BBA - Molecular and Cell Biology of Lipids (v.1851, #10)

Characterization of lipid droplets in steroidogenic MLTC-1 Leydig cells: Protein profiles and the morphological change induced by hormone stimulation by Tomohiro Yamaguchi; Noriyuki Fujikawa; Satomi Nimura; Yutaro Tokuoka; Sonoka Tsuda; Toshihiro Aiuchi; Rina Kato; Takashi Obama; Hiroyuki Itabe (1285-1295).
Lipid droplets (LDs) are functional subcellular organelles involved in multiple intracellular processes. LDs are found in nearly all types of eukaryotic cells, but their properties are highly variable in different types of tissues. Steroidogenic cells synthesize steroid hormones de novo from the cholesterol deposited in cytosolic LDs. However, the roles of LD proteins in steroidogenesis under pituitary hormone stimulation have not been well elucidated. The protein profile of isolated LDs from the mouse Leydig tumor cell line MLTC-1 was distinct from that of hepatic cells or macrophages. By proteomic analysis of the components using mass spectrometry, two enzymes for steroidogenesis, 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1) and 17 β-hydroxysteroid dehydrogenase type 11 (17βHSD11), were identified in two strong bands in the LD fractions. The LD fraction of MLTC-1 cells also included CYP11A1 and CYP17, suggesting that the LDs contain all the enzymes needed for testosterone synthesis. The steroidogenesis in Leydig cells is activated by luteinizing hormone through a PKA-dependent pathway. Stimulation of MLTC-1 cells with luteinizing hormone or 8-bromo-cAMP caused drastic changes in the morphology of the LDs in the MLTC-1 cells. Upon stimulation, large perinuclear LDs are turned into much smaller LDs and dispersed throughout the cytosol. These results raise the possibility that LDs are involved in a regulatory pathway of steroidogenesis, not just by serving as a storage depot for cholesterol esters, but also by providing enzymes and generating sites for enzymatic activity.
Keywords: Lydig cells; Lipid droplets; Steroidogenesis; 17beta-HSD11; 3beta-HSD1; MLTC-1;

Short-chain polyisoprenoids in the yeast Saccharomyces cerevisiae — New companions of the old guys by Liliana Surmacz; Jacek Wojcik; Magdalena Kania; Magnus Bentinger; Witold Danikiewicz; Gustav Dallner; Przemyslaw Surowiecki; Piotr Cmoch; Ewa Swiezewska (1296-1303).
Dolichols are, among others, obligatory cofactors of protein glycosylation in eukaryotic cells. It is well known that yeast cells accumulate a family of dolichols with Dol-15/16 dominating while upon certain physiological conditions a second family with Dol-21 dominating is noted. In this report we identified the presence of additional short-chain length polyprenols — all-trans Pren-7 in three yeast strains (SS328, BY4741 and L5366), Pren-7 was accompanied by traces of putative Pren-6 and -8. Moreover, in two of these strains a single polyprenol mainly-cis-Pren-11 was synthesized at the stationary phase of growth. Identity of polyprenols was confirmed by HR-HPLC/MS, NMR and metabolic labeling. Additionally, simvastatin inhibited their biosynthesis.
Keywords: Polyisoprenoid alcohols; Dolichol; Prenol; Saccharomyces cerevisiae; yeast;

Active autophagy but not lipophagy in macrophages with defective lipolysis by Madeleine Goeritzer; Nemanja Vujic; Stefanie Schlager; Prakash G. Chandak; Melanie Korbelius; Benjamin Gottschalk; Christina Leopold; Sascha Obrowsky; Silvia Rainer; Prakash Doddapattar; Elma Aflaki; Martin Wegscheider; Vinay Sachdev; Wolfgang F. Graier; Dagmar Kolb; Branislav Radovic; Dagmar Kratky (1304-1316).
During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages.Display Omitted
Keywords: Adipose triglyceride lipase; Hormone-sensitive lipase; Lipid droplets; Triglyceride mobilization;

Metabolomic analysis revealed the role of DNA methylation in the balance of arachidonic acid metabolism and endothelial activation by Shan-Shan Xue; Jin-Long He; Xu Zhang; Ya-Jin Liu; Feng-Xia Xue; Chun-Jiong Wang; Ding Ai; Yi Zhu (1317-1326).
Arachidonic acid (AA) metabolism plays an important role in vascular homeostasis. We reported that DNA hypomethylation of EPHX2 induced a pro-inflammatory response in vascular endothelial cells (ECs). However, the change in the whole AA metabolism by DNA methylation is still unknown. Using a metabolomic approach, we investigated the effect of DNA methylation on the balance of AA metabolism and the underlying mechanism. ECs were treated with a DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-AZA), and AA metabolic profiles were analyzed. Levels of prostaglandin D2 (PGD2) and thromboxane B2 (TXB2), metabolites in the cyclooxygenase (COX) pathway, were significantly increased by 5-AZA treatment in ECs resulting from the induction of PGD2 synthase (PTGDS) and thromboxane A synthase 1 (TBXAS1) expression by DNA hypomethylation. This phenomenon was also observed in liver and kidney cell lines, indicating a universal mechanism. Pathophysiologically, homocysteine, known to cause DNA demethylation, induced a similar pattern of the change of AA metabolism. Furthermore, 5-AZA activated ECs, as evidenced by the upregulation of adhesion molecules. Indomethacin, a COX inhibitor, reversed the effects of 5-AZA on the levels of PGD2 and TXB2, EC activation and monocyte adhesion. In vivo, the plasma levels of PGD2 and TXB2 and the expression of In vivo PTGDS and TBXAS1 as well as adhesion molecules were increased in the aorta of the mice injected with 5-AZA. In conclusion, using a metabolomic approach, our study uncovered that DNA demethylation increased AA metabolites PGD2 and TXB2 by upregulating the expression of the corresponding enzymes, which might contribute to the DNA hypomethylation-induced endothelial activation.Display Omitted
Keywords: DNA methylation; Arachidonic acid metabolism; Metabolomics; Endothelial cell;

Hepatic lipase (HL) is an important enzyme in the clearance of triacylglycerol (TAG) from the circulation, and has been proposed to have pro-atherogenic as well as anti-atherogenic properties. It hydrolyzes both phospholipids and TAG of lipoproteins, and its activity is negatively correlated with HDL levels. Although it is known that HL acts preferentially on HDL lipids, the basis for this specificity is not known, since it does not require any specific apoprotein for activity. In this study, we tested the hypothesis that sphingomyelin (SM), whose concentration is much higher in VLDL and LDL compared to HDL, is an inhibitor of HL, and that this could explain the lipoprotein specificity of the enzyme. The results presented show that the depletion of SM from normal lipoproteins activated the HL roughly in proportion to their SM content. SM depletion stimulated the hydrolysis of both phosphatidylcholine (PC) and TAG, although the PC hydrolysis was stimulated more. In the native lipoproteins, HL showed specificity for PC species containing polyunsaturated fatty acids at sn-2 position, and produced more unsaturated lyso PC species. The enzyme also showed preferential hydrolysis of certain TAG species over others. SM depletion affected the specificity of the enzyme towards PC and TAG species modestly. These results show that SM is a physiological inhibitor of HL activity in lipoproteins and that the specificity of the enzyme towards HDL is at least partly due to its low SM content.
Keywords: Hepatic lipase; Sphingomyelin; PC species; TAG species; Substrate specificity; Enzyme inhibition;

Lipidomic and proteomic analysis of Caenorhabditis elegans lipid droplets and identification of ACS-4 as a lipid droplet-associated protein by Tracy L. Vrablik; Vladislav A. Petyuk; Emily M. Larson; Richard D. Smith; Jennifer L. Watts (1337-1345).
Lipid droplets are cytoplasmic organelles that store neutral lipids for membrane synthesis and energy reserves. In this study, we characterized the lipid and protein composition of purified Caenorhabditis elegans lipid droplets. These lipid droplets are composed mainly of triacylglycerols, surrounded by a phospholipid monolayer composed primarily of phosphatidylcholine and phosphatidylethanolamine. The fatty acid composition of the triacylglycerols is rich in fatty acid species obtained from the dietary Escherichia coli, including cyclopropane fatty acids and cis-vaccenic acid. Unlike other organisms, C. elegans lipid droplets contain very little cholesterol or cholesterol esters. Comparison of the lipid droplet proteomes of wild type and high-fat daf-2 mutant strains shows a very similar proteome in both strains, except that the most abundant protein in the C. elegans lipid droplet proteome, MDT-28, is relatively less abundant in lipid droplets isolated from daf-2 mutants. Functional analysis of lipid droplet proteins identified in our proteomic studies indicated an enrichment of proteins required for growth and fat homeostasis in C. elegans. Finally, we confirmed the localization of one of the newly identified lipid droplet proteins, ACS-4. We found that ACS-4 localizes to the surface of lipid droplets in the C. elegans intestine and skin. This study bolsters C. elegans as a model to study the dynamics and functions of lipid droplets in a multicellular organism.Display Omitted
Keywords: C. elegans; Lipid droplets; DAF-2; Lipidomics; Proteomics;

There are many reports of the anti-inflammatory, anti-cancer, and anti-atherosclerotic activities of conjugated linolenic acids (cLNA). They constitute a small percentage of fatty acids in the typical human diet, although up to 80% of the fatty acids in certain fruits such as pomegranate. In the course of studying a bacterial fatty acid dioxygenase (Nostoc linoleate 10S-DOX, an ancient relative of mammalian cyclooxygenases), we detected strong inhibitory activity in a commercial sample of linoleic acid. We identified two cLNA isomers, β-eleostearic (9E,11E,13E-18:3) and β-calendic acid (8E,10E,12E-18:3), as responsible for that striking inhibition with a Ki of ~ 49 nM and ~ 125 nM, respectively, the most potent among eight cLNA tested. We also examined the effects of all eight cLNA on the activity of COX-1 and COX-2. Jacaric acid (8Z,10E,12Z-18:3) and its 12E isomer, 8Z,10E,12E-18:3, strongly inhibit the activity of COX-1 with a Ki of ~ 1.7 and ~ 1.1 μM, respectively. By contrast, COX-2 was ≤ 30% inhibited at 10 μM concentrations of the cLNA. Identifying the activities of the naturally occurring fatty acids is of interest in terms of understanding their interaction with the enzymes, and for explaining the mechanistic basis of their biological effects. The study also highlights the potential presence of inhibitory fatty acids in commercial lipids prepared from natural sources. Analysis of seven commercial samples of linoleic acid by HPLC and UV spectroscopy is illustrated as supplementary data.
Keywords: Fatty acid dioxygenase; Cyclooxygenase; Linoleic acid; Conjugated fatty acid; 10S-DOX; Nostoc punctiforme;

Novel role of TLR4 in NAFLD development: Modulation of metabolic enzymes expression by Darkiane Fernandes Ferreira; Jarlei Fiamoncini; Iryna Hirata Prist; Suely Kubo Ariga; Heraldo Possolo de Souza; Thais Martins de Lima (1353-1359).
The rise in the prevalence of obesity and metabolic syndrome turned NAFLD as the most common cause of chronic liver diseases worldwide. Although the role of toll like receptors, especially TLR4, as activators of inflammatory pathways in liver diseases is well established, our goal was to investigate if TLR4 activation could modulate metabolic lipid pathways and alter the onset of NAFLD. We used LDL receptor-deficient mice (LDLrKO) fed with an atherogenic diet as a model. The role of TLR4 activation was evaluated by crossing LDLrKO mice with the TLR4 knockout mice. Animals were fed for 12 weeks with high-fat high-cholesterol diet (HFD) containing 18% saturated fat and 1.25% cholesterol. TLR4/LDLr KO mice presented lower triacylglyceride (TAG) plasma levels when compared to LDLrKO, despite the type of diet ingested. HFD induced TAG and cholesterol accumulation in the liver of all mice genotypes studied, but TLR4/LDLr KO presented lower TAG accumulation than LDLrKO mice. Gene expression of TAG synthesis enzymes (ApoB100, MTTP, GPAT1 and GPAT4) was not differentially altered in TLR4/LDLr KO and LDLrKO mice. On the other hand, TLR4 deficiency enhanced the expression of several enzymes involved in the oxidation of fatty acids, as follows: ACOX, CPT-1, MTPa, MTBb, PBE and 3-ketoacyl-CoA thiolase. Acyl-carnitine plasma profile showed an increase in C0 and C2 concentration in TLR4/LDLr KO group, corroborating the hypothesis of increased fat oxidation. Our results indicate that TLR4 may have an important role in the onset of steatosis, once its depletion enhances fatty acid oxidation in the liver of mice, preventing triglyceride accumulation.
Keywords: Toll like receptor 4; Non-alcoholic fatty liver disease; Fatty acid oxidation; LDL receptor knockout; High-fat diet; Acyl-carnitine profile;

Evaluating computational models of cholesterol metabolism by Yared Paalvast; Jan Albert Kuivenhoven; Albert K. Groen (1360-1376).
Regulation of cholesterol homeostasis has been studied extensively during the last decades. Many of the metabolic pathways involved have been discovered. Yet important gaps in our knowledge remain. For example, knowledge on intracellular cholesterol traffic and its relation to the regulation of cholesterol synthesis and plasma cholesterol levels is incomplete. One way of addressing the remaining questions is by making use of computational models. Here, we critically evaluate existing computational models of cholesterol metabolism making use of ordinary differential equations and addressed whether they used assumptions and make predictions in line with current knowledge on cholesterol homeostasis. Having studied the results described by the authors, we have also tested their models. This was done primarily by testing the effect of statin treatment in each model. Ten out of eleven models tested have made assumptions in line with current knowledge of cholesterol metabolism. Three out of the ten remaining models made correct predictions, i.e. predicting a decrease in plasma total and LDL cholesterol or increased uptake of LDL upon treatment upon the use of statins.In conclusion, few models on cholesterol metabolism are able to pass a functional test. Apparently most models have not undergone the critical iterative systems biology cycle of validation. We expect modeling of cholesterol metabolism to go through many more model topologies and iterative cycles and welcome the increased understanding of cholesterol metabolism these are likely to bring.
Keywords: Mathematical modeling; Apolipoprotein; Atherosclerosis; Reverse cholesterol transport; Systems biology;

A conserved π–cation and an electrostatic bridge are essential for 11R-lipoxygenase catalysis and structural stability by Priit Eek; Mari-Ann Piht; Margus Rätsep; Arvi Freiberg; Ivar Järving; Nigulas Samel (1377-1382).
Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that consist of a regulatory calcium- and membrane-binding PLAT (polycystin-1, lipoxygenase, α-toxin) domain and a catalytic domain. In a previous study, the crystal structure of an 11R-LOX revealed a conserved π–cation bridge connecting these two domains which could mediate the regulatory effect of the PLAT domain to the active site. Here we analyzed the role of residues Trp107 and Lys172 that constitute the π–cation bridge in 11R-LOX along with Arg106 and Asp173—a potential salt bridge, which could also contribute to the inter-domain communication. According to our kinetic assays and protein unfolding experiments conducted using differential scanning fluorimetry and circular dichroism spectroscopy, mutants with a disrupted link display diminished catalytic activity alongside reduced stability of the protein fold. The results demonstrate that both these bridges contribute to the two-domain interface, and are important for proper enzyme activation.
Keywords: Lipoxygenase; Allosteric regulation; Domain interaction; PLAT domain; Arachidonic acid; Thermal denaturation;

Signaling network of lipids as a comprehensive scaffold for omics data integration in sputum of COPD patients by Sadegh Azimzadeh; Mehdi Mirzaie; Mohieddin Jafari; Hossein Mehrani; Parvin Shariati; Mahvash Khodabandeh (1383-1393).
Chronic obstructive pulmonary disease (COPD) is a heterogeneous and progressive inflammatory condition that has been linked to the dysregulation of many metabolic pathways including lipid biosynthesis. How lipid metabolism could affect disease progression in smokers with COPD remains unclear. We cross-examined the transcriptomics, proteomics, metabolomics, and phenomics data available on the public domain to elucidate the mechanisms by which lipid metabolism is perturbed in COPD. We reconstructed a sputum lipid COPD (SpLiCO) signaling network utilizing active/inactive, and functional/dysfunctional lipid-mediated signaling pathways to explore how lipid-metabolism could promote COPD pathogenesis in smokers. SpLiCO was further utilized to investigate signal amplifiers, distributers, propagators, feed-forward and/or -back loops that link COPD disease severity and hypoxia to disruption in the metabolism of sphingolipids, fatty acids and energy. Also, hypergraph analysis and calculations for dependency of molecules identified several important nodes in the network with modular regulatory and signal distribution activities. Our systems-based analyses indicate that arachidonic acid is a critical and early signal distributer that is upregulated by the sphingolipid signaling pathway in COPD, while hypoxia plays a critical role in the elevated dependency to glucose as a major energy source. Integration of SpLiCo and clinical data shows a strong association between hypoxia and the upregulation of sphingolipids in smokers with emphysema, vascular disease, hypertension and those with increased risk of lung cancer.
Keywords: COPD; Chronic obstructive pulmonary disease; Lipid; Data integration; Multi-omics; Multi-layer; Network analysis;

Phytol is lethal for Amacr-deficient mice by Eija M. Selkälä; Remya R. Nair; Werner Schmitz; Ari-Pekka Kvist; Myriam Baes; J. Kalervo Hiltunen; Kaija J. Autio (1394-1405).
α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of the 25-methyl group in C27-intermediates in bile acid synthesis and in methyl-branched fatty acids such as pristanic acid, a metabolite derived from phytol. Consequently, patients with Amacr deficiency accumulate C27-bile acid intermediates, pristanic and phytanic acid and display sensorimotor neuropathy, seizures and relapsing encephalopathy. In contrast to humans, Amacr-deficient mice are clinically symptomless on a standard laboratory diet, but failed to thrive when fed phytol-enriched chow. In this study, the effect and the mechanisms behind the development of the phytol-feeding associated disease state in Amacr-deficient mice were investigated. All Amacr −/− mice died within 36 weeks on a phytol diet, while wild-type mice survived. Liver failure was the main cause of death accompanied by kidney and brain abnormalities. Histological analysis of liver showed inflammation, fibrotic and necrotic changes, Kupffer cell proliferation and fatty changes in hepatocytes, and serum analysis confirmed the hepatic disease. Pristanic and phytanic acids accumulated in livers of Amacr −/− mice after a phytol diet. Microarray analysis also revealed changes in the expression levels of numerous genes in wild-type mouse livers after two weeks of the phytol diet compared to a control diet. This indicates that detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways at the molecular level and Amacr −/− mice are not able to respond adequately. Phytol causes primary failure in liver leading to death of Amacr −/− mice thus emphasizing the indispensable role of Amacr in detoxification of α-methyl-branched fatty acids.Display Omitted
Keywords: Alpha-methylacyl-CoA racemase; Phytol; Peroxisome; Refsum disease; Knock-out mouse model;

Short branched-chain C6 carboxylic acids result in increased growth, novel ‘unnatural’ fatty acids and increased membrane fluidity in a Listeria monocytogenes branched-chain fatty acid-deficient mutant by Suranjana Sen; Sirisha Sirobhushanam; Michael P. Hantak; Peter Lawrence; J. Thomas Brenna; Craig Gatto; Brian J. Wilkinson (1406-1415).
Listeria monocytogenes is a psychrotolerant food borne pathogen, responsible for the high fatality disease listeriosis, and expensive food product recalls. Branched-chain fatty acids (BCFAs) of the membrane play a critical role in providing appropriate membrane fluidity and optimum membrane biophysics. The fatty acid composition of a BCFA-deficient mutant is characterized by high amounts of straight-chain fatty acids and even-numbered iso fatty acids, in contrast to the parent strain where odd-numbered anteiso fatty acids predominate. The presence of 2-methylbutyrate (C5) stimulated growth of the mutant at 37 °C and restored growth at 10 °C along with the content of odd-numbered anteiso fatty acids. The C6 branched-chain carboxylic acids 2-ethylbutyrate and 2-methylpentanoate also stimulated growth to a similar extent as 2-methylbutyrate. However, 3-methylpentanoate was ineffective in rescuing growth. 2-Ethylbutyrate and 2-methylpentanoate led to novel major fatty acids in the lipid profile of the membrane that were identified as 12-ethyltetradecanoic acid and 12-methylpentadecanoic acid respectively. Membrane anisotropy studies indicated that growth of strain MOR401 in the presence of these precursors increased its membrane fluidity to levels of the wild type. Cells supplemented with 2-methylpentanoate or 2-ethylbutyrate at 10 °C shortened the chain length of novel fatty acids, thus showing homeoviscous adaptation. These experiments use the mutant as a tool to modulate the membrane fatty acid compositions through synthetic precursor supplementation, and show how existing enzymes in L. monocytogenes adapt to exhibit non-native activity yielding unique ‘unnatural’ fatty acid molecules, which nevertheless possess the correct biophysical properties for proper membrane function in the BCFA-deficient mutant.
Keywords: Branched-chain fatty acids; Branched-chain carboxylic acids; Novel fatty acids; Membrane biophysical properties; Fatty acid composition; Membrane fluidity;