BBA - Molecular and Cell Biology of Lipids (v.1736, #3)

Long-chain fatty acid uptake and FAT/CD36 translocation in heart and skeletal muscle by Debby P.Y. Koonen; Jan F.C. Glatz; Arend Bonen; Joost J.F.P. Luiken (163-180).
Cellular long-chain fatty acid (LCFA) uptake constitutes a process that is not yet fully understood. LCFA uptake likely involves both passive diffusion and protein-mediated transport. Several lines of evidence support the involvement of a number of plasma membrane-associated proteins, including fatty acid translocase (FAT)/CD36, plasma membrane-bound fatty acid binding protein (FABPpm), and fatty acid transport protein (FATP). In heart and skeletal muscle primary attention has been given to unravel the mechanisms by which FAT/CD36 expression and function are regulated. It appears that both insulin and contractions induce the translocation of intracellular stored FAT/CD36 to the plasma membrane to increase cellular LCFA uptake. This review focuses on this novel mechanism of regulation of LCFA uptake in heart and skeletal muscle in health and disease. The distinct signaling pathways underlying insulin-induced and contraction-induced FAT/CD36 translocation will be discussed and a comparison will be made with the well-defined glucose transport system involving the glucose transporter GLUT4. Finally, it is hypothesized that malfunctioning of recycling of these transporters may lead to intracellular triacylglycerol (TAG) accumulation and cellular insulin resistance. Current data indicate a pivotal role for FAT/CD36 in the regulation of LCFA utilization in heart and skeletal muscle under normal conditions as well as during the altered LCFA utilization observed in obesity and insulin resistance. Hence, FAT/CD36 might provide a useful therapeutic target for the prevention or treatment of insulin resistance.
Keywords: Long-chain fatty acid uptake; FAT/CD36; Insulin; PI(3)K; Contraction; GLUT4;

The presence of oxidized phosphatidylserine on Fas-mediated apoptotic cell surface by Tatsuya Matsura; Aki Togawa; Masachika Kai; Tadashi Nishida; Junya Nakada; Yuichi Ishibe; Shosuke Kojo; Yorihiro Yamamoto; Kazuo Yamada (181-188).
A growing body of evidence suggests that phosphatidylserine (PS) oxidation is linked with its transmembrane migration from the inner to the outer leaflet of the plasma membrane during apoptosis. However, there is no direct evidence for the presence of oxidized PS (PSox) on the surface of cells undergoing apoptosis. The present study was performed to detect PSox externalized to the cell surface after Fas engagement in Jurkat cells. Treatment of Jurkat cells with anti-Fas antibody induced caspase-3 activation, chromatin condensation, PS externalization, generation of reactive oxygen species, intracellular glutathione depletion, disruption of mitochondrial transmembrane potential and release of cytochrome c from mitochondria. To determine externalized PS and phosphatidylethanolamine (PE), Jurkat cells were treated with anti-Fas antibody and then labeled with membrane-impermeable fluorescamine, a probe for visualizing lipids that contain primary amino groups. Their total lipids were extracted and subjected to two-dimensional high-performance thin-layer chromatography (HPTLC). The HPTLC plate was sprayed with N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride to detect phospholipid hydroperoxides. PSox was present in small amounts within but not on the surface of normal cells. Treatment with anti-Fas antibody increased PSox within the cells and caused PSox to appear on the cell surface. In contrast, PE on the surface of Fas-ligated cells was not oxidized. Thus, the present study demonstrates for the first time the presence of PSox both within and on the surface of apoptotic cells.
Keywords: Apoptosis; Oxidized phosphatidylserine; Phosphatidylserine externalization; Reactive oxygen species; Cytochrome c;

Insulin, glucagon and fatty acid treatment of hepatocytes does not result in phosphorylation or changes in activity of triacylglycerol hydrolase by Dean Gilham; Kathleen R. Perreault; Charles F.B. Holmes; David N. Brindley; Dennis E. Vance; Richard Lehner (189-199).
It is recognized that the majority of very low density lipoprotein (VLDL) associated triacylglycerol (TG) is synthesized from fatty acids and partial acylglycerols generated by lipolysis of intra-hepatic storage rather than made de novo. Triacylglycerol hydrolase (TGH) is involved in mobilizing stored TG. Modulating the ability of TGH to hydrolyze stored lipids represents a potentially regulated and rate limiting step in VLDL assembly. Phosphorylation of lipases and carboxylesterases trigger diverse but functionally significant events. We explored the potential for regulating the mobilization of hepatic TG through phosphorylation of TGH. Insulin is known to suppress VLDL secretion from liver, and glucagon can be considered an opposing hormone. However, neither insulin nor glucagon treatment of hepatocytes led to phosphorylation of TGH or changes in its activity. Augmenting intracellular TG stores by incubations with oleic acid also did not lead to changes in TGH activity. Therefore, changes in phosphorylation state are not a mechanism for regulating TGH activity, access to TG substrate pools or for TGH-mediated contributions to VLDL assembly and secretion.
Keywords: Triacylglycerol; Triacylglycerol hydrolase; Apolipoprotein B; Phosphorylation; VLDL;

Diverse cellular localizations of secretory phospholipase A2 enzymes in several human tissues by Seiko Masuda; Makoto Murakami; Yukio Ishikawa; Toshiharu Ishii; Ichiro Kudo (200-210).
The secretory phospholipase A2 (sPLA2) family in mammals contains more than 10 enzymes. In this study, we examined by immunohistochemistry the localization of six sPLA2s (IIA, IID, IIE, IIF, V and X) in human heart, kidney, liver and stomach. In normal hearts, sPLA2-IIA was detected in coronary vascular smooth muscle cells (VSMC) and sPLA2-V in cardiomyocytes beneath the endocardium. In infarcted hearts, expression of these two enzymes was markedly increased in damaged cardiomyocytes, and expression of sPLA2-IID and-IIE, which was undetectable in normal hearts, was elevated in damaged cardiomyocytes and VSMC, respectively. In infarcted kidneys, sPLA2-IIA and-V were markedly induced in the uriniferous tubular epithelium. In livers affected by viral hepatitis, sPLA2-IIA and-V were expressed in hepatocytes with fatty degeneration. In the gastric glands exhibiting intestinal metaplasia, sPLA2-IIA was localized in the glandular base, sPLA2-IID and-V in the glandular body epithelium, sPLA2-IIE and-IIF in goblet cells in the foveolar epithelium, and sPLA2-X in both glandular body epithelial cells and foveolar epithelial goblet cells. In the gastric submucosal tissues, sPLA2-IIA and-IIE were located in VSMC and sPLA2-V was in the interstitial fibroblasts. In addition, sPLA2-IIA,-IIE,-IIF and-X were highly expressed in gastric signet ring cell carcinoma. Thus, individual sPLA2s exhibit unique cellular localizations in each tissue, suggesting their distinct roles in pathophysiology.
Keywords: Phospholipase A 2 ; Immunohistochemistry; Inflammation; Infarction; Cancer;

Involvement of N-acylethanolamine-hydrolyzing acid amidase in the degradation of anandamide and other N-acylethanolamines in macrophages by Yong-Xin Sun; Kazuhito Tsuboi; Li-Ying Zhao; Yasuo Okamoto; Didier M. Lambert; Natsuo Ueda (211-220).
Bioactive N-acylethanolamines including the endocannabinoid anandamide are known to be hydrolyzed to fatty acids and ethanolamine by fatty acid amide hydrolase (FAAH). In addition, we recently cloned an isozyme termed “N-acylethanolamine-hydrolyzing acid amidase (NAAA)”, which is active only at acidic pH [Tsuboi, Sun, Okamoto, Araki, Tonai, Ueda, J. Biol. Chem. 285 (2005) 11082–11092]. However, physiological roles of NAAA remained unclear. Here, we examined a possible contribution of NAAA to the degradation of various N-acylethanolamines in macrophage cells. NAAA mRNA as well as FAAH mRNA was detected in several macrophage-like cells, including RAW264.7, and mouse peritoneal macrophages. The homogenates of RAW264.7 cells showed both the NAAA and FAAH activities which were confirmed with the aid of their respective specific inhibitors, N-cyclohexanecarbonylpentadecylamine (CCP) and URB597. As analyzed with intact cells, RAW264.7 cells and peritoneal macrophages degraded anandamide, N-palmitoylethanolamine, N-oleoylethanolamine, and N-stearoylethanolamine. Pretreatment of the cells with CCP or URB597 partially inhibited the degradation, and a combination of the two compounds caused more profound inhibition. In contrast, the anandamide hydrolysis in mouse brain appeared to be principally attributable to FAAH despite the expression of NAAA in the brain. These results suggested that NAAA and FAAH cooperatively degraded various N-acylethanolamines in macrophages.
Keywords: N-Acylethanolamine; N-Acylethanolamine-hydrolyzing acid amidase; Anandamide; Endocannabinoid; Macrophage; Fatty acid amide hydrolase;

The pituitary is important in the control of lipid metabolism and studies of hypophysectomized (Hx) rats have shown strong effects of growth hormone (GH) on bile acid synthesis, hepatic LDL receptor (LDLR) expression and on the sensitivity to dietary cholesterol. It is unclear if mice may be used in such studies. The aim of the current study was to evaluate if Hx mice may be used to further explore how GH modulates cholesterol and bile acid metabolism, and to define the importance of the LDLR in this regulation by studying LDLR-deficient mice (LDLRko). Experiments on three mouse strains showed that, following Hx, HDL were reduced and LDL increased. Cholesterol/fat feeding of Hx mice increased serum cholesterol levels 2- to 3-fold. Serum triglycerides were reduced 50% in Hx mice; a further 30% reduction was seen after dietary cholesterol/fat. A serum marker for CYP7A1-mediated bile acid synthesis (C4) increased 2-fold in intact mice on cholesterol/fat diet. In Hx mice C4 levels were reduced by 50% as compared to intact controls, but were unexpectedly increased to levels seen in normal mice upon cholesterol/fat feeding. Hx of LDLRko mice moderately increased LDL-cholesterol and reduced triglycerides and GH treatment attenuated these effects; serum C4 levels were increased by GH treatment in all groups. In conclusion, mice can be used to explore the role of the pituitary in lipid metabolism. CYP7A1 is generally reduced in Hx mice but has a normal stimulatory response following dietary cholesterol suggesting that faulty regulation of CYP7A1 is not important for the reduced resistance to dietary cholesterol in Hx mice. Further, the LDLR is only to a minor part involved in the pituitary regulation of serum cholesterol in mice.
Keywords: Hypophysectomy; Growth hormone; Mouse; Lipid metabolism; Bile acid;

5-Oxo-ETE analogs and the proliferation of cancer cells by Joseph T. O'Flaherty; LeAnn C. Rogers; Christian M. Paumi; Roy R. Hantgan; Lance R. Thomas; Carl E. Clay; Kevin High; Yong Q. Chen; Mark C. Willingham; Pamela K. Smitherman; Timothy E. Kute; Anuradha Rao; Scott D. Cramer; Charles S. Morrow (228-236).
MDA-MB-231, MCF7, and SKOV3 cancer cells, but not HEK-293 cells, expressed mRNA for the leukocyte G protein-coupled 5-oxo-eicosatetraenoate (ETE) OXE receptor. 5-Oxo-ETE, 5-oxo-15-OH-ETE, and 5-HETE stimulated the cancer cell lines but not HEK-293 cells to mount pertussis toxin-sensitive proliferation responses. Their potencies in eliciting this response were similar to their known potencies in activating leukocytes and OXE receptor-transfected cells. However, high concentrations of 5-oxo-ETE and 5-oxo-15-OH-ETE, but not 5-HETE, arrested growth and caused apoptosis in all four cell lines; these responses were pertussis toxin-resistant. The same high concentrations of the oxo-ETEs but again not 5-HETE also activated peroxisome proliferator-activated receptor (PPAR)-γ. Pharmacological studies indicated that this activation did not mediate their effects on proliferation. These results are the first to implicate the OXE receptor in malignant cell growth and to show that 5-oxo-ETEs activate cell death programs as well as PPARγ independently of this receptor.
Keywords: 5-Oxo-ETE; 5-HETE; Cancer cell; Proliferation; Apoptosis; PPAR;

Metal-catalyzed LDL oxidation is enhanced by the presence of homocysteine. In this study, the effectiveness of ascorbic acid against low-density lipoprotein (LDL) oxidation by iron(III) and copper(II) in the presence of homocysteine and the main plasma disulfide cystine was investigated. Relative to the degree of LDL oxidation reached in the absence of antioxidants, ascorbic acid was particularly effective against iron-catalyzed LDL oxidation at pH 6.0. This can be explained from its stability under acidic conditions and is likely to be important in ischemia, in inflammation and exhausting exercise. At pH 7.4, an ascorbic acid concentration at least as high as the concentration of homocysteine might be necessary to efficiently inhibit LDL oxidation by iron(III) and copper(II) in the presence of homocysteine and cystine. Histidine increased the efficiency of ascorbic acid as an antioxidant against copper-mediated oxidation in this system. The capacity of homocysteine to regenerate ascorbic acid from dehydroascorbic acid appeared to play a minor role in inhibition of ascorbic acid oxidation by copper as compared to copper chelation by homocysteine.
Keywords: Ascorbic acid; LDL oxidation; Acidic pH; Homocysteine; Iron; Copper;

Attenuation of breast tumor cell growth by conjugated linoleic acid via inhibition of 5-lipoxygenase activating protein by Jung-Hyun Kim; Neil E. Hubbard; Vincent Ziboh; Kent L. Erickson (244-250).
Conjugated linoleic acid (CLA) consists of a group of linoleic acid geometric isomers that have been shown to reduce tumor growth and metastasis in animal models of breast, prostate and colon cancer. To delineate a possible mechanism of action for CLA, we have recently shown that the 5-lipoxygenase product, 5-hydroxyeicosatetraenoic acid (5-HETE), could play a role in CLA alteration of mammary tumorigenesis. In this study, we determined how CLA could modulate 5-lipoxygenase activity. The t10, c12-CLA isomer reduced production of 5-HETE but not 12- and 15-HETE in MDA-MB-231 human breast tumor cells. That isomer and the c9, t11-CLA isomer decreased 5-HETE production by competition with the lipoxygenase substrate, arachidonic acid (AA). Interestingly, t10, c12-CLA reduced the expression of five-lipoxygenase activating protein (FLAP) but not the 5-lipoxygenase enzyme. Over-expression of FLAP abrogated t10, c12-CLA-reduced viability of MDA-MB-231 cells. These data suggest that the reduction of 5-HETE by t10, c12-CLA was due to competition with AA and the reduction of FLAP expression.
Keywords: Conjugated linoleic acid; 5-hydroxyeicosatetraenoic acid; 5-lipoxygenase; 5-lipoxygenase-activating protein;