Alan N. Hunt

Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes.

Chromatin-associated phospholipids are well recognized. A report that catalytically active endonuclear CTP:choline-phosphate cytidylyltransferase α is necessary for cell survival questions whether endonuclear, CDP-choline pathway phosphatidylcholine synthesis may occur in situ. We report that chromatin from human IMR-32 neuroblastoma cells possesses such a biosynthetic pathway. First, membrane-free nuclei retain all three CDP-choline pathway enzymes in proportions comparable with the content of chromatin-associated phosphatidylcholine. Second, following supplementation of cells with deuterated choline and using electrospray ionization mass spectrometry, both the time course and molecular species labeling pattern of newly synthesized endonuclear and whole cell phosphatidylcholine revealed the operation of spatially separate, compositionally distinct biosynthetic routes. Specifically, endogenous and newly synthesized endonuclear phosphatidylcholine species are both characterized by a high degree of diacyl/alkylacyl chain saturation. This unusual species content and synthetic pattern (evident within 10 min of supplementation) are maintained through cell growth arrest by serum depletion and when proliferation is restored, suggesting that endonuclear disaturated phosphatidylcholine enrichment is essential and closely regulated. We propose that endonuclear phosphatidylcholine synthesis may regulate periodic nuclear accumulations of phosphatidylcholine-derived lipid second messengers. Furthermore, our estimates of saturated phosphatidylcholine nuclear volume occupancy of around 10% may imply a significant additional role in regulating chromatin structure.

Dynamic lipidomics with stable isotope labelling

Incorporation of stable isotope labelled precursors enables estimation of the kinetics of lipid synthesis and turnover (dynamic lipidomics) in the clinical as well the experimental setting. Recent advances in tandem mass spectrometry extend the analytical possibilities from measurements of isotope enrichments to determinations of intact substrates. Incorporations of deuteriated choline, ethanolamine and inositol can be determined by precursor and neutral loss scans of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, respectively. This experimental approach provides information on the kinetics of individual phospholipid molecular species and has considerable potential to probe diseases of lipid metabolism in vivo.

Regulation of lung surfactant phospholipid synthesis and metabolism.

The alveolar type II epithelial (ATII) cell is highly specialised for the synthesis and storage, in intracellular lamellar bodies, of phospholipid destined for secretion as pulmonary surfactant into the alveolus. Regulation of the enzymology of surfactant phospholipid synthesis and metabolism has been extensively characterised at both molecular and functional levels, but understanding of surfactant phospholipid metabolism in vivo in either healthy or, especially, diseased lungs is still relatively poorly understood. This review will integrate recent advances in the enzymology of surfactant phospholipid metabolism with metabolic studies in vivo in both experimental animals and human subjects. It will highlight developments in the application of stable isotope-labelled precursor substrates and mass spectrometry to probe lung phospholipid metabolism in terms of individual molecular lipid species and identify areas where a more comprehensive metabolic model would have considerable potential for direct application to disease states.

Developmental variation in whole human lung phosphatidylcholine molecular species: a comparison with guinea pig and rat

Detailed analysis of the pattern of human and rodent lung phosphatidylcholine (PC) species during fetal development revealed a progressive increase in two disaturated species. The rise in the fractional content of dipalmitoyl PC (PC16:0/16:0) and myristoylpalmitoyl PC (PC14:0/16:0) was accompanied at each time point by a fall of similar magnitude in palmitoyloleoyl PC (PC16:0/18:1). Up to 20% of term lung PC was PC14:0/16:0. The temporal increase in rodent lung PC saturation began later in gestation than the human, and in the rat a significant increase in PC saturation only occurred postnatally. In this respect the guinea pig more closely resembled the human. For each mammal, a ratio of whole lung PC16:0/16:0 to PC16:0/18:1 (the P/O ratio) provided a sensitive marker of fetal lung maturity. The PC composition of whole adult lung and its saturation enrichment in bronchoalveolar lavage samples were similar in human, guinea pig and rat. We propose that the guinea pig provides a useful model for human lung prematurity studies.

Dynamic lipidomics of the nucleus

Once nuclear envelope membranes have been removed from isolated nuclei, around 6% of mammalian cell phospholipid is retained within the nuclear matrix, which calculations suggest may occupy 10% of the volume of this subcellular compartment. It is now acknowledged that endonuclear phospholipid, largely ignored for the past 40 years, provides substrate for lipid‐mediated signaling events. However, given its abundance, it likely also has other as yet incompletely defined roles. Endonuclear phosphatidylcholine is the predominant phospholipid comprising distinct and highly saturated molecular species compared with that of the whole cell. Moreover, this unusual composition is subject to tight homeostatic maintenance even under conditions of extreme dietary manipulation, presumably reflecting a functional requirement for highly saturated endonuclear phosphatidylcholine. Recent application of new lipidomic technologies exploiting tandem electrospray ionization mass spectrometry in conjunction with deuterium stable isotope labeling have permitted us to probe not just molecular species compositions but endonuclear phospholipid acquisition and turnover with unparalleled sensitivity and specificity. What emerges is a picture of a dynamic pool of endonuclear phospholipid subject to autonomous regulation with respect to bulk cellular phospholipid metabolism. Compartmental biosynthesis de novo of endonuclear phosphatidylcholine contrasts with import of phosphatidylinositol synthesized elsewhere. However, irrespective of the precise temporal–spatial–dynamic relationships underpinning phospholipid acquisition, derangement of endonuclear lipid‐mediated signaling from these parental phospholipids halts cell growth and division indicating a pivotal control point. This in turn defines the manipulation of compartmentalized endonuclear phospholipid acquisition and metabolism as intriguing potential targets for the development of future antiproliferative strategies. J. Cell. Biochem.

Conditional deletion of Abca3 in alveolar type II cells alters surfactant homeostasis in newborn and adult mice

ATP-binding cassette A3 (ABCA3) is a lipid transport protein required for synthesis and storage of pulmonary surfactant in type II cells in the alveoli. Abca3 was conditionally deleted in respiratory epithelial cells (Abca3(Δ/Δ)) in vivo. The majority of mice in which Abca3 was deleted in alveolar type II cells died shortly after birth from respiratory distress related to surfactant deficiency. Approximately 30% of the Abca3(Δ/Δ) mice survived after birth. Surviving Abca3(Δ/Δ) mice developed emphysema in the absence of significant pulmonary inflammation. Staining of lung tissue and mRNA isolated from alveolar type II cells demonstrated that ∼50% of alveolar type II cells lacked ABCA3. Phospholipid content and composition were altered in lung tissue, lamellar bodies, and bronchoalveolar lavage fluid from adult Abca3(Δ/Δ) mice. In adult Abca3(Δ/Δ) mice, cells lacking ABCA3 had decreased expression of mRNAs associated with lipid synthesis and transport. FOXA2 and CCAAT enhancer-binding protein-α, transcription factors known to regulate genes regulating lung lipid metabolism, were markedly decreased in cells lacking ABCA3. Deletion of Abca3 disrupted surfactant lipid synthesis in a cell-autonomous manner. Compensatory surfactant synthesis was initiated in ABCA3-sufficient type II cells, indicating that surfactant homeostasis is a highly regulated process that includes sensing and coregulation among alveolar type II cells.

A comparison of the molecular specificities of whole cell and endonuclear phosphatidylcholine synthesis.

Deuterated choline‐d9 labelling of IMR‐32 cells enabled comparison of the molecular specificities of whole cell and endonuclear phosphatidylcholine synthesis after 96 h polyunsaturated fatty acid supplementation. Surprisingly, while cell phosphatidylcholine synthesis and remodelling reflected a pattern of polyunsaturated fatty acid accretion, the saturated endonuclear phosphatidylcholine pool was only transiently labelled with polyunsaturates. Periodic endonuclear accumulations of the lipid second messenger diacylglycerol, mobilised from unsaturated phosphatidylinositol or saturated phosphatidylcholine, accompany cell proliferation. Non‐specific incorporation into endonuclear phosphatidylcholine and selective removal or remodelling of unsaturated molecular species may form part of a single ‘off switch’ recycling all endonuclear diacylglycerol accumulations.

Phosphatidylinositol Transfer Protein, Cytoplasmic 1 (PITPNC1) Binds and Transfers Phosphatidic Acid

Background: Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ) promotes metastatic colonization and angiogenesis in humans. Results: We demonstrate that RdgBβ is a phosphatidic acid (PA)- and phosphatidylinositol-binding protein and binds PA derived from the phospholipase D pathway. Conclusion: RdgBβ is the first lipid-binding protein identified that can bind and transfer PA. Significance: PA bound to RdgBβ is a likely effector downstream of phospholipase D. Phosphatidylinositol transfer proteins (PITPs) are versatile proteins required for signal transduction and membrane traffic. The best characterized mammalian PITPs are the Class I PITPs, PITPα (PITPNA) and PITPβ (PITPNB), which are single domain proteins with a hydrophobic cavity that binds a phosphatidylinositol (PI) or phosphatidylcholine molecule. In this study, we report the lipid binding properties of an uncharacterized soluble PITP, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ), of the Class II family. We show that the lipid binding properties of this protein are distinct to Class I PITPs because, besides PI, RdgBβ binds and transfers phosphatidic acid (PA) but hardly binds phosphatidylcholine. RdgBβ when purified from Escherichia coli is preloaded with PA and phosphatidylglycerol. When RdgBβ was incubated with permeabilized HL60 cells, phosphatidylglycerol was released, and PA and PI were now incorporated into RdgBβ. After an increase in PA levels following activation of endogenous phospholipase D or after addition of bacterial phospholipase D, binding of PA to RdgBβ was greater at the expense of PI binding. We propose that RdgBβ, when containing PA, regulates an effector protein or can facilitate lipid transfer between membrane compartments.

Lysophosphatidic acid attenuates the cytotoxic effects and degree of peroxisome proliferator-activated receptor γ activation induced by 15-deoxyΔ12,14-prostaglandin J2 in neuroblastoma cells

PPARgamma (peroxisome proliferator-activated receptor gamma) is a ligand-activated transcription factor that responds to 15dPGJ2 (15-deoxy-Delta12,14-prostglandin J2). 15dPGJ2, in vitro, halts neuroblastoma cell growth, but reported mechanisms vary. Here we evaluated the modulatory effects of endogenous serum lipid mitogens upon the extent of 15dPGJ2-induced growth inhibition and on the precise cellular responses of neuroblastoma cells to PPARgamma activation. We show that 15dPGJ2 specifically inhibited cell growth in both complete and delipidated media. 15dPGJ2-induced growth inhibition was accompanied by decreased cell viability, although the effect was far more marked in delipidated medium than in complete medium. Incubation with 15dPGJ2 in complete medium resulted in cytoplasmic changes characteristic of type II programmed cell death (autophagy), while prior serum lipid removal resulted in cell death via an apoptotic mechanism. These distinct, serum lipid-dependent cellular responses to 15dPGJ2 were accompanied by increases in the expression of a reporter gene construct containing a PPAR response element of 2.3-fold in complete medium, but of 4.8-fold in delipidated medium. Restoration of the serum lysolipid LPA (lysophosphatidic acid) to cells in delipidated medium reduced 15dPGJ2-mediated PPARgamma activation, growth inhibition and cell death; following addition of S1P (sphingosine 1-phosphate), decreases were apparent but more marginal. Further, while the effects of LPA in delipidated medium were mediated through a G(i)/phosphoinositide 3-kinase/MAPK (mitogen-activated protein kinase) pathway, those of S1P did not involve the MAPK component. These data suggest that the serum lysolipid LPA modulates the degree of PPARgamma activation and the precise cellular response to 15dPGJ2 via activation of a G(i)/phosphoinositide 3-kinase/MAPK pathway.

Activation of Sterol-response Element-binding Proteins (SREBP) in Alveolar Type II Cells Enhances Lipogenesis Causing Pulmonary Lipotoxicity

Background: Lung function is dependent upon the regulation of tissue and alveolar lipids. Results: Activation of SREBP in alveolar cells caused neutral lipid accumulation, inflammation, and tissue remodeling. Conclusion: The accumulation of neutral lipids in the lung caused inflammation consistent with findings in lipid storage disorders. Significance: Pulmonary lipotoxicity may contribute to lung dysfunction associated with diabetes, obesity, and metabolic disorders. Pulmonary inflammation is associated with altered lipid synthesis and clearance related to diabetes, obesity, and various inherited metabolic disorders. In many tissues, lipogenesis is regulated at the transcriptional level by the activity of sterol-response element-binding proteins (SREBP). The role of SREBP activation in the regulation of lipid metabolism in the lung was assessed in mice in which both Insig1 and Insig2 genes, encoding proteins that bind and inhibit SREBPs in the endoplasmic reticulum, were deleted in alveolar type 2 cells. Although deletion of either Insig1 or Insig2 did not alter SREBP activity or lipid homeostasis, deletion of both genes (Insig1/2Δ/Δ mice) activated SREBP1, causing marked accumulation of lipids that consisted primarily of cholesterol esters and triglycerides in type 2 epithelial cells and alveolar macrophages. Neutral lipids accumulated in type 2 cells in association with the increase in mRNAs regulating fatty acid, cholesterol synthesis, and inflammation. Although bronchoalveolar lavage fluid phosphatidylcholine was modestly decreased, lung phospholipid content and lung function were maintained. Insig1/2Δ/Δ mice developed lung inflammation and airspace abnormalities associated with the accumulation of lipids in alveolar type 2 cells, alveolar macrophages, and within alveolar spaces. Deletion of Insig1/2 activated SREBP-enhancing lipogenesis in respiratory epithelial cells resulting in lipotoxicity-related lung inflammation and tissue remodeling.