BBA - Molecular and Cell Biology of Lipids (v.1584, #2-3)

The nuclear phosphoinositide 3-kinase/AKT pathway: a new second messenger system by Luca M Neri; Paola Borgatti; Silvano Capitani; Alberto M Martelli (73-80).
Lipid second messengers, particularly those derived from the polyphosphoinositide cycle, play a pivotal role in several cell signaling networks. Phosphoinositide 3-kinases (PI3Ks) generate specific inositol lipids that have been implicated in a plethora of cell functions. One of the best-characterized targets of PI3K lipid products is the serine/threonine protein kinase Akt. Recent findings have implicated Akt in cancer progression because it stimulates cell proliferation and suppresses apoptosis. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid metabolism, and suggests that lipid molecules are important components of signaling pathways operating within the nucleus. PI3Ks, their lipid products, and Akt have also been identified at the nuclear level. In this review, we shall summarize the most updated findings about these molecules in relationship with the nuclear compartment and provide an overview of the possible mechanisms by which they regulate important cell functions.
Keywords: PI3K; Akt; Nucleus; Signal transduction; Transcription factor; Cell differentiation;

We have shown that overnight lipopolysaccharide (LPS) suppresses alveolar macrophage (AM) leukotriene (LT) synthesis mediated in part by induction of inducible nitric oxide synthase (iNOS) and NO production. Here we examined the possibility that reactive oxygen intermediates (ROI) generated by LPS pretreatment contribute to the suppression of 5-lipoxygenase (5-LO) metabolism. Pretreatment of AM with xanthine/xanthine oxidase, which generates high concentrations of ROI, resulted in suppression of LT synthetic capacity. Since NO and ROI reactive species are known to react and form peroxynitrite (ONOO), we examined the effect of ONOO on 5-LO metabolism. Exogenous ONOO caused a dose-dependent suppression of recombinant 5-LO cell-free activity. ONOO also suppressed LT synthesis in intact AM, which was reversed by the ONOO scavenger tetrakis(4-benzoic acid)porphyrin. ONOO treatment also resulted in dose-dependent nitrotyrosination and S-nitrosylation of the recombinant 5-LO enzyme. Since the direct 5-LO inhibitor zileuton prevents the LPS-induced suppression of LT synthesis, we examined if 5-LO itself was the source of ROI. Zileuton reduced ROI generation in LPS-treated cells. These studies identify an important role for ROI and ONOO in the suppression of 5-LO metabolism by LPS.
Keywords: Leukotriene; Eicosanoid; Reactive oxygen intermediate; Nitric oxide;

A method for quantitative acylcarnitine profiling in human skin fibroblasts using unlabelled palmitic acid: diagnosis of fatty acid oxidation disorders and differentiation between biochemical phenotypes of MCAD deficiency by Jürgen G Okun; Stefan Kölker; Andreas Schulze; Dirk Kohlmüller; Katharina Olgemöller; Martin Lindner; Georg F Hoffmann; Ronald J.A Wanders; Ertan Mayatepek (91-98).
Inherited disorders of fatty acid oxidation are a group of acute life-threatening but treatable disorders, clinically complicated by severe hypoketotic hypoglycemia precipitated by prolonged fasting. Among them, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is by far the most frequent disorder.Here we report a modified method for quantitative acylcarnitine profiling by electrospray ionisation-tandem mass spectrometry (ESI-MS-MS) in human skin fibroblasts using unlabelled palmitic acid as substrate. The reliability of this method was tested in cultured skin fibroblasts from previously diagnosed patients with specific carnitine cycle and fatty acid β-oxidation defects. Furthermore, acylcarnitine profiling was investigated in fibroblasts and dried blood spots from patients with different variants of MCAD deficiency.ESI-MS-MS-based investigation of cultured skin fibroblasts from patients with disorders of fatty acid oxidation revealed a pathognomonic acylcarnitine profiling. In addition, this method delineated different variants of MCAD deficiency, i.e. mild and classical. The octanoylcarnitine (C8)-to-decanoylcarnitine (C10) and C8-to-acetylcarnitine (C2) ratios were the most specific markers to differentiate mild and classical forms of MCAD deficiency in fibroblasts. Similar results were obtained by quantitative acylcarnitine profiling in dried blood spots. In conclusion, this novel technique is a powerful tool for the investigation of fatty acid oxidation disorders under standardized conditions in fibroblasts.
Keywords: Acylcarnitine profiling; Beta-oxidation; Human skin fibroblast; Medium-chain acyl-CoA dehydrogenase deficiency; Palmitic acid; Tandem-mass spectrometry;

Daunorubicin- and Ara-C-induced interphasic apoptosis of human Type II leukemia cells is caspase-8-independent by Anne-Dominique Terrisse; Christine Bezombes; Sandra Lerouge; Guy Laurent; Jean-Pierre Jaffrézou (99-103).
Drug-induced interphasic apoptosis in human leukemia cells is mediated through intracellular signaling pathways, of which the most proximal (initiating) event remains unclear. Indeed, both early ceramide generation and procaspase-8 cleavage have been individually identified as the initial apoptotic signaling events which precede the mitochondrial control of the apoptotic execution phase in Type II cells. In order to evaluate whether or not procaspase-8 cleavage is requisite for initial ceramide generation and rapid interphasic apoptosis, we investigated the chronological ordering of early ceramide generation and caspase-8 cleavage induced by daunorubicin (DNR) and 1-β-d-arabinofuranosylcytosine (Ara-C) in U937 cells. We further evaluated the impact of these two drugs on initial ceramide generation and apoptosis in wild-type Jurkat cells and Jurkat clones mutated for caspase-8 and Fas-associated death domain. We show that while both DNR and Ara-C similarly induced early ceramide generation (within 5–20 min) and interphasic apoptosis in all cell models, caspase-8 cleavage was only observed farther downstream (4.5 h) and only in DNR-treated cells. Furthermore, neither DNR or Ara-C induced caspase-8 activation. These results demonstrate that caspase-8 cleavage is not requisite for the drug-induced activation of the ceramide-mediated interphasic apoptotic pathway in human Type II leukemic cells.
Keywords: Daunorubicin; 1-β-d-arabinofuranosylcytosine; Ceramide; Apoptosis; Caspase-8;

Increase of ceramide in adriamycin-induced HL-60 cell apoptosis: detection by a novel anti-ceramide antibody by Mamoru Kawase; Mitsumasa Watanabe; Tadakazu Kondo; Takeshi Yabu; Yoshimitsu Taguchi; Hisanori Umehara; Takashi Uchiyama; Koji Mizuno; Toshiro Okazaki (104-114).
We recently raised an IgM class of monoclonal antibody (Ab) for ceramide (NHCER-2), and examined its specificity and sensitivity. Enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC) showed that NHCER-2 recognized ceramides but not other sphingolipids such as sphingosine, sphinganine, sphingomyelin, sphingosine-1-phosphate, ceramide-1-phosphate, glucosylceramide and cerebroside. In addition, N-hexanoyl, N-octanoyl and N-palmitoylsphingosine were detected by NHCER-2, but N-acetylsphingosine and dihydroceramide were not. Densities of ceramide detected by NHCER-2 were proportional to the amounts of ceramide standard up to 250 ng. When various concentrations of adriamycin (ADR) was added to induce apoptosis, the amounts of ceramide detected by NHCER-2 time- and dose-dependently increased in apoptosis-sensitive HL-60 cells as well as by DGK assay, but not in apoptosis-resistant HL-60/ADR cells. After cell fractionation, ceramide levels judged not only by diacylglycerol kinase (DGK) assay but also by NHCER-2 were shown to increase in the microsomal and the nuclear fraction in apoptosis-sensitive cells, but not in apoptosis-resistant cells. Moreover, absolute amounts of ceramide determined by NHCER-2 were well correlated with those by DGK assay. These results suggest that increase of ceramide in the nuclear fraction as well as in the microsomal fraction may play a role in ADR-induced apoptosis and that a novel anti-ceramide Ab NHCER-2 could be beneficial to investigate changes of ceramide content in the cells.
Keywords: Ceramide; Adriamycin; Leukemia; Drug resistance; Apoptosis;

Circulating adiponectin levels fall whereas leptin levels rise with obesity, suggesting that regulation of these two adipocyte-derived hormones may be simultaneously influenced by common obesity-related factors. We examined adiponectin mRNA levels in WAT and in some instances, brown adipose tissue (BAT) following fasting and refeeding, acute and chronic administration of a β3-adrenergic agonist, acute treatment with retinoic acid (RA) and a glucocorticoid, and following chronic infusion of leptin and compared the expression of adiponectin to that of leptin in each circumstance. Serum concentrations of adiponectin were also reported for most of the treatments. Fasting diminished and refeeding reversed both adiponectin and leptin gene expression. Peripheral injection of the β3-adrenergic agonist, CL316,243, suppressed both leptin and adiponectin expression in WAT. A small but significant reduction in adiponectin expression in BAT was also observed following this treatment. Although CL316,23 lowered serum leptin levels markedly, it did not affect serum adiponectin levels. A chronic 7-day infustion of CL316,243 resulted in an elevation of adiponectin expression in WAT and serum concentrations in contrast to suppressions in both mRNA and serum levels of leptin by a similar treatment as previously reported. Chronic administration of leptin did not alter adiponectin synthesis in WAT compared to controls, but prevented the reduction in adiponectin synthesis associated with pair feeding. Food restriction through pair feeding also diminished adiponectin expression in BAT. Collectively, although leptin and adiponectin are inversely correlated with obesity, leptin does not appear to participate directly in adiponectin synthesis. The short-term regulation of the two adipokine expression in WAT is somewhat similar, perhaps subjective to common control of energy balance. The long-term regulation of adiponectin expression in WAT appears to be the opposite of that of leptin and may be more sensitive to changes in adiposity or insulin sensitivity.
Keywords: Adiponectin; Leptin; Adipose tissue; Fasting; CL316,243;

Evaluation of apolipoprotein B-100 fragmentation and cross-linkage in serum as an index of atherosclerosis by Ryoko Hashimoto; Nao Matsukawa; Yoko Nariyama; Yukako Ogiri; Eri Hamagawa; Kyoko Tanaka; Yasue Usui; Satomi Nakano; Taro Maruyama; Singo Kyotani; Motoo Tsushima; Shosuke Kojo (123-128).
It is well established that radical reaction of low density lipoprotein (LDL) causes fragmentation and cross-linkage of apolipoprotein B-100 (apoB). Our previous studies demonstrated that fragmented and cross-linked apoB proteins are present in normal human serum and tended to increase with age based on immunoblot analysis. These observations suggest that the fragmentation and cross-linkage pattern of apoB reflects the oxidative stress in an individual and that this pattern is a good atherosclerotic index. In this study, a method was developed to evaluate the fragmentation and conjugation pattern of apoB. A parameter named B-ox was introduced for each serum sample to quantitate the staining bands of the immunoblotting analysis. B-ox represents the relative abundance of radical reaction products (a sum of fragmented and conjugated apoB proteins) based on one control subject. If this value increases, it indicates that radical reaction products have increased, i.e., the oxidative stress has increased in the subject. Based on measurements of subjects in a rural area of Japan, B-ox showed significant positive correlation with intima–media thickness (IMT) of the carotid artery, LDL cholesterol, and age, while it showed significant negative correlation with high density lipoprotein (HDL) cholesterol and vitamin C. These results suggest that B-ox is a reliable indicator of atherosclerosis.
Keywords: Apolipoprotein B; Atherosclerosis; Intima–media thickness; LDL; Protein degradation; Vitamin C;

Author Index (129-130).

Cumulative Contents (131-132).