Quantitative Analysis of Cellular Lipids by Nano-Electrospray Ionization Mass Spectrometry

Özbalci, Cagakan; Sachsenheimer, Timo; Brügger, Britta

doi:10.1007/978-1-62703-487-6_1

Cited by 73 publications

(91 citation statements)

References 33 publications

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“…Fatty acid spectra in the A1AT solutions were determined by nano-electrospray ionization tandem mass spectrometry as previously described (25). The fatty acid anion fragments found at m/z 279 and 281 represent the anions of linoleic acid (LA) and oleic acid (OA), respectively.…”

Section: Resultsmentioning

confidence: 99%

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α1-Antitrypsin Combines with Plasma Fatty Acids and Induces Angiopoietin-like Protein 4 Expression

Frenzel

Wrenger

Brügger

et al. 2015

The Journal of Immunology

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α1-Antitrypsin (A1AT) purified from human plasma upregulates expression and release of angiopoietin-like protein 4 (Angptl4) in adherent human blood monocytes and in human lung microvascular endothelial cells, providing a mechanism for the broad immune-regulatory properties of A1AT independent of its anti-protease activity. Here we demonstrate that A1AT (Prolastin®), a potent inducer of Angptl4, contains significant quantities of the fatty acids (FA): linoleic (LA, C18:2) and oleic (OA, C18:1). However, only trace amounts of FAs were present in preparations that failed to increase Angplt4 expression, for example A1AT (Zemaira) or M-type A1AT purified by affinity chromatography. FA pull-down assays with western blot analysis revealed a FA-binding ability of A1AT. In human blood adherent monocytes A1AT‐FA conjugates up-regulated expression of Angptl4 (54.9-fold, p<0.001), fatty acid binding protein 4 (FABP4) (11.4-fold, p<0.001) and to a lesser degree, fatty acid translocase (CD36) (3.1-fold, p<0.001) relative to A1AT devoid of FA (A1AT‐0). These latter effects of A1AT-FA were blocked by inhibitors of PPARβ/δ (ST247) and PPARγ (GW9662). When compared to controls, cell pre-treatment with ST247 diminished the effect of A1AT-LA on Angptl4 mRNA (11.6- vs 4.1-fold, p<0.001) and FABP4 mRNA (5.4-vs 2.8-fold, p<0.001). Similarly, pre-incubation of cells with GW9662 inhibited inducing effect of A1AT-LA on Angptl4 mRNA (by 2-fold, p<0.001) and FABP4 mRNA (by 3-fold, p<0.001). Thus, A1AT binds to FA, and it is this form of A1AT that induces Angptl4 and FABP4 expression via a PPARs-dependent pathway. These findings provide a mechanism for the unexplored area of A1AT biology independent of its anti-protease properties.

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Section: Resultsmentioning

confidence: 99%

“…Lipid extraction was performed as described previously (25). Briefly, 80 μg protein was subjected to an acidic Bligh&Dyer extraction in the presence of 200 pmol C17:0 fatty acid.…”

Section: Methodsmentioning

confidence: 99%

α1-Antitrypsin Combines with Plasma Fatty Acids and Induces Angiopoietin-like Protein 4 Expression

Frenzel

Wrenger

Brügger

et al. 2015

The Journal of Immunology

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“…As lipid standards, 50 pmol of PE standards and 40–60 pmol of a PE P- standards were used. Standard syntheses were performed as described (Özbalci et al, 2013; Paltauf and Hermetter, 1994). After solvent evaporation under a gentle nitrogen flow at 37°C, lipids were re-suspended in 50 µl 10 mM ammonium acetate in methanol.…”

Section: Materials and Methodsmentioning

confidence: 99%

Peroxisomal dysfunctions cause lysosomal storage and axonal Kv1 channel redistribution in peripheral neuropathy

Kleinecke

Richert

Hoz

et al. 2017

eLife

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Impairment of peripheral nerve function is frequent in neurometabolic diseases, but mechanistically not well understood. Here, we report a novel disease mechanism and the finding that glial lipid metabolism is critical for axon function, independent of myelin itself. Surprisingly, nerves of Schwann cell-specific Pex5 mutant mice were unaltered regarding axon numbers, axonal calibers, and myelin sheath thickness by electron microscopy. In search for a molecular mechanism, we revealed enhanced abundance and internodal expression of axonal membrane proteins normally restricted to juxtaparanodal lipid-rafts. Gangliosides were altered and enriched within an expanded lysosomal compartment of paranodal loops. We revealed the same pathological features in a mouse model of human Adrenomyeloneuropathy, preceding disease-onset by one year. Thus, peroxisomal dysfunction causes secondary failure of local lysosomes, thereby impairing the turnover of gangliosides in myelin. This reveals a new aspect of axon-glia interactions, with Schwann cell lipid metabolism regulating the anchorage of juxtaparanodal Kv1-channels.DOI: http://dx.doi.org/10.7554/eLife.23332.001

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“…Mass spectra were evaluated using LipidView software (AB Sciex). PIP and PIP 2 amounts were normalized to total phospholipids, which was determined in neutral and acidic phases, as described previously (Özbalci et al, 2013). …”

Section: Methodsmentioning

confidence: 99%

PRL-3 disrupts epithelial architecture by altering the post-mitotic midbody position

Luján

Varsano

Rubio

et al. 2016

Journal of Cell Science

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Disruption of epithelial architecture is a fundamental event during epithelial tumorigenesis. We show that the expression of the cancer-promoting phosphatase PRL-3 (PTP4A3), which is overexpressed in several epithelial cancers, in polarized epithelial MDCK and Caco2 cells leads to invasion and the formation of multiple ectopic, fully polarized lumens in cysts. Both processes disrupt epithelial architecture and are hallmarks of cancer. The pathological relevance of these findings is supported by the knockdown of endogenous PRL-3 in MCF-7 breast cancer cells grown in three-dimensional branched structures, showing the rescue from multiple-lumen- to single-lumen-containing branch ends. Mechanistically, it has been previously shown that ectopic lumens can arise from midbodies that have been mislocalized through the loss of mitotic spindle orientation or through the loss of asymmetric abscission. Here, we show that PRL-3 triggers ectopic lumen formation through midbody mispositioning without altering the spindle orientation or asymmetric abscission, instead, PRL-3 accelerates cytokinesis, suggesting that this process is an alternative new mechanism for ectopic lumen formation in MDCK cysts. The disruption of epithelial architecture by PRL-3 revealed here is a newly recognized mechanism for PRL-3-promoted cancer progression.

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Quantitative Analysis of Cellular Lipids by Nano-Electrospray Ionization Mass Spectrometry

Cited by 73 publications

References 33 publications

α1-Antitrypsin Combines with Plasma Fatty Acids and Induces Angiopoietin-like Protein 4 Expression

α1-Antitrypsin Combines with Plasma Fatty Acids and Induces Angiopoietin-like Protein 4 Expression

Peroxisomal dysfunctions cause lysosomal storage and axonal Kv1 channel redistribution in peripheral neuropathy

PRL-3 disrupts epithelial architecture by altering the post-mitotic midbody position

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