-) mice was impaired, as was chondrocyte proliferation and expression of matrix metalloproteinases 9 and 13. In chondrocytes from Chkb(-/-) mice, phosphatidylcholine was slightly lower than in WT mice whereas the amount of phosphocholine was decreased by approximately 75%. In addition, the radius and ulna from Chkb(-/-) mice contained fewer osteoclasts along the cartilage/bone interface. CONCLUSIONS: Chkb has a critical role in the normal embryogenic formation of the radius and ulna in mice. GENERAL SIGNIFICANCE: Our data indicate that choline kinase beta plays an important role in endochondral bone formation by modulating growth plate physiology.

Import of phosphatidylserine to and export of phosphatidylethanolamine molecular species from mitochondria.

Heavy isotope-labeled ethanolamine and serine as well as exogenous PE and PS species were used to study trafficking of phosphatidylethanolamine (PE) and -serine (PS) molecular species between the endoplasmic reticulum (ER) and mitochondria in HeLa cells. Import of both endogenous and exogenous PS to IMM was a relatively slow process (T1/2=several hours), but depended on the acyl chains. In particular, the 38:4 and 38:5 species were imported more efficiently compared to the other PS species. Knock-down of Mitofusin 2 or Mitostatin had no detectable effect on PS import to mitochondria, suggesting that the ER-mitochondria contacts regulated by these proteins are not essential. Knock-down of PS synthase 1 inhibited PS decarboxylation, suggesting that import of PS to mitochondria is coupled to its synthesis. Also the export of PE from IMM to microsomes is a relatively slow process, but again depends markedly on the acyl chain structure. Most notably, the polyunsaturated 38:4 and 38:5 PE species were less efficiently exported, which together with rapid import of the PS precursors most probably explains their enrichment in IMM. PE synthesized via the CDP-ethanolamine was also imported to IMM, but most of the PE in this membrane derives from imported PS. In contrast to PS, all PC species made in Golgi/ER translocated similarly and rapidly to IMM. In conclusion, selective translocation of PS species and PS-derived PE species between ER and mitochondria plays a major role in phospholipid homeostasis of these organelles.

Sustained yield forestry in Sweden and Russia: how does it correspond to sustainable forest management policy?

This paper analyzes how sustained yield (SY) forestry is defined and implemented in Sweden and Russia, two countries with different forest-industrial regimes. We first compare definitions of SY forestry in national legislation and policies. Then we study forest management planning in two large forest management units with respect to: delivered forest products and values, how the harvest level of timber is defined, where the harvest takes place, and what treatments are used to sustain desired forest products and values. In Sweden SY forestry is maximum yield based on high-input forest management, and in Russia it is forestry based on natural regeneration with minimum investments in silviculture. We conclude that how SY forestry contributes to SFM depends on the context. Finally, we discuss the consequences of SY forestry as performed in Sweden and Russia related to its ability to support diverse forest functions, as envisioned in sustainable forest management policy.

Lipoprotein(a) induces caspase-1 activation and IL-1 signaling in human macrophages.

Introduction: Lipoprotein(a) (Lp(a)) is an LDL-like particle with an additional apolipoprotein (apo)(a) covalently attached. Elevated levels of circulating Lp(a) are a risk factor for atherosclerosis. A proinflammatory role for Lp(a) has been proposed, but its molecular details are incompletely defined. Methods and results: To explore the effect of Lp(a) on human macrophages we performed RNA sequencing on THP-1 macrophages treated with Lp(a) or recombinant apo(a), which showed that especially Lp(a) induces potent inflammatory responses. Thus, we stimulated THP-1 macrophages with serum containing various Lp(a) levels to investigate their correlations with cytokines highlighted by the RNAseq, showing significant correlations with caspase-1 activity and secretion of IL-1ß and IL-18. We further isolated both Lp(a) and LDL particles from three donors and then compared their atheroinflammatory potentials together with recombinant apo(a) in primary and THP-1 derived macrophages. Compared with LDL, Lp(a) induced a robust and dose-dependent caspase-1 activation and release of IL-1ß and IL-18 in both macrophage types. Recombinant apo(a) strongly induced caspase-1 activation and IL-1ß release in THP-1 macrophages but yielded weak responses in primary macrophages. Structural analysis of these particles revealed that the Lp(a) proteome was enriched in proteins associated with complement activation and coagulation, and its lipidome was relatively deficient in polyunsaturated fatty acids and had a high n-6/n-3 ratio promoting inflammation. Discussion: Our data show that Lp(a) particles induce the expression of inflammatory genes, and Lp(a) and to a lesser extent apo(a) induce caspase-1 activation and IL-1 signaling. Major differences in the molecular profiles between Lp(a) and LDL contribute to Lp(a) being more atheroinflammatory.

DDA-imaging with structural identification of lipid molecules on an Orbitrap Velos Pro mass spectrometer.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a useful technique for visualizing the spatial distribution of lipid molecules in tissues. Nevertheless, the use of MSI to investigate local lipid metabolic hallmarks has until recently been hampered by a lack of adequate technology that supports confident lipid identification. This limitation was recently mitigated by the development of DDA-imaging technology where high-resolution MSI is combined with parallel acquisition of lipid tandem MS2 spectra on a hybrid ion trap-Orbitrap Elite mass spectrometer featuring a resolving power of 240,000 and a scan time of 1 s. Here, we report the key tenets related to successful transfer of the DDA-imaging technology onto an Orbitrap Velos Pro instrument featuring a resolving power of 120,000 and a scan time of 2 s. Through meticulous performance assessments and method optimization, we tuned the DDA-imaging method to be able to confidently identify 73 molecular lipid species in mouse brain sections and demonstrate that the performance of the technology is comparable with DDA-imaging on the Orbitrap Elite. Altogether, our work shows that DDA-imaging on the Orbitrap Velos Pro instrument can serve as a robust workhorse for lipid imaging in routine applications.

Substrate efflux propensity plays a key role in the specificity of secretory A-type phospholipases.

To better understand the principles underlying the substrate specificity of A-type phospholipases (PLAs), a high throughput mass spectrometric assay was employed to study the effect of acyl chain length and unsaturation of phospholipids on their rate of hydrolysis by three different secretory PLAs in micelles and vesicle bilayers. With micelles, each enzyme responded differently to substrate acyl chain unsaturation and double bond position, probably reflecting differences in the accommodative properties of their substrate binding sites. Experiments with saturated acyl positional isomers indicated that the length of the sn2 chain was more critical than that of the sn1 chain, suggesting tighter association of the former with the enzyme. Only the first 9-10 carbons of the sn2 acyl chain seem to interact intimately with the active site. Strikingly, no discrimination between positional isomers was observed with vesicles, and the rate of hydrolysis decreased far more with increasing chain length than with micelles, suggesting that translocation of the phospholipid substrate to the active site is rate-limiting with bilayers. Supporting this conclusion, acyl chain structure affected hydrolysis and spontaneous intervesicle transfer, which correlates with lipid efflux propensity, analogously. We conclude that substrate efflux propensity plays a more important role in the specificity of secretory PLA(2)s than commonly thought and could also be a key attribute in phospholipid homeostasis in which (unknown) PLA(2)s are key players.

Lipid molecular timeline profiling reveals diurnal crosstalk between the liver and circulation.

Diurnal regulation of whole-body lipid metabolism plays a vital role in metabolic health. Although changes in lipid levels across the diurnal cycle have been investigated, the system-wide molecular responses to both short-acting fasting-feeding transitions and longer-timescale circadian rhythms have not been explored in parallel. Here, we perform time-series multi-omics analyses of liver and plasma revealing that the majority of molecular oscillations are entrained by adaptations to fasting, food intake, and the postprandial state. By developing algorithms for lipid structure enrichment analysis and lipid molecular crosstalk between tissues, we find that the hepatic phosphatidylethanolamine (PE) methylation pathway is diurnally regulated, giving rise to two pools of oscillating phosphatidylcholine (PC) molecules in the circulation, which are coupled to secretion of either very low-density lipoprotein (VLDL) or high-density lipoprotein (HDL) particles. Our work demonstrates that lipid molecular timeline profiling across tissues is key to disentangling complex metabolic processes and provides a critical resource for the study of whole-body lipid metabolism.

Adipose MDM2 regulates systemic insulin sensitivity.

The intimate association between obesity and type II diabetes urges for a deeper understanding of adipocyte function. We and others have previously delineated a role for the tumor suppressor p53 in adipocyte biology. Here, we show that mice haploinsufficient for MDM2, a key regulator of p53, in their adipose stores suffer from overt obesity, glucose intolerance, and hepatic steatosis. These mice had decreased levels of circulating palmitoleic acid [non-esterified fatty acid (NEFA) 16:1] concomitant with impaired visceral adipose tissue expression of Scd1 and Ffar4. A similar decrease in Scd and Ffar4 expression was found in in vitro differentiated adipocytes with perturbed MDM2 expression. Lowered MDM2 levels led to nuclear exclusion of the transcriptional cofactors, MORC2 and LIPIN1, and thereby possibly hampered adipocyte function by antagonizing LIPIN1-mediated PPARγ coactivation. Collectively, these data argue for a hitherto unknown interplay between MDM2 and MORC2/LIPIN1 involved in balancing adipocyte function.

Introduction of phospholipids to cultured cells with cyclodextrin.

Previous studies indicate that methyl-ß-cyclodextrin (meß-CD) can greatly enhance translocation of long-chain phospholipids from vesicles to cells in culture, which is very useful when studying, e.g., phospholipid metabolism and trafficking. However, the parameters affecting the transfer have not been systematically studied. Therefore, we studied the relevant parameters including meß-CD and vesicle concentration, incubation time, phospholipid structure, and cell type. Because meß-CD can extract cholesterol and other lipids from cells, thereby potentially altering cell growth or viability, these issues were studied as well. The results show that efficient incorporation of phospholipid species with hydrophobicity similar to that of natural species can be obtained without significantly compromising cell growth or viability. Cellular content of phosphatidyl-serine, -ethanolamine, and -choline could be increased dramatically, i.e., 400, 125, and 25%, respectively. Depletion of cellular cholesterol could be prevented or alleviated by inclusion of the proper amount of cholesterol in the donor vesicles. In summary, meß-CD mediates efficient transfer of long-chain (phospho) lipids from vesicles to cells without significantly compromising their growth or viability. This lays a basis for detailed studies of phospholipid metabolism and trafficking as well as enables extensive manipulation of cellular phospholipid composition, which is particularly useful when investigating mechanisms underlying phospholipid homeostasis.

Acyl chain-dependent effect of lysophosphatidylcholine on endothelial prostacyclin production.

Previously we identified palmitoyl-lysophosphatidylcholine (16:0 LPC), linoleoyl-LPC (18:2 LPC), arachidonoyl-LPC (20:4 LPC), and oleoyl-LPC (18:1 LPC) as the most prominent LPC species generated by the action of endothelial lipase (EL) on high-density lipoprotein. In the present study, the impact of those LPC on prostacyclin (PGI(2)) production was examined in vitro in primary human aortic endothelial cells (HAEC) and in vivo in mice. Although 18:2 LPC was inactive, 16:0, 18:1, and 20:4 LPC induced PGI(2) production in HAEC by 1.4-, 3-, and 8.3-fold, respectively. LPC-elicited 6-keto PGF1α formation depended on both cyclooxygenase (COX)-1 and COX-2 and on the activity of cytosolic phospholipase type IVA (cPLA2). The LPC-induced, cPLA2-dependent (14)C-arachidonic acid (AA) release was increased 4.5-fold with 16:0, 2-fold with 18:1, and 2.7-fold with 20:4 LPC, respectively, and related to the ability of LPC to increase cytosolic Ca(2+) concentration. In vivo, LPC increased 6-keto PGF(1α) concentration in mouse plasma with a similar order of potency as found in HAEC. Our results indicate that the tested LPC species are capable of eliciting production of PGI(2), whereby the efficacy and the relative contribution of underlying mechanisms are strongly related to acyl-chain length and degree of saturation.

The superlattice model of lateral organization of membranes and its implications on membrane lipid homeostasis.

Most biological membranes are extremely complex structures consisting of hundreds of different lipid and protein molecules. According to the famous fluid-mosaic model lipids and many proteins are free to diffuse very rapidly in the plane of the membrane. While such fast diffusion implies that different membrane lipids would be laterally randomly distributed, accumulating evidence indicates that in model and natural membranes the lipid components tend to adopt regular (superlattice-like) distributions. The superlattice model, put forward based on such evidence, is intriguing because it predicts that 1) there is a limited number of allowed compositions representing local minima in membrane free energy and 2) those energy minima could provide set-points for enzymes regulating membrane lipid compositions. Furthermore, the existence of a discrete number of allowed compositions could help to maintain organelle identity in the face of rapid inter-organelle membrane traffic.