BBA - Molecular and Cell Biology of Lipids (v.1801, #1)

Role of fatty acid uptake and fatty acid β-oxidation in mediating insulin resistance in heart and skeletal muscle by Liyan Zhang; Wendy Keung; Victor Samokhvalov; Wei Wang; Gary D. Lopaschuk (1-22).
Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and β-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial β-oxidation. However, imbalance between fatty acid uptake and β-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid β-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid β-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid β-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.
Keywords: Malonyl CoA; Diacylglycerol; Long chain acyl CoA; Ceramide; Obesity; Diabetes;

Lysophosphatidic acid mediates migration of human mesenchymal stem cells stimulated by synovial fluid of patients with rheumatoid arthritis by Hae Young Song; Mi Jeong Lee; Min Young Kim; Kyung Hye Kim; Il Hwan Lee; Sang Hun Shin; Jung Sub Lee; Jae Ho Kim (23-30).
Migration of mesenchymal stem cells plays a key role in regeneration of injured tissues. Rheumatoid arthritis (RA) is a chronic inflammatory disease and synovial fluid (SF) reportedly contains a variety of chemotactic factors. This study was undertaken to investigate the role of SF in migration of human bone marrow-derived mesenchymal stem cells (hBMSCs) and the molecular mechanism of SF-induced cell migration. SF from RA patients greatly stimulated migration of hBMSCs and the SF-induced migration was completely abrogated by pretreatment of the cells with the lysophosphatidic acid (LPA) receptor antagonist Ki16425 and by small interfering RNA- or lentiviral small hairpin RNA-mediated silencing of endogenous LPA1/Edg2. Moreover, SF from RA patients contains higher concentrations of LPA and an LPA-producing enzyme autotoxin than normal SF. In addition, SF from RA patients increased the intracellular concentration of calcium through a Ki16425-sensitive mechanism and pretreatment of the cells with the calmodulin inhibitor W7 or calmodulin-dependent protein kinase II inhibitor KN93 abrogated the SF-induced cell migration. These results suggest that LPA-LPA1 plays a key role in the migration of hBMSCs induced by SF from RA patients through LPA1-dependent activation of calmodulin-dependent protein kinase II.
Keywords: Lysophosphatidic acid; Mesenchymal stem cell; Migration; Synovial fluid; Arthritis;

Inhibition of carboxylesterase activity of THP1 monocytes/macrophages and recombinant human carboxylesterase 1 by oxysterols and fatty acids by J. Allen Crow; Katye L. Herring; Shuqi Xie; Abdolsamad Borazjani; Philip M. Potter; Matthew K. Ross (31-41).
Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC50  = 33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC50  = 8.1 μM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (K iapp  = 10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (K iapp  = 1.7 μM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which resulted in significantly reduced hydrolysis of the pyrethroid insecticide bioresmethrin, a CES1-specific xenobiotic substrate. Collectively, these findings suggest that CE activity of recombinant CES1, cell lysates, and intact cells can be impaired by naturally occurring lipids, which may compromise the ability of CES1 to both detoxify environmental pollutants and metabolize endogenous compounds in vivo.
Keywords: 27-hydroxycholesterol; Arachidonic acid; Oxysterol; Fatty acid; Cholesteryl ester hydrolase; Carboxylesterase; Macrophage; Xenobiotic biotransformation;

Apolipoprotein C-I reduces cholesteryl esters selective uptake from LDL and HDL by binding to HepG2 cells and lipoproteins by Veneta Krasteva; Mathieu R. Brodeur; Félix-Labonté Tremblay; Louise Falstrault; Louise Brissette (42-48).
Plasma cholesterol from low- and high-density lipoproteins (LDL and HDL) are cleared from the circulation by specific receptors that either totally degrade lipoproteins as the LDL receptor or selectively take up their cholesteryl esters (CE) like the scavenger receptor class B type I (SR-BI). The aim of the present study was to define the effect of apoC-I on the uptake of LDL and HDL3 by HepG2 cells. In experiments conducted with exogenously added purified apoC-I, no significant effect was observed on lipoprotein–protein association and degradation; however, LDL- and HDL3-CE selective uptake was significantly reduced in a dose-dependent manner. This study also shows that apoC-I has the ability to associate with HepG2 cells and with LDL and HDL3. Moreover, pre-incubation of HepG2 cells with apoC-I reduces HDL3-CE selective uptake and pre-incubation of LDL and HDL3 with apoC-I decreases their CE selective uptake by HepG2 cells. Thus, apoC-I can accomplish its inhibitory effect on SR-BI activity by either binding to SR-BI or lipoproteins. We conclude that by reducing hepatic lipoprotein-CE selective uptake, apoC-I has an atherogenic character.
Keywords: High-density lipoprotein; Low-density lipoprotein; Apolipoprotein C-I; Cholesterol ester; Selective uptake;

Role of DNA methylation and methyl-DNA binding proteins in the repression of 5-lipoxygenase promoter activity by Careen Katryniok; Nicole Schnur; Ad Gillis; Andreas von Knethen; Bernd L. Sorg; Leendert Looijenga; Olof Rådmark; Dieter Steinhilber (49-57).
Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of inflammatory leukotrienes. 5-LO gene expression is mainly restricted to B cells and cells of myeloid origin. It is known that basal 5-lipoxygenase promoter activity is regulated by DNA methylation. In this study we investigated the impact of the DNA methylation status of the 5-LO promoter on its activity and the role of methyl DNA binding proteins (MBDs) in transcriptional silencing of the 5-LO promoter. Using ChIP assays, we found that the methyl-DNA binding proteins MBD1, MBD2 and MeCP2 bind to the methylated 5-LO core promoter in U937 cells. Knock down of each of the MBDs upregulates 5-LO mRNA expression in U937 cells indicating that these proteins are involved in silencing of the 5-LO gene. In reporter gene assays with in vitro methylated 5-LO promoter constructs, the extent of 5-LO promoter methylation inversely correlated with its activity. Furthermore, we found that MBD1 overexpression repressed 5-LO promoter activity when the CpG sites at the Sp1 binding site close to the transcriptional start site (GC4) were methylated. Gel shift data indicate that recruitment of Sp1 to this binding site is prevented by methylation.
Keywords: 5‑lipoxygenase; DNA methylation; MBD; Knock down; Chromatin immunoprecipitation; Promoter regulation;

Lipoxygenases (LOX) are found in most organisms that contain polyunsaturated fatty acids, usually existing as individual genes although occasionally encoded as a fusion protein with a catalase-related hemoprotein. Such a fusion protein occurs in the cyanobacterium Acaryochloris marina and herein we report the novel catalytic activity of its LOX domain. The full-length protein and the C-terminal LOX domain were expressed in Escherichia coli, and the catalytic activities characterized by UV, HPLC, GC-MS, and CD. All omega-3 polyunsaturates were oxygenated by the LOX domain at the n-7 position and with R stereospecificity: α-linolenic and the most abundant fatty acid in A. marina, stearidonic acid (C18.4ω3), are converted to the corresponding 12R-hydroperoxides, eicosapentaenoic acid to its 14R-hydroperoxide, and docosahexaenoic acid to its 16R-hydroperoxide. Omega-6 polyunsaturates were oxygenated at the n-10 position, forming 9R-hydroperoxy-octadecadienoic acid from linoleic acid and 11R-hydroperoxy-eicosatetraenoic acid from arachidonic acid. The metabolic transformation of stearidonic acid by the full-length fusion protein entails its 12R oxygenation with subsequent conversion by the catalase-related domain to a novel allene epoxide, a likely precursor of cyclopentenone fatty acids or other signaling molecules (Gao et al, J. Biol. Chem. 284:22087-98, 2009). Although omega-3 fatty acids and lipoxygenases are of widespread occurrence, this appears to be the first description of a LOX-catalyzed oxygenation that specifically utilizes the terminal pentadiene of omega-3 fatty acids.
Keywords: Acaryochloris marina; Lipoxygenase; Hydroperoxide; Omega-3 fatty acid; Linolenic acid, stearidonic acid, GC-MS; Chiral analysis;

Recent models of lipid-free apolipoprotein A-I, including a cross-link/homology model and an X-ray crystal structure have identified two potential functionally relevant “patches” on the protein surface. The first is a hydrophobic surface patch composed of leucine residues 42, 44, 46, and 47 and the second a negatively charged patch composed of glutamic acid residues 179, 191, and 198. To determine if these domains play a functional role, these surface patches were disrupted by site-directed mutagenesis and the bacterially expressed mutants were compared with respect to their ability to bind lipid and stimulate ABCA1-mediated cholesterol efflux. It was found that neither patch plays a significant functional role in the ability of apoA-I to accept cholesterol in an ABCA1-dependent manner, but that the hydrophobic patch did affect the ability of apoA-I to clear DMPC liposomes. Interestingly, contrary to previous predictions, disruption of the hydrophobic surface patch enhanced the lipid binding ability of apoA-I. The hydrophobic surface patch may be important to the structural stability of lipid-free apoA-I or may be a necessary permissive structural element for lipid binding.
Keywords: Apolipoprotein A-I; ATP binding cassette transporter A1; Functionality; Surface patch; Lipid-free model; Lipid-binding;

Protein kinase C (PKC) is a family of serine/threonine kinases involved in various signal transduction pathways. We investigated the roles of PKC in the regulation of group IIA secreted phospholipase A2 (sPLA2-IIA) expression in cytokine-stimulated rat fibroblastic 3Y1 cells. Here we show that the induction of sPLA2-IIA by proinflammatory cytokines was under the control of both classical cPKCα and atypical aPKCλ/ι pathways by using PKC inhibitors, a PKC activator, and PKC knockdowns. Treatment of 3Y1 cells with PKC selective inhibitors having broad specificity, such as chelerythrine chloride and GF109203X, blocked IL-1β/TNFα-dependent induction of sPLA2-IIA protein in a dose-dependent manner. Treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA), which activates cPKC and novel nPKC isoforms, markedly attenuated the cytokine-dependent induction of sPLA2-IIA expression. In comparison, 24-h pretreatment with PMA, which down-regulates these PKC isoforms, markedly enhanced sPLA2-IIA expression. Results with short hairpin RNA (shRNA)-mediated knockdown of PKC isoforms revealed that the cytokine-induced sPLA2-IIA expression was markedly enhanced in cPKCα knockdown cells compared to those in replicate control cells. In contrast, knockdown of the aPKCλ/ι isoform reduced the cytokine-induced expression of sPLA2-IIA. These results suggest that the aPKCλ/ι pathway is required for the induction of sPLA2-IIA expression and that the cPKCα pathway acts as a negative regulator of sPLA2-IIA expression in cytokine-stimulated rat fibroblasts.
Keywords: sPLA2-IIA; PKCα; PKCλ/ι; IL-1β;

In vitro stereoselective hydrolysis of diacylglycerols by hormone-sensitive lipase by Jorge A. Rodriguez; Yassine Ben Ali; Slim Abdelkafi; Lilia D. Mendoza; Julien Leclaire; Frédéric Fotiadu; Gerard Buono; Frédéric Carrière; Abdelkarim Abousalham (77-83).
Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids in adipocytes and shows a substrate preference for the diacylglycerols (DAGs) originating from triacylglycerols. To determine whether HSL shows any stereopreference during the hydrolysis of diacylglycerols, racemic 1,2(2,3)-sn-diolein was used as a substrate and the enantiomeric excess (ee%) of residual 1,2-sn-diolein over 2,3-sn-diolein was measured as a function of DAG hydrolysis. Enantiomeric DAGs were separated by performing chiral-stationary-phase HPLC after direct derivatization from lipolysis product extracts. The fact that the ee% of 1,2-sn-diolein over 2,3-sn-diolein increased with the level of hydrolysis indicated that HSL has a preference for 2,3-sn-diolein as a substrate and therefore a stereopreference for the sn-3 position of dioleoylglycerol. The ee% of 1,2-sn-diolein reached a maximum value of 36% at 42% hydrolysis. Among the various mammalian lipases tested so far, HSL is the only lipolytic carboxylester hydrolase found to have a pronounced stereospecificity for the sn-3 position of dioleoylglycerol.
Keywords: Hormone-sensitive lipase; Stereoselectivity; Enantiomeric excess; Triacylglycerol; Diacylglycerol; Carbamate;

Plasma lecithin:cholesterol acyltransferase activity modifies the inverse relationship of C-reactive protein with HDL cholesterol in nondiabetic men by R.P.F. Dullaart; F. Perton; P.J.W.H. Kappelle; R. de Vries; W.J. Sluiter; A. van Tol (84-88).
Lecithin:cholesterol acyltransferase (LCAT) is instrumental in high-density lipoprotein (HDL) maturation, but high LCAT levels do not predict low cardiovascular risk. LCAT may affect antioxidative or anti-inflammatory properties of HDL. We determined the relationship of plasma high-sensitivity C-reactive protein (CRP) with LCAT activity and evaluated whether LCAT activity modifies the decreasing effect of HDL cholesterol (HDL-C) on CRP, as an estimate of its anti-inflammatory properties. Plasma HDL-C, apolipoprotein (apo) A-I and LCAT activity (exogenous substrate method) were measured in 260 nondiabetic men without cardiovascular disease. CRP was correlated inversely with HDL-C and apo A-I, and positively with LCAT activity (P  < 0.01 to 0.001). Multivariate regression analysis demonstrated that age- and smoking-adjusted plasma CRP levels were associated negatively with HDL-C (β  = − 0.224, P  < 0.001) and positively with LCAT activity (β  = 0.119, P  = 0.034), as well as with the interaction between HDL-C and LCAT activity (β  = 0.123, P  = 0.026). There was also an interaction between apo A-I and LCAT activity on CRP (β  = 0.159, P  = 0.005). These relationships remained similar after adjustment for apo B-containing lipoproteins. In conclusion, the inverse relationship of HDL-C with CRP is attenuated by LCAT activity at higher HDL-C levels. It is hypothesized that LCAT could mitigate HDL's anti-inflammatory or antioxidative properties at higher HDL-C concentrations.
Keywords: Apolipoprotein A-I; High-density lipoprotein cholesterol; High-sensitivity C-reactive protein; Lecithin:cholesterol acyltransferase activity;

The extent to which atorvastatin treatment affects LDL size, LDL subfraction levels and remnant-like particle cholesterol (RLP-C) was determined in type 2 diabetes. We also compared LDL size and RLP-C in relation to guideline cut-off values for LDL cholesterol, non-HDL cholesterol and apolipoprotein (apo) B. Changes in LDL size and RLP-C were determined in fasting plasma from type 2 diabetic patients after 30 weeks administration of atorvastatin (10 mg daily, n  = 65; 80 mg daily, n  = 62) or placebo (n  = 58). LDL subfraction cholesterol was measured in 74 participants. Atorvastatin lowered LDL cholesterol, non-HDL cholesterol, triglycerides, apo B and RLP-C (P  < 0.001 for all at each dose) and LDL mean peak particle diameter remained unchanged. Atorvastatin treatment decreased cholesterol concentrations in all LDL subfractions (P  < 0.001 for each dose). RLP-C at follow-up was lower in those patients achieving the non-HDL cholesterol or the apo B guideline targets (P  < 0.01), but the LDL cholesterol cut-off value failed to discriminate. In conclusion, atorvastatin lowers fasting RLP-C and LDL subfraction cholesterol in diabetes. The proposed guideline cut-off levels for non-HDL cholesterol and apo B may be superior to the LDL cholesterol target in discriminating between higher and lower RLP-C levels.
Keywords: Apolipoprotein B; atorvastatin; Diabetes mellitus; LDL-cholesterol; LDL subfractions; Non-HDL-cholesterol; Remnant-like particle cholesterol; Small dense LDL; Triglycerides;