BBA - Molecular and Cell Biology of Lipids (v.1781, #1-2)

Acknowledgement (I-III).

Antibacterial actions of secreted phospholipases A2. Review by Timo J. Nevalainen; Garry G. Graham; Kieran F. Scott (1-9).
Antibacterial properties of secreted phospholipases A2 (PLA2) have emerged gradually. Group (G) IIA PLA2 is the most potent among mammalian secreted (s) PLA2s against Gram-positive bacteria, but additional antibacterial compounds, e.g. the bactericidal/permeability-increasing protein, are needed to kill Gram-negative bacteria. The mechanisms of binding to the bacterial surface and the killing of bacteria by sPLA2s are based on the positive charge of the PLA2 protein and its phospholipolytic enzymatic activity, respectively. The concentration of GIIA PLA2 is highly elevated in serum of patients with bacterial sepsis, and overexpression of GIIA PLA2 protects transgenic mice against experimental Gram-positive infection. The synthesis and secretion of GIIA PLA2 are stimulated by the cytokines TNF-α, IL-1 and IL-6. Secreted PLA2s may be potentially useful new endogenous antibiotics to combat infections including those caused by antibiotic-resistant bacteria such as methicillin-resistant staphylococci and vancomysin-resistant enterococci.
Keywords: Gram-negative bacteria; Gram-positive bacteria; Group IIA phospholipase A2; Infection; Inflammation; Innate immunity;

A recent population-based study showed that cholesteryl ester transfer protein (CETP) gene variations, which relate to lower plasma CETP, may predict increased cardiovascular risk, in spite of higher HDL cholesterol. Among other functions, CETP activity contributes to cellular cholesterol efflux, an early step in the anti-atherogenic reverse cholesterol transport (RCT) process. We hypothesized that cellular cholesterol efflux stimulating capacity of plasma could be associated with CETP gene variation. In this study, we tested the extent to which the ability of plasma to promote cholesterol efflux from cultured human fibroblasts is associated with CETP gene variation. In 223 men, the − 629C→A CETP promoter polymorphism, plasma lipids, CETP mass, cholesteryl ester transfer (CET), lecithin:cholesterol acyltransferase (LCAT) activity and the ability of plasma to promote cholesterol efflux from human skin fibroblasts, obtained from a single normolipidemic donor, were determined. In − 629CC homozygotes (n  = 52), cholesterol efflux, plasma CETP mass, CET and LCAT activity were higher, whereas HDL cholesterol was lower compared to − 629 AA homozygotes (n  = 62) and − 629CA + AA carriers (n  = 171) (P  < 0.05 to P  < 0.001). Univariate correlation analysis showed that cellular cholesterol efflux was related to CETP genotype (P  = 0.04), plasma CET (P<0.05), LCAT activity (P  < 0.001) and apo A–I (P  < 0.05). Multiple linear regression analysis confirmed the independent association of cellular cholesterol efflux to plasma with CETP genotype. In conclusion, an association of cellular cholesterol efflux with the − 629C→A CETP polymorphism, possibly also involving LCAT activity, could provide a mechanism explaining why CETP gene variation, which relates to lower plasma CETP, does not confer diminished cardiovascular risk.
Keywords: Cellular cholesterol efflux; Cholesteryl ester transfer protein; Cholesteryl ester transfer; Cholesteryl ester transfer protein promoter polymorphism; Lecithin:cholesterol acyltransferase; HDL cholesterol; Reverse cholesterol transport; Triglycerides;

Clostridium scindens baiCD and baiH genes encode stereo-specific 7α/7β-hydroxy-3-oxo-Δ4-cholenoic acid oxidoreductases by Dae-Joong Kang; Jason M. Ridlon; Doyle Ray Moore; Stephen Barnes; Phillip B. Hylemon (16-25).
Secondary bile acids, formed by intestinal bacteria, are suggested to play a significant role in cancers of the gastrointestinal tract in humans. Bile acid 7α/β-dehydroxylation is carried out by a few species of intestinal clostridia which harbor a multi-gene bile acid inducible (bai) operon. Several genes encoding enzymes in this pathway have been cloned and characterized. However, no gene product(s) has yet been assigned to the production of 3-oxo-Δ4-cholenoic acid intermediates of cholic acid (CA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA). We previously reported that the baiH gene encodes an NADH:flavin oxidoreductase (NADH:FOR); however, the role of this protein in bile acid 7-dehydroxylation is unclear. Homology searches and secondary structural alignments suggest this protein to be similar to flavoproteins which reduce α/β-unsaturated carbonyl compounds. The baiH gene product was expressed in Escherichia coli, purified and discovered to be a stereo-specific NAD(H)-dependent 7β-hydroxy-3-oxo-Δ4-cholenoic acid oxidoreductase. Additionally, high sequence similarity between the baiH and baiCD gene products suggests the baiCD gene may encode a 3-oxo-Δ4-cholenoic acid oxidoreductase specific for CDCA and CA. We tested this hypothesis using cell extracts prepared from E. coli overexpressing the baiCD gene and discovered that it encodes a stereo-specific NAD(H)-dependent 7α-hydroxy-3-oxo-Δ4-cholenoic acid oxidoreductase.
Keywords: Bile acid 7-dehydroxylation; Flavoprotein; Old yellow enzyme; Enoate reductase; 2,4-dienoyl-CoA reductase; Ursodeoxycholic acid; Clostridium; baiCD; baiH;

Atorvastatin inhibits GSK-3β phosphorylation by cardiac hypertrophic stimuli by Anna Planavila; Ricardo Rodríguez-Calvo; Xavier Palomer; Teresa Coll; Rosa M. Sánchez; Manuel Merlos; Juan C. Laguna; Manuel Vázquez-Carrera (26-35).
In this study we examined the effect of the statin atorvastatin on the Akt/GSK-3β pathway. Our findings indicate that atorvastatin treatment for 15 days inhibited pressure overload-induced cardiac hypertrophy and prevented nuclear translocation of GATA4 and c-Jun and AP-1 DNA-binding activity. In addition, atorvastatin treatment prevented the increase in the phosphorylation of Akt and GSK-3β caused by cardiac hypertrophy, and this effect correlated with an increase in protein levels of phosphatase and tensin homolog on chromosome 10 (PTEN), which negatively regulates the phosphoinositide-3 kinase/Akt pathway. To test whether the inhibitory effect of atorvastatin on Akt and GSK-3β phosphorylation was direct we performed in vitro studies using embryonic rat heart-derived H9c2 cells, human AC16 cardiomyoblasts and neonatal rat cardiomyocytes. Preincubation of cells with atorvastatin prevented Akt/GSK-3β phosphorylation by different hypertrophic stimuli without affecting PTEN protein levels. However, atorvastatin prevented endogenous reactive oxygen species (ROS) generation and PTEN oxidation, a process that correlates with its inactivation, suggesting that atorvastatin prevents ROS-induced PTEN inactivation in acute treatments. These findings point to a new potential anti-hypertrophic effect of statins, which can prevent activation of the Akt/GSK-3β hypertrophic pathway by modulating PTEN activation by different mechanisms in chronic and acute treatments.
Keywords: Atorvastatin; Cardiac hypertrophy; GSK-3β; PTEN;

Hutchinson–Gilford progeria syndrome (HGPS) is a progeroid syndrome characterized by multiple aging-like disease phenotypes. We recently reported that a protein farnesyltransferase inhibitor (FTI) improved several disease phenotypes in mice with a HGPS mutation (Lmna HG/+). Here, we investigated the impact of an FTI on the survival of Lmna HG/+ mice. The FTI significantly improved the survival of both male and female Lmna HG/+ mice. Treatment with the FTI also improved body weight curves and reduced the number of spontaneous rib fractures. This study provides further evidence for a beneficial effect of an FTI in HGPS.
Keywords: Progeria; Aging; Protein farnesyltransferase inhibitor; Knock-in mice;

Ceramide traffic in C6 glioma cells: Evidence for CERT-dependent and independent transport from ER to the Golgi apparatus by Paola Giussani; Thomas Colleoni; Loredana Brioschi; Rosaria Bassi; Kentaro Hanada; Guido Tettamanti; Laura Riboni; Paola Viani (40-51).
Intracellular movements of ceramide are strongly limited by its hydrophobic nature, and the mechanisms involved in ceramide transport can represent a crucial aspect of sphingolipid metabolism and signaling. The recent identification of the ceramide specific carrier protein CERT has revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin biosynthesis. In this study we investigated the metabolic and functional role of CERT in C6 glioma cells. These cells were found to constitutively express CERT, the protein being mainly associated with the cytosolic fraction. Metabolic experiments performed with different radioactive metabolic precursors of sphingolipids demonstrated that the down regulation of CERT by RNAi technology resulted in a significant but not complete reduction of ceramide metabolism to sphingomyelin, without affecting its utilization for glycosphingolipid biosynthesis. Since nitric oxide is an inhibitor of ceramide ER-to-Golgi traffic and metabolism in C6 glioma cells, we evaluated the possibility that the CERT-mediated transport of ceramide might represent a target for nitric oxide. The data obtained demonstrate that CERT down regulation does not affect the inhibitory activity of nitric oxide on Cer metabolism, and the effects of nitric oxide and CERT silencing on ceramide utilization were additive. These results strongly suggest that a CERT-mediated and a CERT-independent, nitric oxide-sensitive Cer transport coexist in C6 glioma cells and can separately contribute to the control of sphingolipid metabolism and Cer levels in these cells.
Keywords: Ceramide; CERT; Nitric oxide; Glioma cells; Sphingolipid traffic;

Effect of polyunsaturated fatty acids on endocannabinoid and N-acyl-ethanolamine levels in mouse adipocytes by Isabel Matias; Gianfranca Carta; Elisabetta Murru; Stefania Petrosino; Sebastiano Banni; Vincenzo Di Marzo (52-60).
The tissue concentrations of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoyl-ethanolamine (anandamide), are altered in the adipose tissue of mice fed a high fat diet. We have investigated here the effect on endocannabinoid levels of incubation of mouse 3T3-F442A adipocytes with several free polyunstaurated fatty acids (PUFAs), including linolenic acid (LA), α-linolenic acid (ALA), arachidonic acid (AA) and docosahexaenoic acid (DHA), as well as oleic acid (OA) and palmitic acid (PA). By using mass spectrometric methods, we quantified the levels of endocannabinoids, of two anandamide congeners, N-palmitoyl-ethanolamine (PEA) and N-oleoyl-ethanolamine (OEA), and of fatty acids esterified in triacylglycerols or phospholipids, which act as 2-AG and/or N-acyl-ethanolamine precursors. Incubation with AA strongly elevated 2-AG levels and the amounts of AA esterified in triacylglycerols and on glycerol carbon atom 2 (sn-2), but not 1 (sn-1), in phospholipids. Incubation with DHA decreased 2-AG and anandamide levels and the amounts of AA esterified on both the sn-2 and sn-1 position of phospholipids, but not on triacylglycerols. PEA levels augmented following incubation of adipocytes with OA and PA, with no corresponding changes in phospholipids and triacylglycerols. We suggest that dietary PUFAs might modulate the levels of adipocyte phospholipids that act as endocannabinoid precursors.
Keywords: Endocannabinoid; Fatty acid; Polyunsaturated fatty acid; Diet; Obesity; Cannabinoid receptor; Anandamide; 2-arachidonoylglycerol;

Insulin-induced translocation of CD36 to the plasma membrane is reversible and shows similarity to that of GLUT4 by Masja M. van Oort; Jan M. van Doorn; Arend Bonen; Jan F.C. Glatz; Dick J. van der Horst; Kees W. Rodenburg; Joost J.F.P. Luiken (61-71).
In cardiac and skeletal muscles, insulin regulates the uptake of long-chain fatty acid (LCFA) via the putative LCFA transporter CD36. Biochemical studies propose an insulin-induced translocation of CD36 from intracellular pools to the plasma membrane (PM), similar to glucose transporter 4 (GLUT4) translocation. To characterize insulin-induced CD36 translocation in intact cells, Chinese hamster ovary (CHO) cells stably expressing CD36 or myc-tagged GLUT4 (GLUT4myc) were created. Immuno-fluorescence microscopy revealed CD36 to be located both intracellularly (in – at least partially – different compartments than GLUT4myc) and at the PM. Upon stimulation with insulin, CD36 translocated to a PM localization similar to that of GLUT4myc; the increase in PM CD36 content, as quantified by surface-protein biotinylation, amounted to 1.7-fold. The insulin-induced CD36 translocation was shown to be phosphatidylinositol-3 kinase-dependent, and reversible (as evidenced by insulin wash-out) in a similar time frame as that for GLUT4. The expression of GLUT4myc in non-stimulated cells, and the insulin-induced increase in PM GLUT4myc correlated with increased deoxyglucose uptake. By contrast, CD36 expression in non-stimulated cells and the insulin-induced increase in PM CD36 were not paralleled by a rise in LCFA uptake, suggesting that in these cells, such increase requires additional proteins, or a protein activation step. Taken together, this study is the first to present morphological evidence for CD36 translocation, and shows this process to resemble GLUT4 translocation.
Keywords: Insulin; CD36; GLUT4; Translocase; Long-chain fatty acid; Translocation; Signal transduction; CHO cells; Immuno-fluorescence microscopy; Membrane permeabilization;

The concentrations of plasma glucosylceramide (GlcCer) and ceramide (Cer) were determined in a cohort of type 1 Gaucher disease patients. In plasma of untreated patients, GlcCer concentrations were on average 3-fold increased (median Gaucher: 17.5 nmol/ml, range: 6.5–45.5 (n  = 27); median control: 5.9 nmol/ml, range 4.0–8.6 (n  = 15)). Although plasma Cer concentrations were not significantly different between the two groups (median Gaucher: 7.2 nmol/ml, range: 4.2–10.9 (n  = 27); median control: 7.8 nmol/ml, range 5.7–11.9 (n  = 15)) in individual patients plasma GlcCer/Cer ratio yields slightly better discrimination between Gaucher disease patients and normal individuals than the GlcCer levels. Positive correlations were detected between plasma GlcCer concentration and GlcCer/Cer ratio and severity of disease, plasma chitotriosidase and CCL18, surrogate markers of storage cells. Gaucher disease is treated by enzyme replacement and substrate reduction therapy. Both therapies were found to result in decreases in plasma GlcCer already within 6 months, without causing abnormal plasma GlcCer or Cer concentrations. The corrections in plasma GlcCer were most robust in patients with a pronounced clinical response. In conclusion, plasma GlcCer concentration and GlcCer/Cer ratio is of value to monitor Gaucher disease manifestation and response to therapeutic intervention.
Keywords: Glucosylceramide; Ceramide; Chitotriosidase; Enzyme replacement therapy; Gaucher disease type I; Substrate reduction therapy;