Lipid Acquisition Research Articles

Fatty Acids and Breast Cancer: Make Them on Site or Have Them Delivered

Brisk fatty acid (FA) production by cancer cells is accommodated by the Warburg effect. Most breast and other cancer cell types are addicted to fatty acids (FA), which they require for membrane phospholipid synthesis, signaling purposes, and energy production. Expression of the enzymes required for FA synthesis is closely linked to each of the major classes of signaling molecules that stimulate BC cell proliferation. This review focuses on the regulation of FA synthesis in BC cells, and the impact of FA, or the lack thereof, on the tumor cell phenotype. Given growing awareness of the impact of dietary fat and obesity on BC biology, we will also examine the less-frequently considered notion that, in addition to de novo FA synthesis, the lipolytic uptake of preformed FA may also be an important mechanism of lipid acquisition. Indeed, it appears that cancer cells may exist at different points along a "lipogenic-lipolytic axis," and FA uptake could thwart attempts to exploit the strict requirement for FA focused solely on inhibition of de novo FA synthesis. Strategies for clinically targeting FA metabolism will be discussed, and the current status of the medicinal chemistry in this area will be assessed. J. Cell. Physiol. 231: 2128-2141, 2016. © 2016 Wiley Periodicals, Inc.

Fluorescent labeling reliably identifies Chlamydia trachomatis in living human endometrial cells and rapidly and accurately quantifies chlamydial inclusion forming units

Chlamydia replication requires host lipid acquisition, allowing flow cytometry to identify Chlamydia-infected cells that accumulated fluorescent Golgi-specific lipid. Herein, we describe modifications to currently available methods that allow precise differentiation between uninfected and Chlamydia trachomatis-infected human endometrial cells and rapidly and accurately quantify chlamydial inclusion forming units.

Profiling the Essential Nature of Lipid Metabolism in Asexual Blood and Gametocyte Stages of Plasmodium falciparum

During its life cycle, Plasmodium falciparum undergoes rapid proliferation fueled by de novo synthesis and acquisition of host cell lipids. Consistent withthis essential role, Plasmodium lipid synthesis enzymes are emerging as potential drug targets. To explore their broader potential for therapeutic interventions, we assayed the global lipid landscape during P.falciparum sexual and asexual blood stage (ABS) development. Using liquid chromatography-mass spectrometry, we analyzed 304 lipids constituting 24 classes in ABS parasites, infected red blood cell (RBC)-derived microvesicles, gametocytes, and uninfected RBCs. Ten lipid classes were previously uncharacterized in P.falciparum, and 70%-75% of the lipid classes exhibited changes in abundance during ABS and gametocyte development. Utilizing compounds that target lipid metabolism, we affirmed the essentiality of major classes, including triacylglycerols. These studies highlight the interplay between host and parasite lipid metabolism and provide a comprehensive analysis of P.falciparum lipids with candidate pathways for drug discovery efforts.

Chlamydiae interaction with the endoplasmic reticulum: contact, function and consequences.

Chlamydiae and chlamydiae-related organisms are obligate intracellular bacterial pathogens. They reside in a membrane-bound compartment termed the inclusion and have evolved sophisticated mechanisms to interact with cellular organelles. This review focuses on the nature, the function(s) and the consequences of chlamydiae-inclusion interaction with the endoplasmic reticulum (ER). The inclusion membrane establishes very close contact with the ER at specific sites termed ER-inclusion membrane contact sites (MCSs). These MCSs are constituted of a specific set of factors, including the C. trachomatis effector protein IncD and the host cell proteins CERT and VAPA/B. Because CERT and VAPA/B have a demonstrated role in the non-vesicular trafficking of lipids between the ER and the Golgi, it was proposed that Chlamydia establish MCSs with the ER to acquire host lipids. However, the recruitment of additional factors to ER-inclusion MCSs, such as the ER calcium sensor STIM1, may suggest additional functions unrelated to lipid acquisition. Finally, chlamydiae interaction with the ER appears to induce the ER stress response, but this response is quickly dampened by chlamydiae to promote host cell survival.

Chlamydia trachomatis Infection Leads to Defined Alterations to the Lipid Droplet Proteome in Epithelial Cells.

The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen and a main cause of genital and ocular diseases. During its intracellular cycle, C. trachomatis replicates inside a membrane-bound vacuole termed an “inclusion”. Acquisition of lipids (and other nutrients) from the host cell is a critical step in chlamydial replication. Lipid droplets (LD) are ubiquitous, ER-derived neutral lipid-rich storage organelles surrounded by a phospholipids monolayer and associated proteins. Previous studies have shown that LDs accumulate at the periphery of, and eventually translocate into, the chlamydial inclusion. These observations point out to Chlamydia-mediated manipulation of LDs in infected cells, which may impact the function and thereby the protein composition of these organelles. By means of a label-free quantitative mass spectrometry approach we found that the LD proteome is modified in the context of C. trachomatis infection. We determined that LDs isolated from C. trachomatis-infected cells were enriched in proteins related to lipid metabolism, biosynthesis and LD-specific functions. Interestingly, consistent with the observation that LDs intimately associate with the inclusion, a subset of inclusion membrane proteins co-purified with LD protein extracts. Finally, genetic ablation of LDs negatively affected generation of C. trachomatis infectious progeny, consistent with a role for LD biogenesis in optimal chlamydial growth.

Mesothelioma tumor cells modulate dendritic cell lipid content, phenotype and function.

Dendritic cells (DCs) play an important role in the generation of anti-cancer immune responses, however there is evidence that DCs in cancer patients are dysfunctional. Lipid accumulation driven by tumor-derived factors has recently been shown to contribute to DC dysfunction in several human cancers, but has not yet been examined in mesothelioma. This study investigated if mesothelioma tumor cells and/or their secreted factors promote increases in DC lipid content and modulate DC function. Human monocyte-derived DCs (MoDCs) were exposed to human mesothelioma tumor cells and tumor-derived factors in the presence or absence of lipoproteins. The data showed that immature MoDCs exposed to mesothelioma cells or factors contained increased lipid levels relative to control DCs. Lipid accumulation was associated with reduced antigen processing ability (measured using a DQ OVA assay), upregulation of the co-stimulatory molecule, CD86, and production of the tolerogenic cytokine, IL-10. Increases in DC lipid content were further enhanced by co-exposure to mesothelioma-derived factors and triglyceride-rich lipoproteins, but not low-density lipoproteins. In vivo studies using a murine mesothelioma model showed that the lipid content of tumor-infiltrating CD4+CD8α- DCs, CD4-CD8α- DCs DCs and plasmacytoid DCs increased with tumor progression. Moreover, increasing tumor burden was associated with reduced proliferation of tumor-antigen-specific CD8+ T cells in tumor-draining lymph nodes. This study shows that mesothelioma promotes DC lipid acquisition, which is associated with altered activation status and reduced capacity to process and present antigens, which may impair the ability of DCs to generate effective anti mesothelioma T cell responses.

Lipid-free antigen B subunits from echinococcus granulosus: oligomerization, ligand binding, and membrane interaction properties.

BackgroundThe hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied.Methodology/Principal FindingsLipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles.Conclusions/SignificanceWe show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy.

Photoautotrophic and heterotrophic carbon in bleached and non-bleached coral lipid acquisition and storage

Lipids are key biomolecules within the coral holobiont, serving as energy reserves and promoting bleaching tolerance. To better understand the physiological implications of bleaching and recovery on lipid acquisition and storage, an isotope pulse-chase labeling experiment was conducted on treatment (bleached) and control (non-bleached) Hawaiian corals Porites compressa and Montipora capitata after 1 and 11months on the reef. After 1month on the reef, 75% of the carbon in newly acquired lipids in treatment corals of both species was heterotrophic in origin, which is two times higher than that in control corals. However, stored lipids in both treatment and controls were hugely dominated by heterotrophic C in P. compressa (i.e., 75–100%) and by photoautotrophic C in M. capitata (i.e., 90–100%). After 11months on the reef, elevated levels of heterotrophic C input in lipid acquisition and storage were only seen in treatment P. compressa. Many previously measured physiological parameters are recovered in P. compressa after 8months, suggesting that the need for increased heterotrophic C input for lipids appears to last beyond recovery of all other parameters, indicating that P. compressa is not yet fully recovered even after 11months. In M. capitata, lipid acquisition and storage were 28–52% heterotrophic in origin and 48–72% photoautotrophic and, along with many previously measured physiological parameters, were fully recovered after 11months on the reef. However, the evidence suggests that carbohydrate and possibly protein acquisition was not fully recovered even after 11months. These species-specific differences in lipid acquisition and storage mechanisms, and response patterns to temperature stress indicate that corals may take more than 11months to recover from bleaching and that heterotrophic carbon is critical to coral lipids, especially when bleached.

Effect of nitrogenous resource on growth, biochemical composition and ultrastructure of Isochrysis galbana (Isochrysidales, Haptophyta)

SummaryThe nitrogenous resource used to promote algal growth has cost implications for mass culture processes. The present study therefore aimed to determine the effect of different nitrogenous resources (nitrate, ammonium and urea) on various performance parameters (growth, final cell yield, pigmentation, lipid yield and cellular and sub‐cellular characteristics) in Isochrysis galbana. Growth rate was unaffected by nitrogenous resource, but the final cellular yield in the nitrate and urea treatments far exceeded that evident in the ammonium treatments. The reduced cell yield in ammonium treatments and the earlier onset of the stationary phase was brought about by nitrogen‐starvation due to an increase in pH and resultant ammonia volatilization. This starvation initiated an early onset of lipid accumulation, chlorophyll depletion and an increase in the carotenoid to chlorophyll ratio relative to the other nitrogen (N) source treatments. Hence, in spite of being potentially the preferred source of N by algae (due to its reduced state), ammonium‐nitrogen is undesirable for mass culture. The performance parameters of Isochrysis grown in urea (an organic N source) and nitrate (an inorganic N source) were similar, but lipid accrued earlier in cells grown in medium supplemented with urea. This is advantageous for lipid acquisition for the production of biodiesel since it would reduce the duration of photobioreactor runs. Urea is easily available and considerably cheaper than all the other N sources tested and is thus recommended as the nitrogenous resource for large‐scale culture of I. galbana for biodiesel production.

Dynamin-mediated lipid acquisition is essential for Chlamydia trachomatis development.

Chlamydia trachomatis is an obligate intracellular pathogen responsible for a high burden of human disease. Here, a loss-of-function screen using a set of lentivirally transduced shRNAs identified 14 human host cell factors that modulate C. trachomatis infectivity. Notably, knockdown of dynamin, a host GTPase, decreased C. trachomatis infectivity. Dynamin functions in multiple cytoplasmic locations, including vesicle formation at the plasma membrane and the trans-Golgi network. However, its role in C. trachomatis infection remains unclear. Here we report that dynamin is essential for homotypic fusion of C. trachomatis inclusions but not for C. trachomatis internalization into the host cell. Further, dynamin activity is necessary for lipid transport into C. trachomatis inclusions and for normal re-differentiation from reticulate to elementary bodies. Fragmentation of the Golgi apparatus is proposed to be an important strategy used by C. trachomatis for efficient lipid acquisition and replication within the host. Here we show that a subset of C. trachomatis-infected cells displayed Golgi fragmentation, which was concurrent with increased mitotic accumulation. Golgi fragmentation was dispensable for dynamin-mediated lipid acquisition into C. trachomatis inclusions, irrespective of the cell cycle phase. Thus, our study reveals a critical role of dynamin in host-derived lipid acquisition for C. trachomatis development.

Schistosome feeding and regurgitation.

Schistosomes are parasitic flatworms that infect >200 million people worldwide, causing the chronic, debilitating disease schistosomiasis. Unusual among parasitic helminths, the long-lived adult worms, continuously bathed in blood, take up nutrients directly across the body surface and also by ingestion of blood into the gut. Recent proteomic analyses of the body surface revealed the presence of hydrolytic enzymes, solute, and ion transporters, thus emphasising its metabolic credentials. Furthermore, definition of the molecular mechanisms for the uptake of selected metabolites (glucose, certain amino acids, and water) establishes it as a vital site of nutrient acquisition. Nevertheless, the amount of blood ingested into the gut per day is considerable: for males ∼100 nl; for the more actively feeding females ∼900 nl, >4 times body volume. Ingested erythrocytes are lysed as they pass through the specialized esophagus, while leucocytes become tethered and disabled there. Proteomics and transcriptomics have revealed, in addition to gut proteases, an amino acid transporter in gut tissue and other hydrolases, ion, and lipid transporters in the lumen, implicating the gut as the site for acquisition of essential lipids and inorganic ions. The surface is the principal entry route for glucose, whereas the gut dominates amino acid acquisition, especially in females. Heme, a potentially toxic hemoglobin degradation product, accumulates in the gut and, since schistosomes lack an anus, must be expelled by the poorly understood process of regurgitation. Here we place the new observations on the proteome of body surface and gut, and the entry of different nutrient classes into schistosomes, into the context of older studies on worm composition and metabolism. We suggest that the balance between surface and gut in nutrition is determined by the constraints of solute diffusion imposed by differences in male and female worm morphology. Our conclusions have major implications for worm survival under immunological or pharmacological pressure.

Abstract 224: Formation of Cholesterol-Enriched Nascent HDL Requires Helices 5 and 7

Heart disease claims nearly 400,000 individuals in the US per year. Large population studies have repeatedly demonstrated an inverse relationship between HDL concentration and risk. Despite the strong correlation, pharmacological trials that raise plasma HDL have shown little efficacy, suggesting that HDL function should be monitored. Studies were initiated into the mechanism of cholesterol efflux leading to nascent HDL (nHDL) formation. The most productive cholesterol removal occurs when lipid-poor apoA-I, the main structural protein in nHDL, forms particles after interacting with ABCA1. ApoA-I acquires cholesterol and phospholipid at the cell surface forming 10-12 nm diameter nHDL whose composition resembles lipid rafts. Because lipidated apoA-I has a significantly different 3-D conformation compared to its lipid-poor counterpart, the structural modifications leading to nHDL formation were investigated. First, we completed a 3-D solution structure of lipid-poor apoA-I using lysine specific chemical cross-linking in conjunction with specifically engineered double cysteine-containing apoA-I mutants. Strategically placed cysteines form disulfide bonds (locked) when residues are within ~0.5 nm. If no disulfide bond formed (unlocked) then the two cysteines were separated by more than ~0.5 nm. Both methods rely on mass spectrometry to identify amino acid sequence and distance constraints within each 10 repeating helices comprising 80% of full-length apoA-I. By comparing lipid-poor apoA-I conformation to our previously described 3-D structure of apoA-I on 10-12 nm nHDL, we formulated a series of hypothetical helical domain transitions that might drive protein-lipid interaction and particle formation. To test, 10 different double cysteine mutants were employed in either their “locked” (oxidized) or “unlocked” (reduced) state by evaluating their ability to form recombinant HDL using synthetic phospholipid or to form nHDL using HEK cells. After determining HDL particle yield, size and composition from all locked and unlocked double cysteine apoA-I mutants we conclude that both the N- and C-terminal ends of apoA-I are essential for the first steps in lipid acquisition, but that the central helices 5, 6 and 7 are essential for nHDL formation.

Phosphatidylethanolamine Synthesis in the Parasite Mitochondrion Is Required for Efficient Growth but Dispensable for Survival of Toxoplasma gondii

Toxoplasma gondii is a highly prevalent obligate intracellular parasite of the phylum Apicomplexa, which also includes other parasites of clinical and/or veterinary importance, such as Plasmodium, Cryptosporidium, and Eimeria. Acute infection by Toxoplasma is hallmarked by rapid proliferation in its host cells and requires a significant synthesis of parasite membranes. Phosphatidylethanolamine (PtdEtn) is the second major phospholipid class in T. gondii. Here, we reveal that PtdEtn is produced in the parasite mitochondrion and parasitophorous vacuole by decarboxylation of phosphatidylserine (PtdSer) and in the endoplasmic reticulum by fusion of CDP-ethanolamine and diacylglycerol. PtdEtn in the mitochondrion is synthesized by a phosphatidylserine decarboxylase (TgPSD1mt) of the type I class. TgPSD1mt harbors a targeting peptide at its N terminus that is required for the mitochondrial localization but not for the catalytic activity. Ablation of TgPSD1mt expression caused up to 45% growth impairment in the parasite mutant. The PtdEtn content of the mutant was unaffected, however, suggesting the presence of compensatory mechanisms. Indeed, metabolic labeling revealed an increased usage of ethanolamine for PtdEtn synthesis by the mutant. Likewise, depletion of nutrients exacerbated the growth defect (∼56%), which was partially restored by ethanolamine. Besides, the survival and residual growth of the TgPSD1mt mutant in the nutrient-depleted medium also indicated additional routes of PtdEtn biogenesis, such as acquisition of host-derived lipid. Collectively, the work demonstrates a metabolic cooperativity between the parasite organelles, which ensures a sustained lipid synthesis, survival and growth of T. gondii in varying nutritional milieus.

Toxoplasma gondii- skeletal muscle cells interaction increases lipid droplet biogenesis and positively modulates the production of IL-12, IFN-g and PGE2

BackgroundThe interest in the mechanisms involved in Toxoplasma gondii lipid acquisition has steadily increased during the past few decades, but it remains not completely understood. Here, we investigated the biogenesis and the fate of lipid droplets (LD) of skeletal muscle cells (SkMC) during their interaction with T. gondii by confocal and electron microscopy. We also evaluated whether infected SkMC modulates the production of prostaglandin E2 (PGE2), cytokines interleukin-12 (IL-12) and interferon-gamma (INF-g), and also the cyclooxygenase-2 (COX-2) gene induction.MethodsPrimary culture of skeletal muscle cells were infected with tachyzoites of T. gondii and analysed by confocal microscopy for observation of LD. Ultrastructural cytochemistry was also used for lipid and sarcoplasmatic reticulum (SR) detection. Dosage of cytokines (IL-12 and INF-g) by ELISA technique and enzyme-linked immunoassay (EIA) for PGE2 measurement were employed. The COX-2 gene expression analysis was performed by real time reverse transcriptase polymerase chain reaction (qRT-PCR).ResultsWe demonstrated that T. gondii infection of SkMC leads to increase in LD number and area in a time course dependent manner. Moreover, the ultrastructural analysis demonstrated that SR and LD are in direct contact with parasitophorous vacuole membrane (PVM), within the vacuolar matrix, around it and interacting directly with the membrane of parasite, indicating that LD are recruited and deliver their content inside the parasitophorous vacuole (PV) in T. gondii-infected SkMC. We also observed a positive modulation of the production of IL-12 and IFN-g, increase of COX-2 mRNA levels in the first hour of T. gondii-SkMC interaction and an increase of prostaglandin E2 (PGE2) synthesis from 6 h up to 48 h of infection.ConclusionsTaken together, the close association between SR and LD with PV could represent a source of lipids as well as other nutrients for the parasite survival, and together with the increased levels of IL-12, INF-g and inflammatory indicators PGE2 and COX-2 might contribute to the establishment and maintenance of chronic phase of the T. gondii infection in muscle cell.

Acquisition of intact polar lipids from the prymnesiophyte <i>Phaeocystis globosa</i> by its lytic virus PgV-07T

Abstract. Recent studies showed changes in phytoplankton lipid composition during viral infection and have indicated roles for specific lipids in the mechanisms of algal virus-host interaction. To investigate the generality of these findings and obtain a better understanding of the allocation of specific lipids to viruses, we studied the intact polar lipid (IPL) composition of virally infected and non-infected cultures of the prymnesiophyte Phaeocystis globosa G(A) and its lytic virus PgV-07T. The P. globosa IPL composition was relatively stable over a diel cycle and not strongly affected by viral infection. Glycolipids, phospholipids and betaine lipids were present in both the host and virus, although specific groups such as the diacylglyceryl-hydroxymethyltrimethyl-β-alanines and the sulfoquinovosyldiacylglycerols, were present in a lower proportion or were not detected in the virus. Viral glycosphingolipids (vGSLs), which have been shown to play a role in the infection strategy of the virus EhV-86, infecting the prymnesiophyte Emiliania huxleyi CCMP374, were not encountered. Our results show that the involvement of lipids in virus–algal host interactions can be very different amongst virus–algal host systems.

Visualization of Lipid Bodies in <i>Chlamydomonas reinhardtii</i> Living Cells with Fluorescent Microscopy

Acquisition of lipids mainly in a form of triacylglycerol from microalgae is a potential way for biofuels production. Many unicellular microalgae make little triacylglycerol under favorable growth condition, but accumulate large amounts of it under environmental stress conditions (e.g., nutrient depletion, high light). In this paper, the green microalgae Chlamydomonas reinhardtii BAFJ5 was used as a model to study production of lipid bodies (LBs) which are important lipid storage organelles, particularly under stress. We discovered LBs accumulated dramatically when cells were grown in nitrogen-deplete medium. Using fluorescent microscopy and confocal laser scanning microscopy, intracellular LBs can be successfully visualized. The number and size of LBs were also quantified.

Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

The predominant players in membrane fusion events are the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins. We hypothesize that SNARE proteins mediate fusion events at the chlamydial inclusion and are important for chlamydial lipid acquisition. We have previously demonstrated that trans-Golgi SNARE syntaxin 6 localizes to the chlamydial inclusion. To investigate the role of syntaxin 6 at the chlamydial inclusion, we examined the localization and function of another trans-Golgi SNARE and syntaxin 6-binding partner, vesicle-associated membrane protein 4 (VAMP4), at the chlamydial inclusion. In this study, we demonstrate that syntaxin 6 and VAMP4 colocalize to the chlamydial inclusion and interact at the chlamydial inclusion. Furthermore, in the absence of VAMP4, syntaxin 6 is not retained at the chlamydial inclusion. Small interfering RNA (siRNA) knockdown of VAMP4 inhibited chlamydial sphingomyelin acquisition, correlating with a log decrease in infectious progeny. VAMP4 retention at the inclusion was shown to be dependent on de novo chlamydial protein synthesis, but unlike syntaxin 6, VAMP4 recruitment is observed in a species-dependent manner. Notably, VAMP4 knockdown inhibits sphingomyelin trafficking only to inclusions in which it localizes. These data support the hypothesis that VAMP proteins play a central role in mediating eukaryotic vesicular interactions at the chlamydial inclusion and, thus, support chlamydial lipid acquisition and chlamydial development.

Androgens inhibit adipogenesis during human adipose stem cell commitment to preadipocyte formation

Androgens play a pivotal role in the regulation of body fat distribution. Adipogenesis is a process whereby multipotent adipose stem cells (ASCs) initially become preadipocytes (ASC commitment to preadipocytes) before differentiating into adipocytes. Androgens inhibit human (h) subcutaneous (SC) abdominal preadipocyte differentiation in both sexes, but their effects on hASC commitment to preadipocyte formation is unknown. We therefore examined whether androgen exposure to human (h) ASCs, isolated from SC abdominal adipose of nonobese women, impairs their commitment to preadipocyte formation and/or subsequent differentiation into adipocytes. For this, isolated hASCs from SC abdominal lipoaspirate were cultured in adipogenesis-inducing medium for 0.5–14days in the presence of testosterone (T, 0–100nM) or dihydrotestosterone (DHT, 0–50nM). Adipogenesis was determined by immunofluorescence microscopy and by quantification of adipogenically relevant transcriptional factors, PPARγ, C/EBPα and C/EBPβ. We found that a 3-day exposure of hASCs to T (50nM) or DHT (5nM) in adipogenesis-inducing medium impaired lipid acquisition and decreased PPARγ, C/EBPα and C/EBPβ gene expression. The inhibitory effects of T and DHT at this early-stage of adipocyte differentiation, were partially and completely reversed by flutamide (F, 100nM), respectively. The effect of androgens on hASC commitment to a preadipocyte phenotype was examined via activation of Bone Morphogenic Protein 4 (BMP4) signaling. T (50nM) and DHT (5nM) significantly inhibited the stimulatory effect of BMP4-induced ASC commitment to the preadipocyte phenotype, as regards PPARγ and C/EBPα gene expression. Our findings indicate that androgens, in part through androgen receptor action, impair BMP4-induced commitment of SC hASCs to preadipocytes and also reduce early-stage adipocyte differentiation, perhaps limiting adipocyte numbers and fat storage in SC abdominal adipose.

Abstract 1905: Influence of serum and oxygen on lipid biosynthesis and energy metabolism in murine glioblastoma.

Abstract Glucose and glutamine are essential energy metabolites for brain tumor growth and survival under both normoxic and hypoxic conditions. Carbons derived from either metabolite can be used for lipid synthesis. Here, we used uniformly labeled C14-glucose and C14-glutamine to examine de novo lipid synthesis in the VM-M3 murine glioblastoma cells. The major lipids synthesized included phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), phosphatidic acid (PA), cholesterol (Chol), and cardiolipin (CL). De novo lipid synthesis was greater with glucose than with glutamine as the metabolic substrate under normoxia. Conversely, glutamine was the preferred substrate for lipid synthesis under anoxia. Lactate was produced largely from glucose carbons with minimal lactate derived from glutamine. Total lipid synthesis, using either glucose or glutamine, was greater in media without serum than in media containing 10% FBS under normoxia. Significant triglyceride accumulation was observed only under anoxia. Our findings show that lipid biosynthesis using glucose or glutamine is dependent on the type of growth environment, and that the VM-M3 glioblastoma cells can grow under anoxia only through acquisition of lipids, especially cholesterol, from serum. Supported in part from Boston College Res. Expense Fund. Citation Format: Nathan Ta, Thomas Seyfried. Influence of serum and oxygen on lipid biosynthesis and energy metabolism in murine glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1905. doi:10.1158/1538-7445.AM2013-1905

Autophagosome Formation: Tracing the Source

Although it is well appreciated that autophagy begins with phagophore formation and expansion through lipid acquisition to become the autophagosome, this process remains poorly understood, with the source of autophagosome membrane controversial. Reporting recently in Nature, Hamasaki etal. (2013) suggest that the ER-mitochondria contact sites are involved in phagophore assembly.