BBA - Molecular and Cell Biology of Lipids (v.1861, #9PA)

Inflammatory intracellular pathways activated by electronegative LDL in monocytes by Montserrat Estruch; Jose Luis Sanchez-Quesada; Jordi Ordoñez-Llanos; Sonia Benitez (963-969).
Electronegative LDL (LDL(−)) is a plasma LDL subfraction that induces cytokine release in monocytes through toll-like receptor 4 (TLR4) activation. However, the intracellular pathways induced by LDL(−) downstream TLR4 activation are unknown. We aimed to identify the pathways activated by LDL(−) leading to cytokine release in monocytes.We determined LDL(−)-induced activation of several intracellular kinases in protein extracts from monocytes using a multikinase ELISA array. LDL(−) induced higher p38 mitogen-activated protein kinase (MAPK) phosphorylation than native LDL. This was corroborated by a specific cell-based assay and it was dependent on TLR4 and phosphoinositide 3-kinase (PI3k)/Akt pathway. P38 MAPK activation was involved in cytokine release promoted by LDL(−). A specific ELISA showed that LDL(−) activated cAMP response-element binding (CREB) in a p38 MAPK dependent manner. P38 MAPK was also involved in the nuclear factor kappa-B (NF-kB) and activating protein-1 (AP-1) activation by LDL(−). We found that NF-kB, AP-1 and CREB inhibitors decreased LDL(−)-induced cytokine release, mainly on MCP1, IL6 and IL10 release, respectively.LDL(−) promotes p38 MAPK phosphorylation through TLR4 and PI3k/Akt pathways. Phosphorylation of p38 MAPK is involved in NF-kB, AP-1 and CREB activation, leading to LDL(−)-induced cytokine release in monocytes.Display Omitted
Keywords: Electronegative LDL; Cell signaling; Cytokines; MAP kinases; Transcription factors; Toll-like receptor (TLR);

Active site mapping of Loxosceles phospholipases D: Biochemical and biological features by L. Vuitika; D. Chaves-Moreira; I. Caruso; M.A. Lima; F.H. Matsubara; M.T. Murakami; H.K. Takahashi; M.S. Toledo; M.A. Coronado; H.B. Nader; A. Senff-Ribeiro; O.M. Chaim; R.K. Arni; S.S. Veiga (970-979).
Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.
Keywords: Brown spider venom; Phospholipase D; Site-directed mutagenesis; Activity modulation;

Construction of a cyanobacterium synthesizing cyclopropane fatty acids by Shuntaro Machida; Yoshihiro Shiraiwa; Iwane Suzuki (980-987).
Microalgae have received much attention as a next-generation source of biomass energy. However, most of the fatty acids (FAs) from microalgae are multiply unsaturated; thus, the biofuels derived from them are fluid, but vulnerable to oxidation. In this study, we attempted to synthesize cyclopropane FAs in the cyanobacterium Synechocystis sp. PCC 6803 by expressing the cfa gene for cyclopropane FA synthase from Escherichia coli with the aim of producing FAs that are fluid and stable in response to oxidization. We successfully synthesized cyclopropane FAs in Synechocystis with a yield of ~ 30% of total FAs. Growth of the transformants was altered, particularly at low temperatures, but photosynthesis and respiration were not significantly affected. C16:1∆ 9 synthesis in the desA /desD strain by expression of the desC2 gene for sn-2 specific ∆ 9 desaturase positively affected growth at low temperatures via promotion of various cellular processes, with the exceptions of photosynthesis and respiration. Estimation of the apparent activities of desaturases suggested that some acyl-lipid desaturases might recognize the lipid side chain.
Keywords: Biofuel; Microalgae; Cyclopropane fatty acid; Acyl-lipid desaturase; Synechocystis sp. PCC 6803;

SND1 overexpression deregulates cholesterol homeostasis in hepatocellular carcinoma by Hiart Navarro-Imaz; Yuri Rueda; Olatz Fresnedo (988-996).
SND1 is a multifunctional protein participating, among others, in gene transcription and mRNA metabolism. SND1 is overexpressed in cancer cells and promotes viability and tumourigenicity of hepatocellular carcinoma cells. This study shows that cholesterol synthesis is increased in SND1-overexpressing hepatoma cells. Neither newly synthesised nor extracellularly supplied cholesterol are able to suppress this increase; however, inhibition of cholesterol esterification reverted the activated state of sterol-regulatory element-binding protein 2 (SREBP2) and cholesterogenesis. These results highlight SND1 as a potential regulator of cellular cholesterol distribution and homeostasis in hepatoma cells, and support the rationale for the therapeutic use of molecules that influence cholesterol management when SND1 is overexpressed.
Keywords: SND1; Hepatoma; Cholesterol; SREBP2; Cancer metabolism;

Whole-body DHA synthesis-secretion kinetics from plasma eicosapentaenoic acid and alpha-linolenic acid in the free-living rat by Adam H. Metherel; Anthony F. Domenichiello; Alex P. Kitson; Kathryn E. Hopperton; Richard P. Bazinet (997-1004).
Whole body docosahexaenoic acid (DHA, 22:6n-3) synthesis from α-linolenic acid (ALA, 18:3n-3) is considered to be very low, however, the daily synthesis-secretion of DHA may be sufficient to supply the adult brain. The current study aims to assess whether whole body DHA synthesis-secretion kinetics are different when comparing plasma ALA versus eicosapentaenoic acid (EPA, 20:5n-3) as the precursor. Male Long Evans rats (n = 6) were fed a 2% ALA in total fat diet for eight weeks, followed by surgery to implant a catheter into each of the jugular vein and carotid artery and 3 h of steady-state infusion with a known amount of 2H-ALA and 13C-eicosapentaenoic acid (EPA, 20:5n3). Blood samples were collected at thirty-minute intervals and plasma enrichment of 2H- and 13C EPA, n − 3 docosapentaenoic acid (DPAn-3, 22:5n-3) and DHA were determined for assessment of synthesis-secretion kinetic parameters. Results indicate a 13-fold higher synthesis-secretion coefficient for DHA from EPA as compared to ALA. However, after correcting for the 6.6 fold higher endogenous plasma ALA concentration, no significant differences in daily synthesis-secretion (nmol/day) of DHA (97.6 ± 28.2 and 172 ± 62), DPAn-3 (853 ± 279 and 1139 ± 484) or EPA (1587 ± 592 and 1628 ± 366) were observed from plasma unesterified ALA and EPA sources, respectively. These results suggest that typical diets which are significantly higher in ALA compared to EPA yield similar daily DHA synthesis-secretion despite a significantly higher synthesis-secretion coefficient from EPA.
Keywords: Omega-3; Polyunsaturated fatty acid; Metabolism; Lipids; Nutrition; Synthesis-secretion;

Knockdown of triglyceride synthesis does not enhance palmitate lipotoxicity or prevent oleate-mediated rescue in rat hepatocytes by Alexandra K. Leamy; Clinton M. Hasenour; Robert A. Egnatchik; Irina A. Trenary; Cong-Hui Yao; Gary J. Patti; Masakazu Shiota; Jamey D. Young (1005-1014).
Experiments in a variety of cell types, including hepatocytes, consistently demonstrate the acutely lipotoxic effects of saturated fatty acids, such as palmitate (PA), but not unsaturated fatty acids, such as oleate (OA). PA + OA co-treatment fully prevents PA lipotoxicity through mechanisms that are not well defined but which have been previously attributed to more efficient esterification and sequestration of PA into triglycerides (TGs) when OA is abundant. However, this hypothesis has never been directly tested by experimentally modulating the relative partitioning of PA/OA between TGs and other lipid fates in hepatocytes. In this study, we found that addition of OA to PA-treated hepatocytes enhanced TG synthesis, reduced total PA uptake and PA lipid incorporation, decreased phospholipid saturation and rescued PA-induced ER stress and lipoapoptosis. Knockdown of diacylglycerol acyltransferase (DGAT), the rate-limiting step in TG synthesis, significantly reduced TG accumulation without impairing OA-mediated rescue of PA lipotoxicity. In both wild-type and DGAT-knockdown hepatocytes, OA co-treatment significantly reduced PA lipid incorporation and overall phospholipid saturation compared to PA-treated hepatocytes. These data indicate that OA's protective effects do not require increased conversion of PA into inert TGs, but instead may be due to OA's ability to compete against PA for cellular uptake and/or esterification and, thereby, normalize the composition of cellular lipids in the presence of a toxic PA load.
Keywords: Saturated fatty acids; Lipoapoptosis; Endoplasmic reticulum stress; Phospholipid metabolism; Triglycerides; Diacylglycerol acyltransferase;

Thermal stability of human plasma electronegative low-density lipoprotein: A paradoxical behavior of low-density lipoprotein aggregation by Anna Rull; Shobini Jayaraman; Donald L. Gantz; Andrea Rivas-Urbina; Montserrat Pérez-Cuellar; Jordi Ordóñez-Llanos; Jose Luis Sánchez-Quesada; Olga Gursky (1015-1024).
Low-density lipoprotein (LDL) aggregation is central in triggering atherogenesis. A minor fraction of electronegative plasma LDL, termed LDL(−), plays a special role in atherogenesis. To better understand this role, we analyzed the kinetics of aggregation, fusion and disintegration of human LDL and its fractions, LDL(+) and LDL(−). Thermal denaturation of LDL was monitored by spectroscopy and electron microscopy. Initially, LDL(−) aggregated and fused faster than LDL(+), but later the order reversed. Most LDL(+) disintegrated and precipitated upon prolonged heating. In contrast, LDL(−) partially retained lipoprotein morphology and formed soluble aggregates. Biochemical analysis of all fractions showed no significant degradation of major lipids, mild phospholipid oxidation, and an increase in non-esterified fatty acid (NEFA) upon thermal denaturation. The main baseline difference between LDL subfractions was higher content of NEFA in LDL(−). Since NEFA promote lipoprotein fusion, increased NEFA content can explain rapid initial aggregation and fusion of LDL(−) but not its resistance to extensive disintegration. Partial hydrolysis of apoB upon heating was similar in LDL subfractions, suggesting that minor proteins importantly modulate LDL disintegration. Unlike LDL(+), LDL(−) contains small amounts of apoA-I and apoJ. Addition of exogenous apoA-I to LDL(+) hampered lipoprotein aggregation, fusion and precipitation, while depletion of endogenous apoJ had an opposite effect. Therefore, the initial rapid aggregation of LDL(−) is apparently counterbalanced by the stabilizing effects of minor proteins such as apoA-I and apoJ. These results help identify key determinants for LDL aggregation, fusion and coalescence into lipid droplets in vivo.Display Omitted
Keywords: Electronegative LDL; Lipoprotein aggregation, fusion and droplet formation; Thermal denaturation; Atherogenesis; Apolipoprotein A-I; Apolipoprotein J;

Resolution of inflammation is an active process involving a novel category of lipid factors known as specialized pro-resolving lipid mediators, which includes Resolvin D1 (RvD1). While accumulating evidence suggests that RvD1 counteracts proinflammatory signaling and promotes resolution, the specific cellular targets and mechanisms of action of RvD1 remain largely unknown. In the present study, we investigated the role and molecular mechanisms of RvD1 in ischemia/reperfusion (IR)-induced sterile liver inflammation. Male C57BL/6 mice underwent 70% hepatic ischemia for 60 min, followed by reperfusion. RvD1 (5, 10, and 15 μg/kg, i.p.) was administered to the mice 1 h before ischemia and then immediately prior to reperfusion. RvD1 attenuated IR-induced hepatocellular damage and the proinflammatory response. In purified Kupffer cells (KCs) from mice exposed to IR, the levels of M1 marker genes (Nos2a and Cd40) increased, while those of M2 marker genes (Arg1, Cd206, and Mst1r) decreased, demonstrating a proinflammatory shift. RvD1 markedly attenuated these changes. Depletion of KCs by liposome clodronate abrogated the effects of RvD1 on proinflammatory mediators and macrophage polarization. In addition, RvD1 attenuated increases in myeloperoxidase activity and Cxcl1 and Cxcl2 mRNA expression. RvD1 markedly augmented the efferocytic activity of KCs, as indicated by increases in F4/80+  Gr-1+ cells in the liver. However, antagonist pretreatment or gene silencing of the RvD1 receptor, ALX/FPR2, abrogated the anti-inflammatory and pro-resolving actions of RvD1. These data indicate that RvD1 ameliorates IR-induced liver injury, and this protection is associated with enhancement of M2 polarization and efferocytosis via ALX/FPR2 activation.
Keywords: ALX/N-formyl peptide receptor 2 (FPR2); Efferocytosis; Ischemia/reperfusion; Macrophage polarization; Resolution of Inflammation; Resolvin D1;

Mechanisms and metabolic regulation of PPARα activation in Nile tilapia (Oreochromis niloticus) by Li-Jun Ning; An-Yuan He; Jia-Min Li; Dong-Liang Lu; Jian-Gang Jiao; Ling-Yu Li; Dong-Liang Li; Mei-Ling Zhang; Li-Qiao Chen; Zhen-Yu Du (1036-1048).
Although the key metabolic regulatory functions of mammalian peroxisome proliferator-activated receptor α (PPARα) have been thoroughly studied, the molecular mechanisms and metabolic regulation of PPARα activation in fish are less known. In the first part of the present study, Nile tilapia (Nt)PPARα was cloned and identified, and high mRNA expression levels were detected in the brain, liver, and heart. NtPPARα was activated by an agonist (fenofibrate) and by fasting and was verified in primary hepatocytes and living fish by decreased phosphorylation of NtPPARα and/or increased NtPPARα mRNA and protein expression. In the second part of the present work, fenofibrate was fed to fish or fish were fasted for 4 weeks to investigate the metabolic regulatory effects of NtPPARα. A transcriptomic study was also performed. The results indicated that fenofibrate decreased hepatic triglyceride and 18C-series fatty acid contents but increased the catabolic rate of intraperitoneally injected [1-14C] palmitate in vivo, hepatic mitochondrial β-oxidation efficiency, the quantity of cytochrome b DNA, and carnitine palmitoyltransferase-1a mRNA expression. Fenofibrate also increased serum glucose, insulin, and lactate concentrations. Fasting had stronger hypolipidemic and gene regulatory effects than those of fenofibrate. Taken together, we conclude that: 1) liver is one of the main target tissues of the metabolic regulation of NtPPARα activation; 2) dephosphorylation is the basal NtPPARα activation mechanism rather than enhanced mRNA and protein expression; 3) activated NtPPARα has a hypolipidemic effect by increasing activity and the number of hepatic mitochondria; and 4) PPARα activation affects carbohydrate metabolism by altering energy homeostasis among nutrients.
Keywords: Nile tilapia; PPARα activation; Dephosphorylation; Fenofibrate; Fasting; Metabolism;

Mapping a kingdom-specific functional domain of squalene synthase by Kristin B. Linscott; Thomas D. Niehaus; Xun Zhuang; Stephen A. Bell; Joe Chappell (1049-1057).
Squalene synthase catalyzes the first committed step in sterol biosynthesis and consists of both an amino-terminal catalytic domain and a carboxy-terminal domain tethering the enzyme to the ER membrane. While the overall architecture of this enzyme is identical in eukaryotes, it was previously shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implied a unique component of the fungal carboxy-terminal domain was responsible for the complementation phenotype. To identify this motif, we used Saccharomyces cerevisiae with a squalene synthase knockout mutation, and expressed intact and chimeric squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. However, when highly expressed, non-fungal squalene synthases could not complement the yeast mutation and instead led to the accumulation of a toxic intermediate(s) as defined by mutations of genes downstream in the ergosterol pathway. Restoration of the complete complementation phenotype was mapped to a 26-amino acid hinge region linking the catalytic and membrane-spanning domains specific to fungal squalene synthases. Over-expression of the C-terminal domain containing a hinge domain from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Because this hinge region is unique to and highly conserved within each kingdom of life, the data suggests that the hinge domain plays an essential functional role, such as assembly of ergosterol multi-enzyme complexes in fungi.
Keywords: Squalene synthase; Sterol biosynthesis; Genetic complementation; Kingdom-of-life specificity;

The PNPLA-family phospholipases involved in glycerophospholipid homeostasis of HeLa cells by Martin Hermansson; Satu Hänninen; Kati Hokynar; Pentti Somerharju (1058-1065).
Mammalian cells maintain the glycerophospholipid (GPL) compositions of their membranes nearly constant. To achieve this, GPL synthesis and degradation must be coordinated. There is strong evidence that A-type phospholipases (PLAs) are key players in homeostatic degradation of GPLs, but the identities of the PLAs involved have not been established. However, some members of the Patatin-like phospholipase domain-containing proteins (PNPLAs) have been implicated. Accordingly, we knocked down all the PNPLAs significantly expressed in human HeLa cells using RNA interference and then determined whether the turnover of the major glycerophospholipids is affected by using mass spectrometry and metabolic labeling with stable isotope-labeled precursors. Knockdown of PNPLA9, PNPLA6 or PNPLA4 significantly (30–50%) reduced the turnover of phosphatidylcholine, − ethanolamine and –serine. In a notable contrast, turnover of phosphatidylinositol was not significantly affected by the knockdown of any PNPLA. Depletion of PNPLA9 and PNPLA4 also inhibited G0/G1 to S cell cycle progression, which could thus be regulated by GPL turnover. These results strongly suggest that PNPLA9, -6 and -4 play a key role in GPL turnover and homeostasis in human cells. A hypothetical model suggesting how these enzymes could recognize the relative concentration of the different GPLs is proposed.
Keywords: Ca2 +-independent phospholipase A2; Phospholipid; Degradation; Turnover; Lipid metabolism;

Low-density lipoprotein upregulate SR-BI through Sp1 Ser702 phosphorylation in hepatic cells by Fan Yang; Yu Du; Jin Zhang; Zhibo Jiang; Li Wang; Bin Hong (1066-1075).
Scavenger receptor class B type I (SR-BI) is one of the key proteins in the process of reverse cholesterol transport (RCT), and its major function is to uptake high density lipoprotein (HDL) cholesterol from plasma into liver cells. The regulation of SR-BI expression is important for controlling serum lipid content and reducing the risks of cardiovascular diseases. Here we found that SR-BI expression was significantly increased by LDL in vivo and in vitro, and the transcription factor specific protein 1 (Sp1) plays a critical role in this process. Results from co-immunoprecipitation experiments indicate that the activation of SR-BI was associated with Sp1-recruited protein complexes in the promoter region of SR-BI, where histone acetyltransferase p300 was recruited and histone deacetylase HDAC1 was dismissed. As a result, histone acetylation increased, leading to activation of SR-BI transcription. With further investigation, we found that LDL phosphorylated Sp1 through ERK1/2 pathway, which affected Sp1 protein complexes formation in SR-BI promoter. Using mass spectrometry and site directed mutagenesis, a new Sp1 phosphorylation site Ser702 was defined to be associated with Sp1-HDAC1 interaction and may be important in SR-BI activation, shedding light on the knowledge of delicate mechanism of hepatic HDL receptor SR-BI gene modulation by LDL.Display Omitted
Keywords: Low density lipoprotein (LDL); Scavenger receptor class B type I (SR-BI); Specific protein 1 (Sp1); Transcriptional regulation; Histone acetyltransferase/deacetylase; Atherosclerosis;

The cellular lipids of Romboutsia by Ziqiang Guan; Lingli Chen; Jacoline Gerritsen; Hauke Smidt; Howard Goldfine (1076-1082).
We have examined the lipids of three isolates, Romboutsia lituseburensis, Romboutsia ilealis, and Romboutsia sp. strain FRIFI, of the newly described genus Romboutsia by two-dimensional thin-layer chromatography (2D-TLC) and by liquid chromatography/mass spectrometry (LC/MS). We have found three phospholipids, phosphatidylglycerol (PG), cardiolipin and phosphatidic acid in all three species. A fourth phospholipid, lysyl-PG, was found in R. lituseburensis and strain FRIFI. Polyprenyl-phosphates were identified in the lipid extracts of all three species. Three glycolipids, mono-, di- and tri-hexosyldiacylglycerol, were common to all three species. An additional glycolipid, tetrahexosyl-diacylglycerol was identified in strain FRIFI. Acylated trihexosyldiacylglycerol and acyl-tetrahexosydiacylglycerol were also found in R. ilealis and strain FRIFI. Remarkably, no alk-1-enyl ether lipids (plasmalogens) were present in Romboutsia as distinct from bacteria of the related genus Clostridium in which these ether lipids are common. We have compared the lipidome of Romboutsia with that recently described for Clostridium difficile, which has plasmalogens, no lysyl-PG, and no tetrahexosyl-diacylglycerol. According to 16S rRNA gene sequencing, Romboutsia spp. and C. difficile are closely related (> 95% sequence identity).
Keywords: Romboutsia; Glycolipid; Mass spectrometry; Phospholipid; Thin-layer chromatography;

Critical role for cytosolic group IVA phospholipase A2 in early adipocyte differentiation and obesity by Lucía Peña; Clara Meana; Alma M. Astudillo; Gema Lordén; Martín Valdearcos; Hiroyasu Sato; Makoto Murakami; Jesús Balsinde; María A. Balboa (1083-1095).
Adipogenesis is the process of differentiation of immature mesenchymal stem cells into adipocytes. Elucidation of the mechanisms that regulate adipocyte differentiation is key for the development of novel therapies for the control of obesity and related comorbidities. Cytosolic group IVA phospholipase A2 (cPLA2α) is the pivotal enzyme in receptor-mediated arachidonic acid (AA) mobilization and attendant eicosanoid production. Using primary multipotent cells and cell lines predetermined to become adipocytes, we show here that cPLA2α displays a proadipogenic function that occurs very early in the adipogenic process. Interestingly, cPLA2α levels decrease during adipogenesis, but cPLA2α-deficient preadipocytes exhibit a reduced capacity to differentiate into adipocytes, which affects early and terminal adipogenic transcription factors. Additionally, the absence of the phospholipase alters proliferation and cell-cycle progression that takes place during adipogenesis. Preconditioning of preadipocytes with AA increases the adipogenic capacity of these cells. Moreover, animals deficient in cPLA2α show resistance to obesity when fed a high fat diet that parallels changes in the expression of adipogenic transcription factors of the adipose tissue. Collectively, these results show that preadipocyte cPLA2α activation is a hitherto unrecognized factor for adipogenesis in vitro and in vivo.
Keywords: Phospholipase A2; Adipogenesis; Adipocyte; Obesity; Arachidonic acid;

Hepatitis C virus p7 mediates membrane-to-membrane adhesion by Gi Young Lee; Sora Lee; Hye-Ra Lee; Young Do Yoo (1096-1101).
Viroporin p7 of the hepatitis C virus (HCV) acts as an ion channel for pH equilibration to stabilize HCV particles; most studies of p7 have focused on this role. However, pH equilibration by p7 via its ion channel activity does not fully explain the importance of p7 in HCV particle production. Indeed, several researchers have suggested p7 to have an unidentified ion channel-independent function. Here, we show that p7 has a novel role as a lipid raft adhesion factor, which is independent of its ion channel activity. We found that p7 targets not only the liquid-disordered (Ld) phase, but also the negatively-charged liquid-ordered (Lo) phase that can be represented as a lipid raft. p7 clusters at the phase boundary of the neutral Ld phase and the negatively-charged Lo phase. Interestingly, p7 targeting the Lo phase facilitates membrane-to-membrane adhesion, and this activity is not inhibited by p7 ion channel inhibitors. Our results demonstrated that HCV p7 has dual roles as a viroporin and as a lipid raft adhesion factor. This ion channel-independent function of p7 might be an attractive target for development of anti-HCV compounds.
Keywords: Hepatitis C virus p7; Membrane adhesion; Lipid raft;

Listeria monocytogenes, the causative organism of the serious food-borne disease listeriosis, has a membrane abundant in branched-chain fatty acids (BCFAs). BCFAs are normally biosynthesized from branched-chain amino acids via the activity of branched chain α-keto acid dehydrogenase (Bkd), and disruption of this pathway results in reduced BCFA content in the membrane. Short branched-chain carboxylic acids (BCCAs) added as media supplements result in incorporation of BCFAs arising from the supplemented BCCAs in the membrane of L. monocytogenes bkd mutant MOR401. High concentrations of the supplements also effect similar changes in the membrane of the wild type organism with intact bkd. Such carboxylic acids clearly act as fatty acid precursors, and there must be an alternative pathway resulting in the formation of their CoA thioester derivatives. Candidates for this are the enzymes phosphotransbutyrylase (Ptb) and butyrate kinase (Buk), the products of the first two genes of the bkd operon. Ptb from L. monocytogenes exhibited broad substrate specificity, a strong preference for branched-chain substrates, a lack of activity with acetyl CoA and hexanoyl CoA, and strict chain length preference (C3–C5). Ptb catalysis involved ternary complex formation. Additionally, Ptb could utilize unnatural branched-chain substrates such as 2-ethylbutyryl CoA, albeit with lower efficiency, consistent with a potential involvement of this enzyme in the conversion of the carboxylic acid additives into CoA primers for BCFA biosynthesis.
Keywords: Phosphotransbutyrylase; Branched-chain carboxylic acids; Branched-chain fatty acids; Fatty acid biosynthesis; Membrane fatty acid composition; Acyl CoA;

Effect of preduodenal lipase inhibition in suckling rats on dietary octanoic acid (C8:0) gastric absorption and plasma octanoylated ghrelin concentration by F. Lemarié; J.-F. Cavalier; C. Garcia; F. Boissel; V. Point; D. Catheline; P. Legrand; F. Carrière; V. Rioux (1111-1120).
Part of medium chain fatty acids (MCFAs) coming from dietary triglycerides (TGs) can be directly absorbed through the gastric mucosa after the action of preduodenal lipase (lingual lipase in the rat). MCFA gastric absorption, particularly that of octanoic acid (C8:0), may have a physiological importance in the octanoylation of ghrelin, the orexigenic gastric peptide acting as an endogenous ligand of the hypothalamic growth hormone secretagogue receptor 1a (GHSR-1a). However, the amount of C8:0 absorbed in the stomach and its metabolic fate still haven't been clearly characterized. The purpose of the present study was to further characterize and quantify the importance of preduodenal lipase activity on the release and gastric absorption of dietary C8:0 and on the subsequent ghrelin octanoylation in the stomach mucosa. Fifteen days old rats received fat emulsions containing triolein or [1,1,1-13C]-Tri-C8:0 and a specific inhibitor of preduodenal lipase, 5-(2-(benzyloxy)ethoxy)-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one or BemPPOX. The fate of the 13C-C8:0 was followed in rat tissues after 30 and 120 min of digestion and octanoylated ghrelin was measured in the plasma. This work (1) demonstrates that part of C8:0 coming from Tri-C8:0 is directly absorbed at the gastric level, (2) allows the estimation of C8:0 gastric absorption level (1.3% of the 13C-C8:0 in sn-3 position after 30 min of digestion), as well as (3) the contribution of rat lingual lipase to total lipolysis and to duodenal absorption of dietary FAs (at least 30%), (4) shows no short-term effect of dietary Tri-C8:0 consumption and subsequent increase of C8:0 gastric tissue content on plasma octanoylated ghrelin concentration.
Keywords: Octanoic acid; Lingual lipase; Preduodenal lipase; Gastric absorption; Gastric mucosa; Ghrelin; Ghrelin O-acyltransferase; Acylation; Octanoylation;

Regulation of adipogenesis by paracrine factors from adipose stromal-vascular fraction - a link to fat depot-specific differences by Bettina Meissburger; Aliki Perdikari; Hansjörg Moest; Sebastian Müller; Matthias Geiger; Christian Wolfrum (1121-1131).
Visceral and subcutaneous adipose tissue depots have distinct features and contribute differentially to the development of metabolic dysfunction. We show here that adipocyte differentiation in subcutaneous stromal-vascular fraction (SVF) is increased compared to visceral SVF, however this increased differentiation capacity seems not to be due to changes in the number of adipocyte precursor cells. Rather, we demonstrate that secreted heat-sensitive factors from the SVF can inhibit adipocyte differentiation and that this effect is higher in visceral than in subcutaneous SVF, suggesting that visceral SVF is a source of secreted factors that can inhibit adipocyte formation. In order to explore secreted proteins that potentially inhibit differentiation in visceral preadipocytes we analyzed the secretome of both SVFs which led to the identification of 113 secreted proteins with an overlap of 42%. Further expression analysis in both depots revealed 16 candidates that were subsequently analyzed in a differentiation screen using an adenoviral knockdown system. From this analysis we were able to identify two potential inhibitory candidates, namely decorin (Dcn) and Sparc-like 1 (Sparcl1). We could show that ablation of either candidate enhanced adipogenesis in visceral preadipocytes, while treatment of primary cultures with recombinant Sparcl1 and Dcn blocked adipogenesis in a dose dependent manner. In conclusion, our data suggests that the differences in adipogenesis between depots might be due to paracrine and autocrine feedback mechanisms which could in turn contribute to metabolic homeostasis.
Keywords: Adipogenesis; Preadipocyte; Decorin; Sparc like 1;

Novel role of a triglyceride-synthesizing enzyme: DGAT1 at the crossroad between triglyceride and cholesterol metabolism by Vinay Sachdev; Christina Leopold; Raimund Bauer; Jay V. Patankar; Jahangir Iqbal; Sascha Obrowsky; Renze Boverhof; Marcela Doktorova; Bernhard Scheicher; Madeleine Goeritzer; Dagmar Kolb; Andrew V. Turnbull; Andreas Zimmer; Gerald Hoefler; M. Mahmood Hussain; Albert K. Groen; Dagmar Kratky (1132-1141).
Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol (TG) biosynthesis. Here we show that genetic deficiency and pharmacological inhibition of DGAT1 in mice alters cholesterol metabolism. Cholesterol absorption, as assessed by acute cholesterol uptake, was significantly decreased in the small intestine and liver upon DGAT1 deficiency/inhibition. Ablation of DGAT1 in the intestine (I-DGAT1−/−) alone is sufficient to cause these effects. Consequences of I-DGAT1 deficiency phenocopy findings in whole-body DGAT1−/− and DGAT1 inhibitor-treated mice. We show that deficiency/inhibition of DGAT1 affects cholesterol metabolism via reduced chylomicron size and increased trans-intestinal cholesterol excretion. These effects are independent of cholesterol uptake at the apical surface of enterocytes but mediated through altered dietary fatty acid metabolism. Our findings provide insight into a novel role of DGAT1 and identify a pathway by which intestinal DGAT1 deficiency affects whole-body cholesterol homeostasis in mice. Targeting intestinal DGAT1 may represent a novel approach for treating hypercholesterolemia.Display Omitted
Keywords: Cholesterol absorption; Chylomicron size; DGAT1 inhibition; Intestinal DGAT1 deficiency; Trans-intestinal cholesterol excretion;

Hormone-sensitive lipase deficiency disturbs lipid composition of plasma membrane microdomains from mouse testis by María E. Casado; Oscar Pastor; David García-Seisdedos; Lydia Huerta; Fredric B. Kraemer; Miguel A. Lasunción; Antonia Martín-Hidalgo; Rebeca Busto (1142-1150).
Hormone-sensitive lipase (HSL) is an intracellular neutral lipase capable of hydrolyzing acylglycerols, as well as cholesteryl and retinyl esters. In mice, HSL deficiency results in male sterility. Lipid rafts, a plasma membrane microdomain enriched in cholesterol, sphingolipids and saturated glycerophospholipids, comprise a highly dynamic clustering of proteins and lipids that play a central role in signal transduction and intercellular communication. In the present work, we examined the effect of HSL deficiency in the lipid composition of plasma membrane microdomains in mouse testis. The lack of HSL affected the density of lipid rafts, as indicated by the shifting of caveolin 1 to denser fractions in the sucrose-gradient fractionation, and altered the sterol and phospholipid composition of both lipid raft and non-raft fractions, the biochemical differences among them being less obvious in HSL−/− than in HSL+/+ mice. Compared to HSL+/+, the lipid rafts from HSL−/− mice had significantly less desmosterol and T-MAS, while the non-raft domain had increased cholesterol content. Lipid rafts from HSL−/− mice had reduced PUFA-containing phospholipid species but increased phosphatidylcholine and phosphatidylethanolamine species with monounsaturated fatty acid moieties, while the non-raft domain was enriched in phosphatidylethanolamine and phosphatidylserine species having four double bonds. The changes in both the sterols and phospholipid composition of lipid raft and non-raft microdomains may have consequences in signal transduction and contribute to the sterility observed in HSL-deficient male mice.
Keywords: Hormone sensitive lipase; Sterol; Phospholipids; Lipid raft; Testis; Fertility;

Oleate protects beta-cells from the toxic effect of palmitate by activating pro-survival pathways of the ER stress response by Ernest Sargsyan; Konstantin Artemenko; Levon Manukyan; Jonas Bergquist; Peter Bergsten (1151-1160).
Long-term exposure of beta cells to saturated fatty acids impairs insulin secretion and increases apoptosis. In contrast, unsaturated fatty acids protect beta-cells from the long-term negative effects of saturated fatty acids. We aimed to identify the mechanisms underlying this protective action of unsaturated fatty acids. To address the aim, insulin-secreting MIN6 cells were exposed to palmitate in the absence or presence of oleate and analyzed by using nano-LC MS/MS based proteomic approach. Important findings were validated by using alternative approaches. Proteomic analysis identified 34 proteins differentially expressed in the presence of palmitate compared to control samples. These proteins play a role in insulin processing, mitochondrial function, metabolism of biomolecules, calcium homeostasis, exocytosis, receptor signaling, ER protein folding, antioxidant activity and anti-apoptotic function. When oleate was also present during culture, expression of 15 proteins was different from the expression in the presence of palmitate alone. Most of the proteins affected by oleate are targets of the ER stress response and play a pro-survival role in beta cells such as protein folding and antioxidative defence. We conclude that restoration of pro-survival pathways of the ER stress response is a major mechanism underlying the protective effect of unsaturated fatty acids in beta-cells treated with saturated fatty acids.Display Omitted
Keywords: Palmitate; Oleate; Insulin secretion; Apoptosis; ER stress;

Phosphatidylcholine passes through lateral tight junctions for paracellular transport to the apical side of the polarized intestinal tumor cell-line CaCo2 by Wolfgang Stremmel; Simone Staffer; Hongying Gan-Schreier; Andreas Wannhoff; Margund Bach; Annika Gauss (1161-1169).
Phosphatidylcholine (PC) is the most abundant phospholipid in intestinal mucus, indicative of a specific transport system across the mucosal epithelium to the intestinal lumen. To elucidate this transport mechanism, we employed a transwell tissue culture system with polarized CaCo2 cells. It was shown that PC could not substantially be internalized by the cells. However, after basal application of increasing PC concentrations, an apical transport of 47.1 ± 6.3 nmol h− 1  mM PC− 1 was observed. Equilibrium distribution studies with PC applied in equal concentrations to the basal and apical compartments showed a 1.5-fold accumulation on the expense of basal PC. Disruption of tight junctions (TJ) by acetaldehyde or PPARγ inhibitors or by treatment with siRNA to TJ proteins suppressed paracellular transport by at least 50%. Transport was specific for the choline containing the phospholipids PC, lysoPC and sphingomyelin. We showed that translocation is driven by an electrochemical gradient generated by apical accumulation of Cl and HCO3 through CFTR. Pretreatment with siRNA to mucin 3 which anchors in the apical plasma membrane of mucosal cells inhibited the final step of luminal PC secretion. PC accumulates in intestinal mucus using a paracellular, apically directed transport route across TJs.
Keywords: Ulcerative colitis; Tight junctions; Phosphatidylcholine; Mucosal barrier; Mucus; Mucins; CFTR;

Integrin-mediated cell migration is blocked by inhibitors of human neuraminidase by Feng Jia; Md. Amran Howlader; Christopher W. Cairo (1170-1179).
Integrins are critical receptors in cell migration and adhesion. A number of mechanisms are known to regulate the function of integrins, including phosphorylation, conformational change, and cytoskeletal anchoring. We investigated whether native neuraminidase (Neu, or sialidase) enzymes which modify glycolipids could play a role in regulating integrin-mediated cell migration. Using a scratch assay, we found that exogenously added Neu3 and Neu4 activity altered rates of cell migration. We observed that Neu4 increased the rate of migration in two cell lines (HeLa, A549); while Neu3 only increased migration in HeLa cells. A bacterial neuraminidase was able to increase the rate of migration in HeLa, but not in A549 cells. Treatment of cells with complex gangliosides (GM1, GD1a, GD1b, and GT1b) resulted in decreased cell migration rates, while LacCer was able to increase rates of migration in both lines. Importantly, our results show that treatment of cells with inhibitors of native Neu enzymes had a dramatic effect on the rates of cell migration. The most potent compound tested targeted the human Neu4 isoenzyme, and was able to substantially reduce the rate of cell migration. We found that the lateral mobility of integrins was reduced by treatment of cells with Neu3, suggesting that Neu3 enzyme activity resulted in changes to integrin–co-receptor or integrin–cytoskeleton interactions. Finally, our results support the hypothesis that inhibitors of human Neu can be used to investigate mechanisms of cell migration and for the development of anti-adhesive therapies.Display Omitted
Keywords: Glycolipid; Ganglioside; Neuraminidase; Sialidase; Integrin; Cell migration;

MEK1/2 inhibitors activate macrophage ABCG1 expression and reverse cholesterol transport—An anti-atherogenic function of ERK1/2 inhibition by Ling Zhang; Yuanli Chen; Xiaoxiao Yang; Jie Yang; Xingyue Cao; Xiaoju Li; Luyuan Li; Qing Robert Miao; David P. Hajjar; Yajun Duan; Jihong Han (1180-1191).
Expression of ATP-binding cassette transporter G1 (ABCG1), a molecule facilitating cholesterol efflux to HDL, is activated by liver X receptor (LXR). In this study, we investigated if inhibition of ERK1/2 can activate macrophage ABCG1 expression and functions. MEK1/2 inhibitors, PD98059 and U0126, increased ABCG1 mRNA and protein expression, and activated the natural ABCG1 promoter but not the promoter with the LXR responsive element (LXRE) deletion. Inhibition of ABCG1 expression by ABCG1 siRNA did enhance the formation of macrophage/foam cells and it attenuated the inhibitory effect of MEK1/2 inhibitors on foam cell formation. MEK1/2 inhibitors activated macrophage cholesterol efflux to HDL in vitro, and they enhanced reverse cholesterol transport (RCT) in vivo. ApoE deficient (apoE−/−) mice receiving U0126 treatment had reduced sinus lesions in the aortic root which was associated with activated macrophage ABCG1 expression in the lesion areas. MEK1/2 inhibitors coordinated the RXR agonist, but not the LXR agonist, to induce ABCG1 expression. Furthermore, induction of ABCG1 expression by MEK1/2 inhibitors was associated with activation of SIRT1, a positive regulator of LXR activity, and inactivation of SULT2B1 and RIP140, two negative regulators of LXR activity. Taken together, our study suggests that MEK1/2 inhibitors activate macrophage ABCG1 expression/RCT, and inhibit foam cell formation and lesion development by multiple mechanisms, supporting the concept that ERK1/2 inhibition is anti-atherogenic.
Keywords: ABCG1; Atherosclerosis; LXR; MEK1/2; RCT; SIRT1;

Membrane topology of human monoacylglycerol acyltransferase-2 and identification of regions important for its localization to the endoplasmic reticulum by Pamela J. McFie; Sabrina Izzard; Huyen Vu; Youzhi Jin; Erwan Beauchamp; Luc G. Berthiaume; Scot J. Stone (1192-1204).
Acyl CoA:2-monoacylglycerol acyltransferase (MGAT)-2 has an important role in dietary fat absorption in the intestine. MGAT2 resides in the endoplasmic reticulum and catalyzes the synthesis of diacylglycerol which is then utilized as a substrate for triacylglycerol synthesis. This triacylglycerol is then incorporated into chylomicrons which are released into the circulation.In this study, we determined the membrane topology of human MGAT2. Protease protection experiments showed that the C-terminus is exposed to the cytosol, while the N-terminus is partially buried in the ER membrane. MGAT2, like murine DGAT2, was found to have two transmembrane domains. We also identified a region of MGAT2 associated with the ER membrane that contains the histidine–proline–histidine–glycine sequence present in all DGAT2 family members that is thought to comprise the active site.Proteolysis experiments demonstrated that digestion of total cellular membranes from cells expressing MGAT2 with trypsin abolished MGAT activity, indicating that domains that are important for catalysis face the cytosol. We also explored the role that the five cysteines residues present in MGAT2 have in catalysis. MGAT activity was sensitive to two thiol modifiers, N-ethylmaleimide and 5,5′-dithiobis-(2-nitrobenzoic acid). Furthermore, mutation of four cysteines resulted in a reduction in MGAT activity. However, when the C-terminal cysteine (C334) was mutated, MGAT activity was actually higher than that of wild-type FL-MGAT2. Lastly, we determined that both transmembrane domains of MGAT2 are important for its ER localization, and that MGAT2 is present in mitochondrial-associated membranes.
Keywords: MGAT; Topology; Diacylglycerol; Triacylglycerol; Cysteine; Mitochondrial-associated membranes;