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BBA - Molecular and Cell Biology of Lipids (v.1801, #5)

Editorial Board (pp. i).

Evidence for proteolytic processing and stimulated organelle redistribution of iPLA₂β by Haowei Song; Shunzhong Bao; Xiaoyong Lei; Chun Jin; Sheng Zhang; John Turk; Sasanka Ramanadham (pp. 547-558).

Over the past decade, important roles for the 84–88kDa Group VIA Ca²⁺-independent phospholipase A₂ (iPLA₂β) in various organs have been described. We demonstrated that iPLA₂β participates in insulin secretion, insulinoma cells and native pancreatic islets express full-length and truncated isoforms of iPLA₂β, and certain stimuli promote perinuclear localization of iPLA₂β. To gain a better understanding of its mobilization, iPLA₂β was expressed in INS-1 cells as a fusion protein with EGFP, enabling detection of subcellular localization of iPLA₂β by monitoring EGFP fluorescence. Cells stably-transfected with fusion protein expressed nearly 5-fold higher catalytic iPLA₂β activity than control cells transfected with EGFP cDNA alone, indicating that co-expression of EGFP does not interfere with manifestation of iPLA₂β activity. Dual fluorescence monitoring of EGFP and organelle Trackers combined with immunoblotting analyses revealed expression of truncated iPLA₂β isoforms in separate subcellular organelles. Exposure to secretagogues and induction of ER stress are known to activate iPLA₂β in β-cells and we find here that these stimuli promote differential localization of iPLA₂β in subcellular organelles. Further, mass spectrometric analyses identified iPLA₂β variants from which N-terminal residues were removed. Collectively, these findings provide evidence for endogenous proteolytic processing of iPLA₂β and redistribution of iPLA₂β variants in subcellular compartments. It might be proposed that in vivo processing of iPLA₂β facilitates its participation in multiple biological processes.

Keywords: Golgi; ER; Mitochondria; Fusion protein; Truncation; Mass spectrometry

HIV-protease inhibitors suppress skeletal muscle fatty acid oxidation by reducing CD36 and CPT1 fatty acid transporters by Scott R. Richmond; Michael J. Carper; Xiaoyong Lei; Sheng Zhang; Kevin E. Yarasheski; Sasanka Ramanadham (pp. 559-566).

Infection with human immunodeficiency virus (HIV) and treatment with HIV-protease inhibitor (PI)-based highly active antiretroviral therapies (HAART) is associated with dysregulated fatty acid and lipid metabolism. Enhanced lipolysis, increased circulating fatty acid levels, and hepatic and intramuscular lipid accumulation appear to contribute to insulin resistance in HIV-infected people treated with PI-based HAART. However, it is unclear whether currently prescribed HIV-PIs directly alter skeletal muscle fatty acid transport, oxidation, and storage. We find that ritonavir (r, 5µmol/l) plus 20µmol/l of atazanavir (ATV), lopinavir (LPV), or darunavir (DRV) reduce palmitate oxidation(16–21%) in differentiated C2C12 myotubes. Palmitate oxidation was increased following exposure to high fatty acid media but this effect was blunted when myotubes were pre-exposed to the HIV-PIs. However, LPV/r and DRV/r, but not ATV/r suppressed palmitate uptake into myotubes. We found no effect of the HIV-PIs on FATP1, FATP4, or FABPpm but both CD36/FAT and carnitine palmitoyltransferase 1 (CPT1) were reduced by all three regimens though ATV/r caused only a small decrease in CPT1, relative to LPV/r or DRV/r. In contrast, sterol regulatory element binding protein-1 was increased by all 3 HIV-PIs. These findings suggest that HIV-PIs suppress fatty acid oxidation in murine skeletal muscle cells and that this may be related to decreases in cytosolic- and mitochondrial-associated fatty acid transporters. HIV-PIs may also directly impair fatty acid handling and partitioning in skeletal muscle, and this may contribute to the cluster of metabolic complications that occur in people living with HIV.

Keywords: HIV-PIs; Fatty acid uptake; Fatty acid oxidation; CD36; CPT1; Metabolic dysregulation

Tryptase promotes human monocyte-derived macrophage foam cell formation by suppressing LXRα activation by Pohsheng Yeong; Yanxia Ning; Yali Xu; Xiaobo Li; Lianhua Yin (pp. 567-576).

Accumulated mast cells in atherosclerotic plaques secrete a high level of tryptase that may participate in the pathogenesis of atherosclerotic disease by diverse pathways. However, the role of tryptase in the lipid metabolism of macrophages remains to be defined. In the present study, we found that the addition of tryptase into THP-1-derived macrophages increased both intracellular lipid accumulation and total cholesterol level. Tryptase promoting foam cell formation was also observed by transmission electron microscope. These effects were resisted by APC366, a selective inhibitor of mast cell tryptase. Tryptase dramatically resisted 22RHC induced activation of LXRα protein expression, which can be reversed by SAM-11 (a PAR-2-specific neutralizing antibody) and reduced LXRα, ABCG1, ABCA1 and SREBP-1c mRNA levels and ABCG1 protein level, which were all blocked by APC366. PAR-2 agonist also redeemed 22RHC stimulation to activate LXRα, ABCG1 protein expression, and mRNA levels of LXRα and its target genes in both THP-1-derived macrophages and primary human monocyte-derived macrophages. In primary macrophages that were first transfected with PAR-2 siRNA and then treated with tryptase, both the ABCG1 protein level and mRNA levels of LXRα and ABCG1 were higher than those in the control siRNA-treated cells. Taken together, our data clarified the PAR-2 expression of human macrophages and suggested that tryptase might promote lipid accumulation in macrophages and foam cell formation by suppressing LXRα activation via PAR-2/LXRα/LXRα target genes signaling pathway. This investigation sheds a new light on the role of tryptase in foam cell formation and pathogenesis of atherosclerosis.

Keywords: Tryptase; Macrophage; Foam cell; PAR-2; LXR

Control of cholesterol biosynthesis, uptake and storage in hepatocytes by Cideb by John Zhong Li; Yao Lei; Yue Wang; Yinxin Zhang; Jing Ye; Xiayu Xia; Xianming Pan; Peng Li (pp. 577-586).

Cideb, a member of CIDE family proteins, has emerged as an important regulator in the development of obesity and diabetes by controlling fatty acid synthesis and VLDL secretion in hepatocytes. Here, we investigated the role of Cideb in cholesterol biosynthesis, uptake and storage in the liver by using Cideb-null mice as a model system. Cideb-null mice and wild-type mice were treated with normal diet (ND) or high cholesterol diet (HCD) for one month. The metabolic parameters of cholesterol metabolism and expression profiles of genes in cholesterol biosynthesis and storage were measured. Cideb-null mice had lower levels of plasma cholesterol and LDL when fed with both ND and HCD and increased rate of cholesterol absorption. Furthermore, the liver of Cideb-null mice has lower rates of cholesterol biosynthesis and reduced expression levels of sterol response element-binding protein (SREBP) cleavage-activation protein (SCAP), and lower levels of nuclear form of SREBP2 and its downstream target genes in cholesterol biosynthesis pathway under a normal diet treatment. On the contrary, hepatic cholesterol biosynthesis rate between wild-type and Cideb-null mice was similar after high cholesterol diet treatment. Interestingly, hepatic cholesterol storage in the liver of Cideb-null mice was significantly increased due to its increased LDL receptor (LDLR) and acyl-CoA cholesterol acyltransferase (ACAT) expression. Finally, we observed drastically reduced cholesterol levels in the heart of Cideb-null mice fed with a high cholesterol diet. Overall, our data suggest that Cideb is a novel regulator in controlling cholesterol homeostasis in the liver. Therefore, Cideb could serve as an important therapeutical target for the treatment of atherosclerosis and cardiovascular diseases.

Keywords: Cideb; Liver; Cholesterol synthesis; SCAP; SREBP2; ACAT; LDL receptor

Activation of TM7SF2 promoter by SREBP-2 depends on a new sterol regulatory element, a GC-box, and an inverted CCAAT-box by Gianluca Schiavoni; Anna Maria Bennati; Marilena Castelli; Maria Agnese Della Fazia; Tommaso Beccari; Giuseppe Servillo; Rita Roberti (pp. 587-592).

TM7SF2 gene encodes 3β-hydroxysterol Δ¹⁴-reductase, responsible for the reduction of C14-unsaturated sterols in cholesterol biosynthesis. TM7SF2 gene expression is controlled by cell sterol levels through the SREBP-2. The motifs of TM7SF2 promoter responsible for activation by SREBP-2 have not been characterized. Using electrophoretic mobility shift assays and mutation analysis, we identified a new SRE motif, 60% identical to an inverted SRE-3, able to bind SREBP-2 in vitro and in vivo. Co-transfection of promoter–luciferase reporter constructs in HepG2 cells showed that the binding of SREBP-2 to SRE produced approximately 26-fold promoter activation, whereas mutation of the SRE motif caused a dramatic decrease of transactivation by SREBP-2. The function of additional motifs that bind transcription factors cooperating with SREBP-2 was investigated. An inverted CCAAT-box, that binds nuclear factor Y (NF-Y), cooperates with SREBP-2 in TM7SF2 promoter activation. Deletion of this motif resulted in the loss of promoter induction by sterol starvation in HepG2 cells, as well as a decrease in fold activation by SREBP-2 in co-transfection experiments. Moreover, co-transfection of the promoter with a plasmid expressing dominant negative NF-YA did not permit full activation by SREBP-2. Three GC-boxes (1, 2, 3), known to bind Sp1 transcription factor, were also investigated. The mutagenesis of each of them produced a decrease in SREBP-2-dependent activation, the most powerful being GC-box2. A triple mutagenized promoter construct did not have an additive effect. We conclude that, besides the SRE motif, both the inverted CCAAT-box and GC-box2 are essential for full promoter activation by SREBP-2.

Keywords: Abbreviations; SRE; sterol regulatory element; SREBP-2; SRE-binding protein 2; NF-Y; nuclear factor Y; LDLR; low density lipoprotein receptor; FBS; foetal bovine serum; Lov; lovastatin; 25-OH chol; 25-hydroxycholesterol; LPDS; lipoprotein deficient serum; EMSA; electrophoretic mobility shift assay; ChIP; chromatin immunoprecipitationCholesterol; TM7SF2; 3beta-hydoxysterol delta14-reductase; SREBP-2; Promoter

Altered lipid A structures and polymyxin hypersensitivity of Rhizobium etli mutants lacking the LpxE and LpxF phosphatases by Brian O. Ingram; Christian Sohlenkamp; Otto Geiger; Christian R.H. Raetz (pp. 593-604).

The lipid A of Rhizobium etli, a nitrogen-fixing plant endosymbiont, displays significant structural differences when compared to that of Escherichia coli. An especially striking feature of R. etli lipid A is that it lacks both the 1- and 4′-phosphate groups. The 4′-phosphate moiety of the distal glucosamine unit is replaced with a galacturonic acid residue. The dephosphorylated proximal unit is present as a mixture of the glucosamine hemiacetal and an oxidized 2-aminogluconate derivative. Distinct lipid A phosphatases directed to the 1 or the 4′-positions have been identified previously in extracts of R. etli and Rhizobium leguminosarum. The corresponding structural genes, lpxE and lpxF, respectively, have also been identified. Here, we describe the isolation and characterization of R. etli deletion mutants in each of these phosphatase genes and the construction of a double phosphatase mutant. Mass spectrometry confirmed that the mutant strains completely lacked the wild-type lipid A species and accumulated the expected phosphate-containing derivatives. Moreover, radiochemical analysis revealed that phosphatase activity was absent in membranes prepared from the mutants. Our results indicate that LpxE and LpxF are solely responsible for selectively dephosphorylating the lipid A molecules of R. etli. All the mutant strains showed an increased sensitivity to polymyxin relative to the wild-type. However, despite the presence of altered lipid A species containing one or both phosphate groups, all the phosphatase mutants formed nitrogen-fixing nodules on Phaseolus vulgaris. Therefore, the dephosphorylation of lipid A molecules in R. etli is not required for nodulation but may instead play a role in protecting the bacteria from cationic antimicrobial peptides or other immune responses of plants.

Keywords: Abbreviations; ACP; acyl carrier protein; BCA; bicinchoninic acid; CL; cardiolipin; DEAE-cellulose; diethylaminoethyl cellulose; DMPE; dimethyl PE; EDTA; ethylenediaminetetraacetic acid; ESI; electrospray ionization; Kdo; 3-deoxy-; d; -; manno; -oct-2-ulosonic acid; LPS; lipopolysaccharide; MES; 2-(; N; -morpholino)-ethanesulfonic acid; MS; mass spectrometry; OL; ornithine-containing lipid; PBS; phosphate-buffered saline; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; SL; sulfolipid; TLC; thin-layer chromatography; TLR-4; Toll-like receptor-4Gram-negative bacteria; Outer membranes; Rhizobium etli; Lipopolysaccharide; Lipid A phosphatases; LpxE; LpxF

Apolipoprotein A-V associates with intrahepatic lipid droplets and influences triglyceride accumulation by Xiao Shu; Lisa Nelbach; Robert O. Ryan; Trudy M. Forte (pp. 605-608).

Apolipoprotein A-V (apoA-V), secreted solely by the liver, is a low abundance protein that strongly influences plasma triglyceride (TG) levels. In vitro, in transfected hepatoma cell lines apoA-V is largely retained within the cell in association with cytosolic lipid droplets (LD). To evaluate if this is true in vivo, in the present study the amount of apoA-V in the plasma compartment versus liver tissue was determined in APOA5 transgenic (Tg) mice. The majority of total apoA-V (∼80%) was in the plasma compartment. Injection of APOA5 Tg mice with heparin increased plasma apoA-V protein levels by ∼25% indicating the existence of a heparin-releasable pool. Intrahepatic apoA-V was associated with LD isolated from livers of wild type (WT) and APOA5 Tg mice. Furthermore, livers from APOA5 Tg mice contained significantly higher amounts of TG than livers from WT or apoa5 knockout mice suggesting that apoA-V influences intrahepatic TG levels.

Keywords: Apolipoprotein A-V; APOA5; transgenic mouse; Liver; Lipid droplet; Triglyceride

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BBA - Molecular and Cell Biology of Lipids (v.1801, #5)