BBA - Molecular and Cell Biology of Lipids (v.1821, #12)

The role of acid sphingomyelinase and caspase 5 in hypoxia-induced HuR cleavage and subsequent apoptosis in hepatocytes by Qun Zhu; Lianku Lin; Qi Cheng; Qing Xu; Jingmei Zhang; Stephen Tomlinson; Junfei Jin; Xiaoping Chen; Songqing He (1453-1461).
A previous data showed that the hypoxia mimetic compound CoCl2 induced cleavage of HuR and subsequent apoptosis in human oral cancer cells. We also previously demonstrated that exposure of NT-2 human neuronal precursor cells to hypoxia resulted in changes in sphingolipid levels and apoptosis. Since it is known that CoCl2 induces cleavage of HuR, we investigated whether there is a link between HuR cleavage and the observed sphingolipid changes in cells exposed to hypoxia, and whether this link is associated with the induction of apoptosis. Exposure of hepatocytes to direct hypoxia by means of a hypoxic chamber resulted in acid sphingomyelinase activation and ceramide elevation. The elevation in ceramide levels was associated with activation of caspase 5 and the subsequent cleavage of HuR and apoptotic cell death. These data raise the possibility that acid sphingomyelinase and caspase 5 are each potential targets for treating hypoxia (ischemia)-induced liver injury.► Hypoxia led acid sphingomyelinase activation and ceramide increase in hepatocytes. ► Ceramide led to activation of caspase 5 in hepatocyte exposure to hypoxia. ► Activation of caspase 5 led to HuR cleavage that resulted in cell apoptosis. ► Acid sphingomyelinase and caspase 5 may be targets for liver damage by hypoxia.
Keywords: Acid sphingomyelinase; Ceramide; Caspase 5; HuR cleavage; Apoptosis;

Although triacylglycerol (TAG) stores play a critical role in organisms, mechanisms underlying TAG synthesis are poorly understood in invertebrates. In mammals, the synthesis of glycerolipids, including TAG, diacylglycerol (DAG) and phospholipids (PL), occurs predominantly by the glycerol-3-phosphate (G3P) pathway in most cell types, except for in enterocytes. In these cells, the monoacylglycerol (MAG) pathway accounts for the majority of glycerolipid production. The insect Rhodnius prolixus, a vector of Chagas' disease, exhibits a high capacity to produce glycerolipids in the midgut after a blood meal, providing substrates that are transferred to other organs, such as the fat body, which is specialized in TAG production and storage. In this report, the genes required for TAG synthesis were identified in the R. prolixus genome. The genomic data indicated that TAG is synthesized by the G3P pathway, which is the sole pathway for TAG synthesis in this organism. Furthermore, transcription of both the RpGpat and RpDgat genes were upregulated in a diverse number of organs at moments of highest lipid production. In the midgut and fat body, in vitro synthesis of glycerolipids required G3P, but not MAG, as the initial substrate. These results indicate that the G3P pathway is the only route for TAG synthesis in R. prolixus, and its regulation at the transcriptional level can be a determinant of glycerolipid synthesis and TAG formation in insect organs.► Genes required for triacylglycerol biosynthesis were identified. ► Enzymes of glycerol-3-phosphate pathway are transcriptionally regulated by feeding. ► Triacylglycerol biosynthesis depends on glycerol-3-phosphate as an initial substrate.
Keywords: Triacylglycerol synthesis; GPAT; AGPAT; DGAT; Phosphatidate phosphohydrolase;

ORP10, a cholesterol binding protein associated with microtubules, regulates apolipoprotein B-100 secretion by Eija Nissilä; Yuki Ohsaki; Marion Weber-Boyvat; Julia Perttilä; Elina Ikonen; Vesa M. Olkkonen (1472-1484).
ORP10/OSBPL10 is a member of the oxysterol-binding protein family, and genetic variation in OSBPL10 is associated with dyslipidemias and peripheral artery disease. In this study we investigated the ligand binding properties of ORP10 in vitro as well as its localization and function in human HuH7 hepatocytes. The pleckstrin homology (PH) domain of ORP10 selectively interacts with phosphatidylinositol-4-phosphate, while the C-terminal ligand binding domain binds cholesterol and several acidic phospholipids. Full-length ORP10 decorates microtubules (MT), while the ORP10 N-terminal fragment (aa 1–318) localizes at Golgi membranes. Removal of the C-terminal aa 712–764 of ORP10 containing a predicted coiled-coil segment abolishes the MT association, but allows partial Golgi targeting. A PH domain-GFP fusion protein is distributed mainly in the cytosol and the plasma membrane, indicating that the Golgi affinity of ORP10 involves other determinants in addition to the PH domain. HuH7 cells expressing ORP10-specific shRNA display increased accumulation of apolipoprotein B-100 (apoB-100), but not of albumin, in culture medium, and contain reduced levels of intracellular apoB-100. Pulse-chase analysis of cellular [35S]apoB-100 demonstrates enhanced apoB-100 secretion by cells expressing ORP10-specific shRNA. The apoB-100 secretion phenotype is replicated in HepG2 cells transduced with the ORP10 shRNA lentiviruses. As a conclusion, the present study dissects the determinants of ORP10 association with MT and the Golgi complex and provides evidence for a specific role of this protein in β-lipoprotein secretion by human hepatocytes.► The ORP10 PH domain binds PtdIns(4)P, while its C-terminal domain binds cholesterol. ► ORP10 associates with microtubules, but its N-terminal domain targets the Golgi. ► Silencing ORP10 in hepatic cells results in enhanced apoB-100 secretion.
Keywords: apoB-100; Golgi; Microtubule; ORP10; Oxysterol-binding protein;

Advanced glycated albumin impairs HDL anti-inflammatory activity and primes macrophages for inflammatory response that reduces reverse cholesterol transport by Ligia S. Okuda; Gabriela Castilho; Debora D.F.M. Rocco; Edna R. Nakandakare; Sergio Catanozi; Marisa Passarelli (1485-1492).
Objective: We investigated the effect of advanced glycated albumin (AGE-albumin) on macrophage sensitivity to inflammation elicited by S100B calgranulin and lipopolysaccharide (LPS) and the mechanism by which HDL modulates this response. We also measured the influence of the culture medium, isolated from macrophages treated with AGE-albumin, on reverse cholesterol transport (RCT). Methods and results: Macrophages were incubated with control (C) or AGE-albumin in the presence or absence of HDL, followed by incubations with S100B or LPS. Also, culture medium obtained from cells treated with C- or AGE-albumin, following S100B or LPS stimulation was utilized to treat naive macrophages in order to evaluate cholesterol efflux and the expression of HDL receptors. In comparison with C-albumin, AGE-albumin, promoted a greater secretion of cytokines after stimulation with S100B or LPS. A greater amount of cytokines was also produced by macrophages treated with AGE-albumin even in the presence of HDL. Cytokine-enriched medium, drawn from incubations with AGE-albumin and S100B or LPS impaired the cholesterol efflux mediated by apoA-I (23% and 37%, respectively), HDL2 (43% and 47%, respectively) and HDL3 (20% and 8.5%, respectively) and reduced ABCA-1 protein level (16% and 26%, respectively). Conclusions: AGE-albumin primes macrophages for an inflammatory response impairing the RCT. Moreover, AGE-albumin abrogates the anti-inflammatory role of HDL, which may aggravate the development of atherosclerosis in DM.► AGE-albumin primes macrophages for inflammation induced by S100B-calgranulin or LPS; ► AGE-albumin impairs the anti-inflammatory property of HDL; ► Inflammation induced by AGE-albumin reduces ABCA-1 and cell cholesterol efflux.
Keywords: Diabetes mellitus; Advanced glycated albumin; Inflammation; Atherosclerosis; Reverse cholesterol transport;

The cholesterol content of the erythrocyte membrane is an important determinant of phosphatidylserine exposure by Rob van Zwieten; Andrea E. Bochem; Petra M. Hilarius; Robin van Bruggen; Ferry Bergkamp; G. Kees Hovingh; Arthur J. Verhoeven (1493-1500).
Maintenance of the asymmetric distribution of phospholipids across the plasma membrane is a prerequisite for the survival of erythrocytes. Various stimuli have been shown to induce scrambling of phospholipids and thereby exposure of phosphatidylserine (PS). In two types of patients, both with aberrant plasma cholesterol levels, we observed an aberrant PS exposure in erythrocytes upon stimulation. We investigated the effect of high and low levels of cholesterol on the ATP-dependent flippase, which maintains phospholipid asymmetry, and the ATP-independent scrambling activity, which breaks down phospholipid asymmetry. We analyzed erythrocytes of a patient with spur cell anemia, characterized by elevated plasma cholesterol, and the erythrocytes of Tangier disease patients with very low levels of plasma cholesterol. In normal erythrocytes, loaded with cholesterol or depleted of cholesterol in vitro, the same analyses were performed. Changes in the cholesterol/phospholipid ratio of erythrocytes had marked effects on PS exposure upon cell activation. Excess cholesterol profoundly inhibited PS exposure, whereas cholesterol depletion led to increased PS exposure. The activity of the ATP‐dependent flippase was not changed, suggesting a major influence of cholesterol on the outward translocation of PS. The effects of cholesterol were not accompanied by eminent changes in cytoskeletal and membrane proteins. These findings emphasize the importance of cholesterol exchange between circulating plasma and the erythrocyte membrane as determinant for phosphatidylserine exposure in erythrocytes.► Changes in the cholesterol/phospholipid ratio of erythrocytes has marked effects on PS exposure upon cell activation. ► This was studied in patients and in normal erythrocytes, loaded with cholesterol or depleted of cholesterol in vitro. ► Excess cholesterol profoundly inhibits PS exposure, whereas cholesterol depletion leads to increased PS exposure. ► The activity of the ATP‐dependent flippase is not changed, suggesting the scramblase activity to be influenced by cholesterol. ► Cholesterol exchange between plasma and the erythrocyte membrane is a determinant for phosphatidylserine exposure.
Keywords: Spur cell anemia; Tangier disease; Cholesterol; Flippase; PS exposure; Phospholipid scrambling;

Phosphatidylethanolamine is externalized at the surface of microparticles by Michael C. Larson; Jeffrey E. Woodliff; Cheryl A. Hillery; Tyce J. Kearl; Ming Zhao (1501-1507).
Microparticles (MPs) are membrane-bound vesicles shed normally or as a result of various (pathological) stimuli. MPs contain a wealth of bio-active macromolecules. Aminophospholipid phosphatidylserine (PS) is present on the surface of many MPs. As PS and phosphatidylethanolamine (PE) are related, yet distinct aminophospholipids, the purpose of this study was to systematically and directly assess PE exposure on MPs. We examined MPs from various human cellular sources (human breast cancer, endothelial, red and white blood cells) by flow cytometry using a PE-specific probe, duramycin, and two PS-specific probes, annexin V and lactadherin. PS and PE exposure percentage was comparable on vascular and blood cell-derived MPs (80–90% of MP-gated events). However, the percentage of malignant breast cancer MPs exposing PE (~ 90%) was significantly higher than PS (~ 50%). Thus, while PS and PE exposure can result from a general loss of membrane asymmetry, there may also be distinct mechanisms of PE and PS exposure on MPs that vary by cellular source.► Phosphatidylethanolamine (PE) is detectable using duramycin at the surface of microparticles (MPs). ► Surface PE was systematically characterized in MPs from different sources. ► Different types of MPs consistently express PE ± PS. ► Externalized PE is a reliable molecular marker for detecting MPs. ► Coagulation time is prolonged by blocking MP surface PE.
Keywords: Microparticles; Microvesicles; Phosphatidylethanolamine; Duramycin; Membrane asymmetry;

Cyclooxygenases (COX) and 8R-dioxygenase (8R-DOX) activities of linoleate diol synthases (LDS) are homologous heme-dependent enzymes that oxygenate fatty acids by a tyrosyl radical-mediated hydrogen abstraction and antarafacial insertion of O2. Soybean lipoxygenase-1 (sLOX-1) contains non-heme iron and oxidizes 18:2n − 6 with a large deuterium kinetic isotope effect (D-KIE). The aim of the present work was to obtain further mechanistic insight into the action of these enzymes by using a series of n − 6 and n − 9 fatty acids and by analysis of D-KIE. COX-1 oxidized C20 and C18 fatty acids in the following order of rates: 20:2n − 6 > 20:1n − 6 > 20:3n − 9 > 20:1n − 9 and 18:3n − 3 ≥ 18:2n − 6 > 18:1n − 6. 18:2n − 6 and its geometrical isomer (9E,12Z)18:2 were both mainly oxygenated at C-9 by COX-1, but the 9Z,12E isomer was mostly oxygenated at C-13. A cis-configured double bond in the n − 6 position therefore seems important for substrate positioning. 8R-DOX oxidized (9Z,12E)18:2 at C-8 in analogy with 18:2n − 6, but the 9E,12Z isomer was mainly subject to hydrogen abstraction at C-11 and oxygen insertion at C-9 by 8R-DOX of 5,8-LDS. sLOX-1 and 13R-MnLOX oxidized [11S-2H]18:2n − 6 with similar D-KIE (~ 53), which implies that the catalytic metals did not alter the D-KIE. Oxygenation of 18:2n − 6 by COX-1 and COX-2 took place with a D-KIE of 3–5 as probed by incubations of [11,11-2H2]- and [11S-2H]18:2n − 6. In contrast, the more energetically demanding hydrogen abstractions of the allylic carbons of 20:1n − 6 by COX-1 and 18:1n − 9 by 8R-DOX were both accompanied by large D-KIE (> 20).► Deuterium kinetic isotope effect (D-KIE) larger than 20 can be explained by hydrogen tunneling. ► Fe- and Mn-LOX oxidize bis-allylic carbons with large D-KIE (> 50) and COX-1 with small (D-KIE < 4). ► COX-1 and 8R-DOX oxidize allylic carbons efficiently, but with large D-KIE (> 20). ► Hydrogen tunneling contributes to reaction rates of energetically demanding enzymatic reactions.
Keywords: Animal heme peroxidase; Chiral phase HPLC; Fatty acid oxygenation; Kinetic isotope effect; Mass spectrometry; Oxygenation mechanism;