Mass spectrometry data: glycerol 3-phosphate and glycerolipids

MSMS identification of phospholipids and glycerol 3-phosphate using collision induced dissociation (CID) fragmentation.

Negative ion mass spectrometry (phospholipid) analysis of lipid extracts from HL60 and K562 cells after 24 hr BaP treatment (0.5 mM bezafibrate, BEZ, and 5 µM medroxyprogesterone acetate, MPA).

Mass spectrometry data: free fatty acids and phospholipids from cells that were drug treated in the presence of 13C D-glucose

Table S1: Peaks used for the calibration of mass spectral data sets related to the Materials and Methods section. Table S2: Peaks used for the calibration of mass spectral data sets related to the Materials and Methods section. Table S3: Peaks used for the calibration of mass spectral data sets related to the Materials and Methods section. Table S4: Peaks used for the calibration of mass spectral data sets related to the Materials and Methods section. Figure S1: Schematic showing the method for the MSMS-stitch technique related to the Materials and Methods section. Table S8: Mass spectrometry 13C-glucose tracer lipidomics phosphatidylcholine data related to the "BaP treatment decreases fatty acid and phospholipid synthesis and increases triacylglycerol levels" results section. Figure S3A: The absolute mass spectral intensities of 13C labelled phosphatidylcholine (PC) species in HL60 cells after BaP treatment in the presence of 1-13C D-glucose. This is related to the "BaP treatment decreases fatty acid and phospholipid synthesis and increases triacylglycerol levels" results section. Figure S3B: The absolute mass spectral intensities of 12C phosphatidylcholine (PC) species in HL60 cells after BaP treatment in the presence of 1-13C D-glucose. This is related to the "BaP treatment decreases fatty acid and phospholipid synthesis and increases triacylglycerol levels" results section. Figure S4: Heatmaps showing the fold changes of individual phosphatidylcholine (PC) species in HL60 cells after BaP treatment in the presence of 1-13C D-glucose. This is related to the "BaP treatment decreases fatty acid and phospholipid synthesis and increases triacylglycerol levels" results section. ( Figure S5A: The absolute mass spectral intensities of 13C labelled free fatty acids in HL60 cells after BaP treatment in the presence of 13C D-glucose. This is related to the "A commercial SCD1 inhibitor can partially recapitulate the effect of BaP on lipogenesis" section. Figure S5B: The absolute mass spectral intensities of 13C labelled saturated (SFA: C16:0 and C18:0) and monounsaturated (MUFA: C16:1 and C18:1) free fatty acids in HL60 cells after BaP treatment in the presence of 13C D-glucose. This is related to the "A commercial SCD1 inhibitor can partially recapitulate the effect of BaP on lipogenesis" section. Figure S6A: The absolute mass spectral intensities of 13C labelled phosphatidylcholine (PC) in HL60 cells after BaP treatment in the presence of 13C D-glucose. This is related to the "A commercial SCD1 inhibitor can partially recapitulate the effect of BaP on lipogenesis" section. Figure S6B: The absolute mass spectral intensities of 12C labelled phosphatidylcholine (PC) in HL60 cells after BaP treatment in the presence of 13C D-glucose. This is related to the "A commercial SCD1 inhibitor can partially recapitulate the effect of BaP on lipogenesis" section. Figure S7: Images of cells supplemented with oleic acid related to the "Oleate decreases the levels of BaP-induced reactive oxygen species (ROS)" results section. "

There is much discussion in the media and some of the scientific literature of how many of the conclusions from scientific research should be doubted. These critiques often focus on studies - typically in non-experimental spheres of biomedical and social sciences - that search large datasets for novel correlations, with a risk that inappropriate statistical evaluations might yield dubious conclusions. By contrast, results from experimental biological research can often be interpreted largely without statistical analysis. Typically: novel observation(s) are reported, and an explanatory hypothesis is offered; multiple labs undertake experiments to test the hypothesis; interpretation of the results may refute the hypothesis, support it or provoke its modification; the test/revise sequence is reiterated many times; and the field moves forward. I illustrate this experimental/non-experimental dichotomy by examining the contrasting recent histories of: (a) our remarkable and growing understanding of how several inositol-containing phospholipids contribute to the lives of eukaryote cells; and (b) the difficulty of achieving any agreed mechanistic understanding of why consuming dietary supplements of inositol is clinically beneficial in some metabolic diseases.

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum. PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.

This review attempts to explain why consuming extra myoinositol (Ins), an essential component of membrane phospholipids, is often beneficial for patients with conditions characterised by insulin resistance, non-alcoholic fatty liver disease and endoplasmic reticulum (ER) stress. For decades we assumed that most human diets provide an adequate Ins supply, but newer evidence suggests that increasing Ins intake ameliorates several disorders, including polycystic ovary syndrome, gestational diabetes, metabolic syndrome, poor sperm development and retinopathy of prematurity. Proposed explanations often suggest functional enhancement of minor facets of Ins Biology such as insulin signalling through putative inositol-containing 'mediators', but offer no explanation for this selectivity. It is more likely that eating extra Ins corrects a deficiency of an abundant Ins-containing cell constituent, probably phosphatidylinositol (PtdIns). Much of a cell's PtdIns is in ER membranes, and an increase in ER membrane synthesis, enhancing the ER's functional capacity, is often an important part of cell responses to ER stress. This review: (a) reinterprets historical information on Ins deficiency as describing a set of events involving a failure of cells adequately to adapt to ER stress; (b) proposes that in the conditions that respond to dietary Ins there is an overstretching of Ins reserves that limits the stressed ER's ability to make the 'extra' PtdIns needed for ER membrane expansion; and (c) suggests that eating Ins supplements increases the Ins supply to Ins-deficient and ER-stressed cells, allowing them to make more PtdIns and to expand the ER membrane system and sustain ER functions.

Abstract The redeployed drug combination of bezafibrate and medroxyprogesterone acetate (designated BaP) has potent in vivo anticancer activity in acute myelogenous leukemia (AML) and endemic Burkitt lymphoma (eBL) patients; however, its mechanism-of-action is unclear. Given that elevated fatty acid biosynthesis is a hallmark of many cancers and that these drugs can affect lipid metabolism, we hypothesized that BaP exerts anticancer effects by disrupting lipogenesis. We applied mass spectrometry–based lipidomics and gene and protein expression measurements of key lipogenic enzymes [acetyl CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and stearoyl CoA desaturase 1 (SCD1)] to AML and eBL cell lines treated with BaP. BaP treatment decreased fatty acid and phospholipid biosynthesis from 13C D-glucose. The proportion of phospholipid species with saturated and monounsaturated acyl chains was also decreased after treatment, whereas those with polyunsaturated chains increased. BaP decreased SCD1 protein levels in each cell line (0.46- to 0.62-fold; P < 0.023) and decreased FASN protein levels across all cell lines (0.87-fold decrease; P = 1.7 × 10−4). Changes to ACC1 protein levels were mostly insignificant. Supplementation with the SCD1 enzymatic product, oleate, rescued AML and e-BL cells from BaP cell killing and decreased levels of BaP-induced reactive oxygen species, whereas supplementation with the SCD1 substrate (and FASN product), palmitate, did not rescue cells. In conclusion, these data suggest that the critical anticancer actions of BaP are decreases in SCD1 levels and monounsaturated fatty acid synthesis. To our knowledge, this is the first time that clinically available antileukemic and antilymphoma drugs targeting SCD1 have been reported. Cancer Res; 75(12); 2530–40. ©2015 AACR.

The last couple of decades have seen an extraordinary transformation in our knowledge and understanding of the multifarious biological roles of inositol phospholipids. Herein, I briefly consider two topics. The first is the role that recently acquired biochemical and genomic information-especially from archaeons-has played in illuminating the possible evolutionary origins of the biological employment of inositol in lipids, and some questions that these studies raise about the classical' biosynthetic route to phosphatidylinositol. The second is the growing recognition of the importance in eukaryotic cells of phosphatidylinositol 3,5-bisphosphate. Phosphatidylinositol 3,5-bisphosphate only entered our phosphoinositide consciousness quite recently, but it is speedily gathering a plethora of roles in diverse cellular processes and diseases thereof. These include: control of endolysosomal vesicular trafficking and of the activity of ion channels and pumps in the endolysosomal compartment; control of constitutive and stimulated protein traffic to and from plasma membrane subdomains; control of the nutrient and stress-sensing target of rapamycin complex1 pathway (TORC1); and regulation of key genes in some central metabolic pathways.