Publications by authors named "Jos F Brouwers"

67 Publications

Lipidome of cricket species used as food.

Food Chem 2021 Jul 19;349:129077. Epub 2021 Jan 19.

Animal Nutrition Group, Wageningen University & Research Centre, 6700 EV Wageningen, The Netherlands; Laboratory of Entomology, Wageningen University & Research Centre, 6700 EV Wageningen, The Netherlands.

The variation in lipidome of house cricket, banded cricket, Jamaican field cricket and two-spotted cricket was studied using high-throughput screening techniques for fingerprinting (MALDI TOF MS, GC-MS and LC MS-MS) and well-stablished chromatographic techniques for quantification (HPLC-ELSD, GC- FID). Although the four cricket species were reared in identical conditions, two-spotted & banded crickets had a lipid content 1.5 fold higher than house cricket. The lipids were high in UFA (>63%) and unsaturated TAG (>98%) making them liquid at room temperature, thus an oil. Cholesterol and several phytosterols were profiled finding high cholesterol concentration which is a point of concern. Eight phospholipid types (211 species) were identified with no major differences among cricket species. Using high-throughput screening techniques we demonstrate the complexity of cricket lipidome. Information on the lipidome of these crickets with high commercial value is important to estimate its nutritional value and their potential food applications.
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http://dx.doi.org/10.1016/j.foodchem.2021.129077DOI Listing
July 2021

Phosphatidylinositol synthesis, its selective salvage, and inter-regulation of anionic phospholipids in Toxoplasma gondii.

Commun Biol 2020 Dec 10;3(1):750. Epub 2020 Dec 10.

Department of Molecular Parasitology, Faculty of Life Sciences, Humboldt University, Berlin, Germany.

Phosphatidylinositol (PtdIns) serves as an integral component of eukaryotic membranes; however, its biosynthesis in apicomplexan parasites remains poorly understood. Here we show that Toxoplasma gondii-a common intracellular pathogen of humans and animals-can import and co-utilize myo-inositol with the endogenous CDP-diacylglycerol to synthesize PtdIns. Equally, the parasite harbors a functional PtdIns synthase (PIS) containing a catalytically-vital CDP-diacylglycerol phosphotransferase motif in the Golgi apparatus. Auxin-induced depletion of PIS abrogated the lytic cycle of T. gondii in human cells due to defects in cell division, gliding motility, invasion, and egress. Isotope labeling of the PIS mutant in conjunction with lipidomics demonstrated de novo synthesis of specific PtdIns species, while revealing the salvage of other lipid species from the host cell. Not least, the mutant showed decline in phosphatidylthreonine, and elevation of selected phosphatidylserine and phosphatidylglycerol species, indicating a rerouting of CDP-diacylglycerol and homeostatic inter-regulation of anionic phospholipids upon knockdown of PIS. In conclusion, strategic allocation of own and host-derived PtdIns species to gratify its metabolic demand features as a notable adaptive trait of T. gondii. Conceivably, the dependence of T. gondii on de novo lipid synthesis and scavenging can be exploited to develop new anti-infectives.
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http://dx.doi.org/10.1038/s42003-020-01480-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728818PMC
December 2020

Dataset of the phospholipidome and transcriptome of under different growth conditions.

Data Brief 2020 Dec 28;33:106349. Epub 2020 Sep 28.

Department of Biomolecular Health Sciences, Utrecht University, 3584 CL Utrecht, the Netherlands.

The membrane phospholipid composition is not a stable bacterial characteristic but can change in response to altered environmental conditions. Here we provide the dataset of the phospholipidome and transcriptome of the microaerophilic human pathogen under different environmental conditions. These data have been used in (2020), The unique phospholipidome of the enteric pathogen : Lysolipids are required for motility at low oxygen availability. Here the abundance of each phospholipid is shown during the growth of for 0-108 h under low and high oxygen conditions (0.3 vs 10% O). The phospholipid data were obtained by applying high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS). The transcriptomic data obtained by RNA-seq show the differential expressed genes between logarithmic and stationary grown bacteria. In addition, our data might serve as a reference information for further in-depth investigation to understand the relation between specific phospholipids and the activity of membrane associated proteins.
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http://dx.doi.org/10.1016/j.dib.2020.106349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7548934PMC
December 2020

Explorative Combined Lipid and Transcriptomic Profiling of Substantia Nigra and Putamen in Parkinson's Disease.

Cells 2020 08 26;9(9). Epub 2020 Aug 26.

Department Molecular Animal Physiology, Donders Institute for Brain, Cognition and Behaviour, Donders Centre for Neuroscience, Faculty of Science, Geert Grooteplein Zuid 26-28, 6525 GA Nijmegen, The Netherlands.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons from the substantia nigra (SN) that project to the dorsal striatum (caudate-putamen). To better understand the molecular mechanisms underlying PD, we performed combined lipid profiling and RNA sequencing of SN and putamen samples from PD patients and age-matched controls. SN lipid analysis pointed to a neuroinflammatory component and included elevated levels of the endosomal lipid Bis (Monoacylglycero)Phosphate 42:8, while two of the three depleted putamen lipids were saturated sphingomyelin species. Remarkably, we observed gender-related differences in the SN and putamen lipid profiles. Transcriptome analysis revealed that the top-enriched pathways among the 354 differentially expressed genes (DEGs) in the SN were "protein folding" and "neurotransmitter transport", and among the 261 DEGs from putamen "synapse organization". Furthermore, we identified pathways, e.g., "glutamate signaling", and genes, encoding, e.g., an angiotensin receptor subtype or a proprotein convertase, that have not been previously linked to PD. The identification of 33 genes that were common among the SN and putamen DEGs, which included the α-synuclein paralog β-synuclein, may contribute to the understanding of general PD mechanisms. Thus, our proof-of-concept data highlights new genes, pathways and lipids that have not been explored before in the context of PD.
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http://dx.doi.org/10.3390/cells9091966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564986PMC
August 2020

Sensitization of drug resistant sarcoma tumors by membrane modulation via short chain sphingolipid-containing nanoparticles.

Nanoscale 2020 Aug 11;12(32):16967-16979. Epub 2020 Aug 11.

Laboratory Experimental Oncology, Department of Surgery, Erasmus Medical Center, Rotterdam, The Netherlands.

Nanoparticles such as liposomes are able to overcome cancer treatment challenges such as multidrug resistance by increasing the bioavailability of the encapsulated drug, bypassing drug pumps or through targeting resistant cells. Here, we merge enhanced drug delivery by nanotechnology with tumor cell membrane modulation combined in a single formulation. This is achieved through the incorporation of Short chain sphingolipids (SCSs) in the liposomal composition, which permeabilizes cell membranes to amphiphilic drugs such as Doxorubicin (Dxr). To study the mechanism and capability of SCS-containing nanodevices to overcome Dxr resistance, a sensitive uterine sarcoma cell line, MES-SA, and a resistant derived cell line, MES-SA/MX2, were used. The mechanism of resistance was explored by lipidomics and flow cytometry, revealing significant differences in lipid composition and in P glycoprotein (Pgp) expression. In vitro assays show that SCS liposomes were able to reverse cell resistance, and importantly, display a higher net effect on resistant than sensitive cells. SCS lipids modulated the cell membrane of MES-SA/MX2 drug resistant cells, while Pgp expression was not affected. Furthermore, SCS-modified liposomes were evaluated in a sarcoma xenograft model on drug accumulation, pharmacokinetics and efficacy. SCS liposomes improved Dxr levels in tumor nuclei of MES-SA/MX2 tumor cells, which was accompanied by a delay in tumor growth of the resistant model. Here we show that Dxr accumulation in tumor cells by SCS-modified liposomes was especially improved in Dxr resistant cells, rendering Dxr as effective as in sensitive cells. Moreover, this phenomenon translated to improved efficacy when Dxr liposomes where modified with SCSs in the drug resistant tumor model, while no benefit was seen in the sensitive tumors.
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http://dx.doi.org/10.1039/d0nr02257hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497538PMC
August 2020

The Unique Phospholipidome of the Enteric Pathogen Campylobacter jejuni: Lysophosholipids Are Required for Motility at Low Oxygen Availability.

J Mol Biol 2020 09 22;432(19):5244-5258. Epub 2020 Jul 22.

Department Biomolecular Health Sciences, Utrecht University, Utrecht, the Netherlands. Electronic address:

In response to changes in their environment bacteria need to change both their protein and phospholipid repertoire to match environmental requirements, but the dynamics of bacterial phospholipid composition under different growth conditions is still largely unknown. In the present study, we investigated the phospholipidome of the bacterial pathogen Campylobacter jejuni. Transcription profiling on logarithmic and stationary phase grown cells of the microaerophilic human pathogen C. jejuni using RNA-seq revealed differential expression of putative phospholipid biosynthesis genes. By applying high-performance liquid chromatography tandem-mass spectrometry, we identified 203 phospholipid species representing the first determination of the phospholipidome of this pathogen. We identified nine different phospholipid classes carrying between one and three acyl chains. Phospholipidome analysis on bacteria of different ages (0-5 days) showed rapid changes in the ratio of phospholipids containing ethanolamine, or glycerol as phospholipid head group and in the number of cyclopropane bond containing fatty acids. Oxygen concentration influenced the percentage of lysophospholipids, and cyclo-propane bonds containing acyl chains. We show that large amounts of the phospholipids are lysophospholipids (30-45%), which mutant studies reveal are needed for normal C. jejuni motility at low oxygen conditions. C. jejuni possesses an unusual phospholipidome that is highly dynamic in response to environmental changes.
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http://dx.doi.org/10.1016/j.jmb.2020.07.012DOI Listing
September 2020

Schistosoma mansoni infection affects the proteome and lipidome of circulating extracellular vesicles in the host.

Mol Biochem Parasitol 2020 07 27;238:111296. Epub 2020 Jun 27.

Department of Medical Microbiology and Infectious Diseases, Erasmus MC University Medical Center Rotterdam, Rotterdam, the Netherlands. Electronic address:

Eggs, schistosomula and adult Schistosoma worms are known to release extracellular vesicles (EV) during in vitro incubations and these EVs are postulated to affect the host responses. So far only those EVs released during in vitro incubations of schistosomes have been studied and it is unknown whether in blood of infected hosts the schistosomal EVs can be detected amidst all the circulating EVs of the host itself. In this study we analyzed the protein as well as the phospholipid composition of EVs circulating in blood plasma of S. mansoni infected hamsters and compared those with the EVs circulating in blood of non-infected hamsters. Although neither proteins nor lipids specific for schistosomes could be detected in the circulating EVs of the infected hamsters, the infection with schistosomes had a marked effect on the circulating EVs of the host, as the protein as well as the lipid composition of EVs circulating in infected hamsters were different from the EVs of uninfected hamsters. The observed changes in the EV lipid and protein content suggest that more EVs are released by the diseased liver, the affected erythrocytes and activated immune cells.
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http://dx.doi.org/10.1016/j.molbiopara.2020.111296DOI Listing
July 2020

Lipid Analysis of the 6-Hydroxydopamine-Treated SH-SY5Y Cell Model for Parkinson's Disease.

Mol Neurobiol 2020 Feb 6;57(2):848-859. Epub 2019 Sep 6.

Department of Molecular Animal Physiology, Faculty of Science, Donders Institute for Brain, Cognition and Behaviour, Donders Centre for Neuroscience, Geert Grooteplein Zuid 26-28, 6525 GA, Nijmegen, The Netherlands.

Parkinson's disease (PD) is a highly prevalent neurodegenerative disease for which no disease-modifying treatments are available, mainly because knowledge about its pathogenic mechanism is still incomplete. Recently, a key role for lipids emerged, but lipid profiling of brain samples from human subjects is demanding. Here, we used an unbiased approach, lipidomics, to determine PD-linked changes in the lipid profile of a well-established cell model for PD, the catecholaminergic neuronal cell line SH-SY5Y treated with the neurotoxin 6-hydroxydopamine (6-OHDA). We observed changes in multiple lipid classes, including phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), and total cholesterol, in 6-OHDA-treated SH-SY5Y cells. Furthermore, we found differences in the length and degree of unsaturation of the fatty acyl chains, indicating changes in their metabolism. Except for the observed decreased PS levels, the alterations in PC, PG, PI, and cholesterol levels are in agreement with the results of previous studies on PD-patient material. Opposite to what has been previously described, the cholesterol-lowering drug statins did not have a protective effect, while low doses of cholesterol supplementation partially protected SH-SY5Y cells from 6-OHDA toxicity. However, cholesterol supplementation triggered neuronal differentiation, which could have confounded the results of cholesterol modulation. Taken together, our results show that 6-OHDA-treated SH-SY5Y cells display many lipid changes also found in PD patient and animal model brains, although the SH-SY5Y cell model seems less suitable to study the involvement of cholesterol in PD initiation and progression.
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http://dx.doi.org/10.1007/s12035-019-01733-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031185PMC
February 2020

Liver phosphorus content and liver function in states of phosphorus deficiency in transition dairy cows.

PLoS One 2019 22;14(7):e0219546. Epub 2019 Jul 22.

Clinic for Cattle, University of Veterinary Medicine Hannover, Foundation, Hanover, Germany.

Phosphorus (P) deficiency in early lactating dairy cows is receiving increased attention because of incentives aiming at curtailing environmental pollution with P by reducing dietary P in ruminant diets. An in-vitro study using bovine hepatocytes incubated for 7 days with phosphate (Pi) concentrations of 0.9, 1.8 or 2.7 mmol/L, and an in-vivo study feeding late pregnant dairy cows diets with either adequate (0.28% and 0.44% in DM ante-partum and post-partum respectively) or low P content (0.15% and 0.20% in DM ante-partum and post-partum respectively) from 4 weeks before to 4 weeks after calving were conducted to explore effects of P deprivation on liver function. In vitro the relative abundance of mRNA of key enzymes of the carbohydrate metabolism in incubated hepatocytes and liver metabolites in culture medium were determined. In vivo health and productivity of experimental cows on low and adequate dietary P supply were monitored, and liver tissue and blood samples were obtained repeatedly. Liver tissue was assayed for its triacylglycerol-, mineral and water content as well as for the relative abundance of mRNA of enzymes of the carbohydrate-, fat- and protein metabolism. Reduced Pi-availability was not associated with altered enzyme transcription rates or metabolic activity in-vitro. The most prominent clinical finding associated with P deprivation in-vivo was feed intake depression developing after the first week of lactation. Accordingly cows on low P diets had lower milk yield and showed more pronounced increases in liver triacylglycerol after calving. Although the liver P content decreased in P deficient cows, neither negative effects on enzyme transcription rates nor on blood parameters indicative of impaired liver metabolic activity or liver injury were identified. These results indicate the P deprivation only indirectly affects the liver through exacerbation of the negative energy balance occurring as P deficient cows become anorectic.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0219546PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6645509PMC
February 2020

Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead.

Int J Parasitol 2019 07 3;49(8):647-656. Epub 2019 Jun 3.

Dept. Medical Microbiology & Infectious Diseases, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. Electronic address:

Adult schistosomes, parasitic flatworms that cause the tropical disease schistosomiasis, have always been considered to be homolactic fermenters and, in their energy metabolism, strictly dependent on carbohydrates. However, more recent studies suggested that fatty acid β-oxidation is essential for egg production by adult female Schistosoma mansoni. To address this conundrum, we performed a comprehensive study on the lipid metabolism of S. mansoni. Incubations with [C]-labelled fatty acids demonstrated that adults, eggs and miracidia of S. mansoni did not oxidise fatty acids, as no CO production could be detected. We then re-examined the S. mansoni genome using the genes known to be involved in fatty acid oxidation in six eukaryotic model reference species. This showed that the earlier automatically annotated genes for fatty acid oxidation were in fact incorrectly annotated. In a further analysis we could not detect any genes encoding β-oxidation enzymes, which demonstrates that S. mansoni cannot use this pathway in any of its lifecycle stages. The same was true for Schistosoma japonicum and all other schistosome species that have been sequenced. Absence of β-oxidation, however, does not imply that fatty acids from the host are not metabolised by schistosomes. Adult schistosomes can use and modify fatty acids from their host for biosynthetic purposes and incorporate those in phospholipids and neutral lipids. Female worms deposit large amounts of these lipids in the eggs they produce, which explains why interference with the lipid metabolism in females will disturb egg formation, even though fatty acid β-oxidation does not occur in schistosomes. Our analyses of S. mansoni further revealed that during the development and maturation of the miracidium inside the egg, changes in lipid composition occur which indicate that fatty acids deposited in the egg by the female worm are used for phospholipid biosynthesis required for membrane formation in the developing miracidium.
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http://dx.doi.org/10.1016/j.ijpara.2019.03.005DOI Listing
July 2019

LION/web: a web-based ontology enrichment tool for lipidomic data analysis.

Gigascience 2019 06;8(6)

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM, Utrecht, The Netherlands.

Background: A major challenge for lipidomic analyses is the handling of the large amounts of data and the translation of results to interpret the involvement of lipids in biological systems.

Results: We built a new lipid ontology (LION) that associates >50,000 lipid species to biophysical, chemical, and cell biological features. By making use of enrichment algorithms, we used LION to develop a web-based interface (LION/web, www.lipidontology.com) that allows identification of lipid-associated terms in lipidomes. LION/web was validated by analyzing a lipidomic dataset derived from well-characterized sub-cellular fractions of RAW 264.7 macrophages. Comparison of isolated plasma membranes with the microsomal fraction showed a significant enrichment of relevant LION-terms including "plasma membrane", "headgroup with negative charge", "glycerophosphoserines", "above average bilayer thickness", and "below average lateral diffusion". A second validation was performed by analyzing the membrane fluidity of Chinese hamster ovary cells incubated with arachidonic acid. An increase in membrane fluidity was observed both experimentally by using pyrene decanoic acid and by using LION/web, showing significant enrichment of terms associated with high membrane fluidity ("above average", "very high", and "high lateral diffusion" and "below average transition temperature").

Conclusions: The results demonstrate the functionality of LION/web, which is freely accessible in a platform-independent way.
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http://dx.doi.org/10.1093/gigascience/giz061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541037PMC
June 2019

A Comprehensive Functional Characterization of Escherichia coli Lipid Genes.

Cell Rep 2019 04;27(5):1597-1606.e2

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584CM Utrecht, the Netherlands. Electronic address:

Lipid membranes are the border between living cells and their environments. The membrane's lipid composition defines fluidity, thickness, and protein activity and is controlled by the intricate actions of lipid gene-encoded enzymes. However, a comprehensive analysis of each protein's contribution to the lipidome is lacking. Here, we present such a comprehensive and functional overview of lipid genes in Escherichia coli by individual overexpression or deletion of these genes. We developed a high-throughput lipidomic platform, combining growth analysis, one-step lipid extraction, rapid LC-MS, and bioinformatic analysis into one streamlined procedure. This allowed the processing of more than 300 samples per day and revealed interesting functions of known enzymes and distinct effects of individual proteins on the phospholipidome. Our data demonstrate the plasticity of the phospholipidome and unexpected relations between lipid classes and cell growth. Modeling of lipidomic responses to short-chain alcohols provides a rationale for targeted membrane engineering.
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http://dx.doi.org/10.1016/j.celrep.2019.04.018DOI Listing
April 2019

Comparison of the systemic phospholipid profile in dogs diagnosed with idiopathic inflammatory bowel disease or food-responsive diarrhea before and after treatment.

PLoS One 2019 16;14(4):e0215435. Epub 2019 Apr 16.

Division of Small Animal Internal Medicine, Department for Companion Animals and Horses, Vetmeduni Vienna, Vienna, Austria.

Background: Inflammatory bowel disease (IBD) and food-responsive diarrhea (FRD) are common chronic enteropathies in dogs, of which the exact pathogenesis has not been fully understood. In people dyslipidemia has been reported in patients with IBD, and potential therapeutic benefits of polyunsaturated fatty acids (PUFA) in the treatment of IBD have been investigated. Studies on the phospholipid profile in dogs with IBD and FRD are still lacking.

Aim: To investigate the systemic phospholipid profile of dogs with IBD or FRD and to evaluate possible differences in phospholipids before and after treatment.

Methods: The phospholipids in whole blood and EDTA plasma of 32 dogs diagnosed with either IBD (n = 16) or FRD (n = 16) were analyzed by hydrophilic interaction liquid chromatography (HILIC) prior to and after initiation of treatment, which included an elimination diet enriched with PUFAs.

Results: A clear separation of the phospholipids between whole blood and plasma was demonstrated on principal component analysis plots. In addition to the type of specimen, treatment and disease severity were the most significant factors determining the variance of the phospholipid profile. An increase in lysolipids was observed after treatment. The phosphatidylcholine (PC) species changed from PC 38:4 before treatment to mainly lysophosphatidylcholine 18:0 after treatment. Furthermore, several differences in the abundance of individual phospholipids were identified between dogs with IBD and dogs with FRD and between treatment statuses using random forest analysis.

Conclusion: Significant variances were identified in the phospholipid profiles of dogs with IBD and FRD. These were particularly determined by type of specimen used, disease severity and treatment status. After treatment, a shift of phospholipid species towards lysophosphatidylcholine 18:0 was observed. Future studies should further investigate the role of lipids in the pathophysiology of IBD and FRD as well as their potential therapeutic benefits.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0215435PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467395PMC
January 2020

Cholesterol Metabolism Is a Druggable Axis that Independently Regulates Tau and Amyloid-β in iPSC-Derived Alzheimer's Disease Neurons.

Cell Stem Cell 2019 03 24;24(3):363-375.e9. Epub 2019 Jan 24.

Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Sanford Consortium for Regenerative Medicine, La Jolla, CA 92037, USA. Electronic address:

Genetic, epidemiologic, and biochemical evidence suggests that predisposition to Alzheimer's disease (AD) may arise from altered cholesterol metabolism, although the molecular pathways that may link cholesterol to AD phenotypes are only partially understood. Here, we perform a phenotypic screen for pTau accumulation in AD-patient iPSC-derived neurons and identify cholesteryl esters (CE), the storage product of excess cholesterol, as upstream regulators of Tau early during AD development. Using isogenic induced pluripotent stem cell (iPSC) lines carrying mutations in the cholesterol-binding domain of APP or APP null alleles, we found that while CE also regulate Aβ secretion, the effects of CE on Tau and Aβ are mediated by independent pathways. Efficacy and toxicity screening in iPSC-derived astrocytes and neurons showed that allosteric activation of CYP46A1 lowers CE specifically in neurons and is well tolerated by astrocytes. These data reveal that CE independently regulate Tau and Aβ and identify a druggable CYP46A1-CE-Tau axis in AD.
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http://dx.doi.org/10.1016/j.stem.2018.12.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414424PMC
March 2019

High-Throughput Screening of Lipidomic Adaptations in Cultured Cells.

Biomolecules 2019 01 24;9(2). Epub 2019 Jan 24.

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, 3584CM Utrecht, The Netherlands.

High-throughput screening of biologically active substances in cell cultures remains challenging despite great progress in contemporary lipidomic techniques. These experiments generate large amounts of data that are translated into lipid fingerprints. The subsequent visualization of lipidomic changes is key to meaningful interpretation of experimental results. As a demonstration of a rapid and versatile pipeline for lipidomic analysis, we cultured HeLa cells in 96-well format for four days in the presence or absence of various inhibitors of lipid metabolic pathways. Visualization of the data by principle component analysis revealed a high reproducibility of the method, as well as drug specific changes to the lipidome. Construction of heatmaps and networks revealed the similarities and differences between the effects of different drugs at the lipid species level. Clusters of related lipid species that might represent distinct membrane domains emerged after correlation analysis of the complete dataset. Taken together, we present a lipidomic platform for high-throughput lipidomic analysis of cultured cell lines.
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http://dx.doi.org/10.3390/biom9020042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407004PMC
January 2019

Chronic dietary changes in n-6/n-3 polyunsaturated fatty acid ratios cause developmental delay and reduce social interest in mice.

Eur Neuropsychopharmacol 2019 01 15;29(1):16-31. Epub 2018 Dec 15.

Department of Translational Neuroscience, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands; Groningen Institute for Evolutionary Life Sciences, University of Groningen, The Netherlands. Electronic address:

Polyunsaturated fatty acids (PUFAs) are one of the main cellular building blocks, and dietary changes in PUFA composition are proposed as a potential route to influence brain development. For example, initial studies indicated that there is a relation between blood omega-6(n-6)/omega-3(n-3) PUFA ratios and neurodevelopmental disease diagnosis. To study the consequences of dietary n-6/n-3 PUFA ratio changes, we investigated the impact of a n-3 supplemented and n-3 deficient diet in developing BTBR T + Itpr3tf/J (BTBR) - a mouse inbred strain displaying Autism Spectrum Disorder (ASD)-like symptomatology - and control C57BL/6J mice. This study showed that pre- and postnatal changed dietary n-6/n-3 ratio intake has a major impact on blood and brain PUFA composition, and led to delayed physical development and puberty onset in both strains. The PUFA induced developmental delay did not impact adult cognitive performance, but resulted in reduced social interest, a main ASD behavioral feature. Thus, both chronic dietary n-3 PUFA supplementation and depletion may not be beneficial.
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http://dx.doi.org/10.1016/j.euroneuro.2018.11.1106DOI Listing
January 2019

The glycerophosphocholine acyltransferase Gpc1 is part of a phosphatidylcholine (PC)-remodeling pathway that alters PC species in yeast.

J Biol Chem 2019 01 4;294(4):1189-1201. Epub 2018 Dec 4.

From the Departments of Biological Sciences and

Phospholipase B-mediated hydrolysis of phosphatidylcholine (PC) results in the formation of free fatty acids and glycerophosphocholine (GPC) in the yeast GPC can be reacylated by the glycerophosphocholine acyltransferase Gpc1, which produces lysophosphatidylcholine (LPC), and LPC can be converted to PC by the lysophospholipid acyltransferase Ale1. Here, we further characterized the regulation and function of this distinct PC deacylation/reacylation pathway in yeast. Through and experiments, we show that Gpc1 and Ale1 are the major cellular GPC and LPC acyltransferases, respectively. Importantly, we report that Gpc1 activity affects the PC species profile. Loss of Gpc1 decreased the levels of monounsaturated PC species and increased those of diunsaturated PC species, whereas Gpc1 overexpression had the opposite effects. Of note, Gpc1 loss did not significantly affect phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine profiles. Our results indicate that Gpc1 is involved in postsynthetic PC remodeling that produces more saturated PC species. qRT-PCR analyses revealed that mRNA abundance is regulated coordinately with PC biosynthetic pathways. Inositol availability, which regulates several phospholipid biosynthetic genes, down-regulated expression at the mRNA and protein levels and, as expected, decreased levels of monounsaturated PC species. Finally, loss of decreased stationary phase viability in inositol-free medium. These results indicate that Gpc1 is part of a postsynthetic PC deacylation/reacylation remodeling pathway (PC-DRP) that alters the PC species profile, is regulated in coordination with other major lipid biosynthetic pathways, and affects yeast growth.
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http://dx.doi.org/10.1074/jbc.RA118.005232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349126PMC
January 2019

Lipids Are the Preferred Substrate of the Protist Naegleria gruberi, Relative of a Human Brain Pathogen.

Cell Rep 2018 10;25(3):537-543.e3

Department of Medical Microbiology and Infectious Diseases, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands; Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands. Electronic address:

Naegleria gruberi is a free-living non-pathogenic amoeboflagellate and relative of Naegleria fowleri, a deadly pathogen causing primary amoebic meningoencephalitis (PAM). A genomic analysis of N. gruberi exists, but physiological evidence for its core energy metabolism or in vivo growth substrates is lacking. Here, we show that N. gruberi trophozoites need oxygen for normal functioning and growth and that they shun both glucose and amino acids as growth substrates. Trophozoite growth depends mainly upon lipid oxidation via a mitochondrial branched respiratory chain, both ends of which require oxygen as final electron acceptor. Growing N. gruberi trophozoites thus have a strictly aerobic energy metabolism with a marked substrate preference for the oxidation of fatty acids. Analyses of N. fowleri genome data and comparison with those of N. gruberi indicate that N. fowleri has the same type of metabolism. Specialization to oxygen-dependent lipid breakdown represents an additional metabolic strategy in protists.
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http://dx.doi.org/10.1016/j.celrep.2018.09.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205838PMC
October 2018

Structural and functional characterization of protein-lipid interactions of the Salmonella typhimurium melibiose transporter MelB.

BMC Biol 2018 08 3;16(1):85. Epub 2018 Aug 3.

Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX, 79430, USA.

Background: Membrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelB) is a symporter coupling melibiose translocation with a cation (Na, Li, or H). We present an extensive study on the effects of specific phospholipids on the structure of MelB and the melibiose transport catalyzed by this protein.

Results: Lipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelB at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelB; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H- and Na-coupled melibiose active transport to different extents. Interestingly, neither the co-substrate binding (melibiose or Na) nor MelB folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelB with only 2-3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy.

Conclusions: (1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelB. (2) The headgroups of PE and PG, but not of CL, play important roles in melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelB
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http://dx.doi.org/10.1186/s12915-018-0553-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091025PMC
August 2018

Cardiolipin synthases of Escherichia coli have phospholipid class specific phospholipase D activity dependent on endogenous and foreign phospholipids.

Biochim Biophys Acta Mol Cell Biol Lipids 2018 10 19;1863(10):1345-1353. Epub 2018 Jun 19.

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584CM Utrecht, the Netherlands. Electronic address:

E. coli has three Cls-isoenzymes for cardiolipin (CL) synthesis but the differences between these three enzymes remain unresolved. All three Cls enzymes contain the phospholipase D (PLD) characteristic HKD motive and synthesize CL using PLD activity. Here, using LC-MS we show the effect of overexpressing or deletion of the three individual Cls enzymes on the lipidome, which included changes in lipid class distribution and CL species profiles. We demonstrate, for the first time, that overexpression of only ClsB resulted in the appreciable synthesis of a variety of phosphatidylalcohols, thereby establishing a 'classic' PLD activity for this enzyme: phospholipid headgroup exchange. Endogenous E. coli lipids and primary alcohols were substrates for this trans-phosphatidylation reaction. Furthermore, we show that endogenous levels of ClsA mediated a similar trans-phosphatidylation reaction to form phosphatidylalcohols, however this reaction was dependent on the presence of the foreign phospholipid class phosphatidylcholine (PC). This allows us to clarify the different specificities of the cardiolipin synthases.
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http://dx.doi.org/10.1016/j.bbalip.2018.06.017DOI Listing
October 2018

Potential Health and Environmental Risks of Three-Dimensional Engineered Polymers.

Environ Sci Technol Lett 2018 Feb 5;5(2):80-85. Epub 2018 Jan 5.

Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht 3584CM, The Netherlands.

Polymer engineering, such as in three-dimensional (3D) printing, is rapidly gaining popularity, not only in the scientific and medical fields but also in the community in general. However, little is known about the toxicity of engineered materials. Therefore, we assessed the toxicity of 3D-printed and molded parts from five different polymers commonly used for prototyping, fabrication of organ-on-a-chip platforms, and medical devices. Toxic effects of PIC100, E-Shell200, E-Shell300, polydimethylsiloxane, and polystyrene (PS) on early bovine embryo development, on the transactivation of estrogen receptors were assessed, and possible polymer-leached components were identified by mass spectrometry. Embryo development beyond the two-cell stage was inhibited by PIC100, E-Shell200, and E-Shell300 and correlated to the released amount of diethyl phthalate and polyethylene glycol. Furthermore, all polymers (except PS) induced estrogen receptor transactivation. The released materials from PIC100 inhibited embryo cleavage across a confluent monolayer culture of oviduct epithelial cells and also inhibited oocyte maturation. These findings highlight the need for cautious use of engineered polymers for household 3D printing and bioengineering of culture and medical devices and the need for the safe disposal of used devices and associated waste.
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http://dx.doi.org/10.1021/acs.estlett.7b00495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997463PMC
February 2018

Lipid analysis of sporozoites reveals exclusive phospholipids, a phylogenetic mosaic of endogenous synthesis, and a host-independent lifestyle.

Cell Discov 2018 22;4:24. Epub 2018 May 22.

1Department of Molecular Parasitology, Faculty of Life Sciences, Humboldt University, Berlin, 10115 Germany.

Successful inter-host transmission of most apicomplexan parasites requires the formation of infective sporozoites within the oocysts. Unlike all other infective stages that are strictly intracellular and depend on host resources, the sporozoite stage develops outside the host cells, but little is known about its self-governing metabolism. This study deployed , a parasite infecting the mouse as its natural host, to investigate the process of phospholipid biogenesis in sporozoites. Lipidomic analyses demonstrated the occurrence of prototypical phospholipids along with abundant expression of at least two exclusive lipids, phosphatidylthreonine (PtdThr) and inositol phosphorylceramide with a phytosphingosine backbone, in sporozoites. To produce them de novo, the parasite harbors nearly the entire biogenesis network, which is an evolutionary mosaic of eukaryotic-type and prokaryotic-type enzymes. Notably, many have no phylogenetic counterpart or functional equivalent in the mammalian host. Using as a gene-tractable surrogate to examine enzymes, we show a highly compartmentalized network of lipid synthesis spread primarily in the apicoplast, endoplasmic reticulum, mitochondrion, and Golgi complex. Likewise, trans-genera complementation of a mutant with the PtdThr synthase from reveals a convergent role of PtdThr in fostering the lytic cycle of coccidian parasites. Taken together, our work establishes a model of autonomous membrane biogenesis involving significant inter-organelle cooperation and lipid trafficking in sporozoites. Phylogenetic divergence of certain pathways offers attractive drug targets to block the sporulation and subsequent transmission. Not least, our results vindicate the possession of an entire de novo lipid synthesis network in a representative protist adapted to an obligate intracellular parasitic lifestyle.
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http://dx.doi.org/10.1038/s41421-018-0023-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964319PMC
May 2018

Aberrant hepatic lipid storage and metabolism in canine portosystemic shunts.

PLoS One 2017 19;12(10):e0186491. Epub 2017 Oct 19.

Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

Non-alcoholic fatty liver disease (NAFLD) is a poorly understood multifactorial pandemic disorder. One of the hallmarks of NAFLD, hepatic steatosis, is a common feature in canine congenital portosystemic shunts. The aim of this study was to gain detailed insight into the pathogenesis of steatosis in this large animal model. Hepatic lipid accumulation, gene-expression analysis and HPLC-MS of neutral lipids and phospholipids in extrahepatic (EHPSS) and intrahepatic portosystemic shunts (IHPSS) was compared to healthy control dogs. Liver organoids of diseased dogs and healthy control dogs were incubated with palmitic- and oleic-acid, and lipid accumulation was quantified using LD540. In histological slides of shunt livers, a 12-fold increase of lipid content was detected compared to the control dogs (EHPSS P<0.01; IHPSS P = 0.042). Involvement of lipid-related genes to steatosis in portosystemic shunting was corroborated using gene-expression profiling. Lipid analysis demonstrated different triglyceride composition and a shift towards short chain and omega-3 fatty acids in shunt versus healthy dogs, with no difference in lipid species composition between shunt types. All organoids showed a similar increase in triacylglycerols after free fatty acids enrichment. This study demonstrates that steatosis is probably secondary to canine portosystemic shunts. Unravelling the pathogenesis of this hepatic steatosis might contribute to a better understanding of steatosis in NAFLD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186491PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648188PMC
November 2017

Lysosome-mediated degradation of a distinct pool of lipid droplets during hepatic stellate cell activation.

J Biol Chem 2017 07 14;292(30):12436-12448. Epub 2017 Jun 14.

From the Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, 3584 CM, Utrecht, The Netherlands,

Activation of hepatic stellate cells (HSCs) is a critical step in the development of liver fibrosis. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerols (TAGs), cholesteryl esters, and retinyl esters (REs). We previously provided evidence for the presence of two distinct LD pools, a preexisting and a dynamic LD pool. Here we investigate the mechanisms of neutral lipid metabolism in the preexisting LD pool. To investigate the involvement of lysosomal degradation of neutral lipids, we studied the effect of lalistat, a specific lysosomal acid lipase (LAL/Lipa) inhibitor on LD degradation in HSCs during activation The LAL inhibitor increased the levels of TAG, cholesteryl ester, and RE in both rat and mouse HSCs. Lalistat was less potent in inhibiting the degradation of newly synthesized TAG species as compared with a more general lipase inhibitor orlistat. Lalistat also induced the presence of RE-containing LDs in an acidic compartment. However, targeted deletion of the Lipa gene in mice decreased the liver levels of RE, most likely as the result of a gradual disappearance of HSCs in livers of Lipa mice. Lalistat partially inhibited the induction of activation marker α-smooth muscle actin (α-SMA) in rat and mouse HSCs. Our data suggest that LAL/Lipa is involved in the degradation of a specific preexisting pool of LDs and that inhibition of this pathway attenuates HSC activation.
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http://dx.doi.org/10.1074/jbc.M117.778472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535019PMC
July 2017

Oligodendroglial myelination requires astrocyte-derived lipids.

PLoS Biol 2017 05 26;15(5):e1002605. Epub 2017 May 26.

Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University Amsterdam, Amsterdam, the Netherlands.

In the vertebrate nervous system, myelination of axons for rapid impulse propagation requires the synthesis of large amounts of lipids and proteins by oligodendrocytes and Schwann cells. Myelin membranes are thought to be cell-autonomously assembled by these axon-associated glial cells. Here, we report the surprising finding that in normal brain development, a substantial fraction of the lipids incorporated into central nervous system (CNS) myelin are contributed by astrocytes. The oligodendrocyte-specific inactivation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), an essential coactivator of the transcription factor SREBP and thus of lipid biosynthesis, resulted in significantly retarded CNS myelination; however, myelin appeared normal at 3 months of age. Importantly, embryonic deletion of the same gene in astrocytes, or in astrocytes and oligodendrocytes, caused a persistent hypomyelination, as did deletion from astrocytes during postnatal development. Moreover, when astroglial lipid synthesis was inhibited, oligodendrocytes began incorporating circulating lipids into myelin membranes. Indeed, a lipid-enriched diet was sufficient to rescue hypomyelination in these conditional mouse mutants. We conclude that lipid synthesis by oligodendrocytes is heavily supplemented by astrocytes in vivo and that horizontal lipid flux is a major feature of normal brain development and myelination.
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http://dx.doi.org/10.1371/journal.pbio.1002605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446120PMC
May 2017

Stearoyl-CoA desaturase activity in bovine cumulus cells protects the oocyte against saturated fatty acid stress.

Biol Reprod 2017 May;96(5):982-992

Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

Metabolic rich and poor conditions are both characterized by elevated free fatty acid levels and have been associated with impaired female fertility. In particular, saturated free fatty acids have a dose-dependent negative impact on oocyte developmental competence, while monounsaturated free fatty acids appear less harmful. Cumulus cells seem to protect the oocyte against free fatty acids, and the aim of this study was to determine the mechanism behind this protection In particular, the role of the enzyme stearoyl-CoA desaturase (SCD) that converts saturated into monounsaturated fatty acids was investigated. SCD gene and protein were abundantly expressed in cumulus cells, but expression was low in oocytes. The level of SCD protein expression in cumulus cells did not change when COCs were exposed to saturated stearic acid during maturation. SCD inhibition in the presence of stearic acid significantly reduced the developmental competence of oocytes and increased the incidence of apoptosis in cumulus cells. The esterified oleic/stearic acid ratio of the neutral lipid fraction in cumulus cells decreased in the presence of SCD inhibitors when COCs were exposed to saturated free fatty acids during maturation, indicating the SCD-specific conversion of saturated fatty acids under noninhibiting conditions. The observation that cumulus cells can desaturate the potentially toxic stearic acid into oleic acid via SCD activity provides a mechanistic insight into how the cumulus cells protect the oocyte against toxicity by saturated fatty acid.
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http://dx.doi.org/10.1095/biolreprod.116.146159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803780PMC
May 2017

Two phylogenetically and compartmentally distinct CDP-diacylglycerol synthases cooperate for lipid biogenesis in .

J Biol Chem 2017 04 17;292(17):7145-7159. Epub 2017 Mar 17.

From the Department of Molecular Parasitology, Humboldt University, Berlin 10115, Germany,

is among the most prevalent protozoan parasites, which infects a wide range of organisms, including one-third of the human population. Its rapid intracellular replication within a vacuole requires efficient synthesis of glycerophospholipids. Cytidine diphosphate-diacylglycerol (CDP-DAG) serves as a major precursor for phospholipid synthesis. Given the peculiarities of lipid biogenesis, understanding the mechanism and physiological importance of CDP-DAG synthesis is particularly relevant in Here, we report the occurrence of two phylogenetically divergent CDP-DAG synthase (CDS) enzymes in the parasite. The eukaryotic-type CDS1 and the prokaryotic-type CDS2 reside in the endoplasmic reticulum and apicoplast, respectively. Conditional knockdown of CDS1 severely attenuated the parasite growth and resulted in a nearly complete loss of virulence in a mouse model. Moreover, mice infected with the CDS1 mutant became fully resistant to challenge infection with a hyper-virulent strain of The residual growth of the CDS1 mutant was abolished by consecutive deletion of CDS2. Lipidomic analyses of the two mutants revealed significant and specific declines in phosphatidylinositol and phosphatidylglycerol levels upon repression of CDS1 and after deletion of CDS2, respectively. Our data suggest a "division of labor" model of lipid biogenesis in in which two discrete CDP-DAG pools produced in the endoplasmic reticulum and apicoplast are subsequently used for the synthesis of phosphatidylinositol in the Golgi bodies and phosphatidylglycerol in the mitochondria. The essential and divergent nature of CDP-DAG synthesis in the parasite apicoplast offers a potential drug target to inhibit the asexual reproduction of .
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http://dx.doi.org/10.1074/jbc.M116.765487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409480PMC
April 2017

Astrocyte lipid metabolism is critical for synapse development and function in vivo.

Glia 2017 04 7;65(4):670-682. Epub 2017 Feb 7.

Department of Molecular & Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University Amsterdam, De Boelelaan 1085, Amsterdam, 1081 HV, The Netherlands.

The brain is considered to be autonomous in lipid synthesis with astrocytes producing lipids far more efficiently than neurons. Accordingly, it is generally assumed that astrocyte-derived lipids are taken up by neurons to support synapse formation and function. Initial confirmation of this assumption has been obtained in cell cultures, but whether astrocyte-derived lipids support synapses in vivo is not known. Here, we address this issue and determined the role of astrocyte lipid metabolism in hippocampal synapse formation and function in vivo. Hippocampal protein expression for the sterol regulatory element-binding protein (SREBP) and its target gene fatty acid synthase (Fasn) was found in astrocytes but not in neurons. Diminishing SREBP activity in astrocytes using mice in which the SREBP cleavage-activating protein (SCAP) was deleted from GFAP-expressing cells resulted in decreased cholesterol and phospholipid secretion by astrocytes. Interestingly, SCAP mutant mice showed more immature synapses, lower presynaptic protein SNAP-25 levels as well as reduced numbers of synaptic vesicles, indicating impaired development of the presynaptic terminal. Accordingly, hippocampal short-term and long-term synaptic plasticity were defective in mutant mice. These findings establish a critical role for astrocyte lipid metabolism in presynaptic terminal development and function in vivo. GLIA 2017;65:670-682.
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http://dx.doi.org/10.1002/glia.23120DOI Listing
April 2017

Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT.

Biochim Biophys Acta Mol Cell Biol Lipids 2017 Feb 1;1862(2):176-187. Epub 2016 Nov 1.

Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, and Institute of Biomembranes, Utrecht University, P.O. Box 80176, 3508 TD Utrecht, The Netherlands. Electronic address:

Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC-MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT HSCs (1080nm) is significantly smaller than in wild type HSCs (1618nm). This is a consequence of an altered lipid droplet size distribution with 50.5±9.0% small (≤700nm) lipid droplets in LRAT HSCs and 25.6±1.4% large (1400-2100nm) lipid droplets in wild type HSC cells. Upon prolonged (24h) incubation, the amounts of small (≤700nm) lipid droplets strongly increased both in wild type and in LRAT HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.
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http://dx.doi.org/10.1016/j.bbalip.2016.10.013DOI Listing
February 2017

ATGL and DGAT1 are involved in the turnover of newly synthesized triacylglycerols in hepatic stellate cells.

J Lipid Res 2016 07 14;57(7):1162-74. Epub 2016 May 14.

Departments of Biochemistry and Cell Biology Faculty of Veterinary Medicine and Institute of Biomembranes, Utrecht University, 3584 CM Utrecht, The Netherlands

Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerol (TAG), cholesteryl esters (CEs), and retinyl esters (REs). Here we aimed to investigate which enzymes are involved in LD turnover in HSCs during activation in vitro. Targeted deletion of the Atgl gene in mice HSCs had little effect on the decrease of the overall TAG, CE, and RE levels during activation. However, ATGL-deficient HSCs specifically accumulated TAG species enriched in PUFAs and degraded new TAG species more slowly. TAG synthesis and levels of PUFA-TAGs were lowered by the diacylglycerol acyltransferase (DGAT)1 inhibitor, T863. The lipase inhibitor, Atglistatin, increased the levels of TAG in both WT and ATGL-deficient mouse HSCs. Both Atglistatin and T863 inhibited the induction of activation marker, α-smooth muscle actin, in rat HSCs, but not in mouse HSCs. Compared with mouse HSCs, rat HSCs have a higher turnover of new TAGs, and Atglistatin and the DGAT1 inhibitor, T863, were more effective. Our data suggest that ATGL preferentially degrades newly synthesized TAGs, synthesized by DGAT1, and is less involved in the breakdown of preexisting TAGs and REs in HSCs. Furthermore a large change in TAG levels has modest effect on rat HSC activation.
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http://dx.doi.org/10.1194/jlr.M066415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918846PMC
July 2016