Journal of Lipid Research Current Content

In Memoriam: William E. Connor (1921-2009) [Tribute]

Patel, S. B., Steiner, R. D. — Tue, 09 Mar 2010 11:16:07 PST

The acute phase response inhibits reverse cholesterol transport [Commentary]

Feingold, K. R., Grunfeld, C. — Tue, 09 Mar 2010 11:16:07 PST

Lipid second messengers and related enzymes in vertebrate rod outer segments [Thematic Reviews]

Giusto, N. M., Pasquare, S. J., Salvador, G. A., Ilincheta de Boschero, M. G. — Tue, 09 Mar 2010 11:16:07 PST

Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.

INSIG1 influences obesity-related hypertriglyceridemia in humans [Research Articles]

Tue, 09 Mar 2010 11:16:07 PST

In our analysis of a quantitative trait locus (QTL) for plasma triglyceride (TG) levels [logarithm of odds (LOD) = 3.7] on human chromosome 7q36, we examined 29 single nucleotide polymorphisms (SNPs) across INSIG1, a biological candidate gene in the region. Insulin-induced genes (INSIGs) are feedback mediators of cholesterol and fatty acid synthesis in animals, but their role in human lipid regulation is unclear. In our cohort, the INSIG1 promoter SNP rs2721 was associated with TG levels (P = 2 x 10^–3 in 1,560 individuals of the original linkage cohort, P = 8 x 10^–4 in 920 unrelated individuals of the replication cohort, combined P = 9.9 x 10^–6). Individuals homozygous for the T allele had 9% higher TG levels and 2-fold lower expression of INSIG1 in surgical liver biopsy samples when compared with individuals homozygous for the G allele. Also, the T allele showed additional binding of nuclear proteins from HepG2 liver cells in gel shift assays. Finally, the variant rs7566605 in INSIG2, the only homolog of INSIG1, enhances the effect of rs2721 (P = 0.00117). The variant rs2721 alone explains 5.4% of the observed linkage in our cohort, suggesting that additional, yet-undiscovered genes and sequence variants in the QTL interval also contribute to alterations in TG levels in humans.

CGI-58/ABHD5 is a coenzyme A-dependent lysophosphatidic acid acyltransferase [Research Articles]

Tue, 09 Mar 2010 11:16:07 PST

Mutations in human CGI-58/ABHD5 cause Chanarin-Dorfman syndrome (CDS), characterized by excessive storage of triacylglycerol in tissues. CGI-58 is an /β-hydrolase fold enzyme expressed in all vertebrates. The carboxyl terminus includes a highly conserved consensus sequence (HXXXXD) for acyltransferase activity. Mouse CGI-58 was expressed in Escherichia coli as a fusion protein with two amino terminal 6-histidine tags. Recombinant CGI-58 displayed acyl-CoA-dependent acyltransferase activity to lysophosphatidic acid, but not to other lysophospholipid or neutral glycerolipid acceptors. Production of phosphatidic acid increased with time and increasing concentrations of recombinant CGI-58 and was optimal between pH 7.0 and 8.5. The enzyme showed saturation kinetics with respect to 1-oleoyl-lysophosphatidic acid and oleoyl-CoA and preference for arachidonoyl-CoA and oleoyl-CoA. The enzyme showed slight preference for 1-oleoyl lysophosphatidic acid over 1-palmitoyl, 1-stearoyl, or 1-arachidonoyl lysophosphatidic acid. Recombinant CGI-58 showed intrinsic fluorescence for tryptophan that was quenched by the addition of 1-oleoyl-lysophosphatidic acid, oleoyl-CoA, arachidonoyl-CoA, and palmitoyl-CoA, but not by lysophosphatidyl choline. Expression of CGI-58 in fibroblasts from humans with CDS increased the incorporation of radiolabeled fatty acids released from the lipolysis of stored triacylglycerols into phospholipids. CGI-58 is a CoA-dependent lysophosphatidic acid acyltransferase that channels fatty acids released from the hydrolysis of stored triacylglycerols into phospholipids.

Modulation of the activity of cytosolic phospholipase A2{alpha} (cPLA2{alpha}) by cellular sphingolipids and inhibition of cPLA2{alpha} by sphingomyelin [Research Articles]

Nakamura, H., Wakita, S., Suganami, A., Tamura, Y., Hanada, K., Murayama, T. — Tue, 09 Mar 2010 11:16:08 PST

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2 (cPLA2) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca²⁺ ionophore A23187 increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2 inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2. Phosphatidylcholine vesicles containing SM reduced cPLA2 activity. Furthermore, SM disturbed the binding of cPLA2 to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2-dependent release of AA by inhibiting the binding of cPLA2 to glycerophospholipids.

A novel role for ABCA1-generated large pre-{beta} migrating nascent HDL in the regulation of hepatic VLDL triglyceride secretion [Research Articles]

Chung, S., Gebre, A. K., Seo, J., Shelness, G. S., Parks, J. S. — Tue, 09 Mar 2010 11:16:08 PST

In Tangier disease, absence of ATP binding cassette transporter A1 (ABCA1) results in reduced plasma HDL and elevated triglyceride (TG) levels. We hypothesized that hepatocyte ABCA1 regulates VLDL TG secretion through nascent HDL production. Silencing of ABCA1 expression in oleate-stimulated rat hepatoma cells resulted in: 1) decreased large nascent HDL (>10 nm diameter) and increased small nascent HDL (<10 nm) formation, 2) increased large buoyant VLDL1 particle secretion, and 3) decreased phosphatidylinositol-3 (PI3) kinase activation. Nascent HDL-containing conditioned medium from rat hepatoma cells or HEK293 cells transfected with ABCA1 was effective in increasing PI3 kinase activation and reducing VLDL TG secretion in ABCA1-silenced hepatoma cells. Addition of isolated large nascent HDL particles to ABCA1-silenced hepatoma cells inhibited VLDL TG secretion to a greater extent than small nascent HDL. Similarly, addition of recombinant HDL, but not human plasma HDL, was effective in attenuating TG secretion and increasing PI3 kinase activation in ABCA1-silenced cells. Collectively, these data suggest that large nascent HDL particles, assembled by hepatic ABCA1, generate a PI3 kinase-mediated autocrine signal that attenuates VLDL maturation and TG secretion. This pathway may explain the elevated plasma TG concentration that occurs in most Tangier subjects and may also account, in part, for the inverse relationship between plasma HDL and TG concentrations in individuals with compromised ABCA1 function.

Myeloperoxidase and serum amyloid A contribute to impaired in vivo reverse cholesterol transport during the acute phase response but not group IIA secretory phospholipase A2 [Research Articles]

Tue, 09 Mar 2010 11:16:08 PST

Atherosclerosis is linked to inflammation. HDL protects against atherosclerotic cardiovascular disease, mainly by mediating cholesterol efflux and reverse cholesterol transport (RCT). The present study aimed to test the impact of acute inflammation as well as selected acute phase proteins on RCT with a macrophage-to-feces in vivo RCT assay using intraperitoneal administration of [³H]cholesterol-labeled macrophage foam cells. In patients with acute sepsis, cholesterol efflux toward plasma and HDL were significantly decreased (P < 0.001). In mice, acute inflammation (75 µg/mouse lipopolysaccharide) decreased [³H]cholesterol appearance in plasma (P < 0.05) and tracer excretion into feces both within bile acids (–84%) and neutral sterols (–79%, each P < 0.001). In the absence of systemic inflammation, overexpression of serum amyloid A (SAA, adenovirus) reduced overall RCT (P < 0.05), whereas secretory phospholipase A₂ (sPLA₂, transgenic mice) had no effect. Myeloperoxidase injection reduced tracer appearance in plasma (P < 0.05) as well as RCT (–36%, P < 0.05). Hepatic expression of bile acid synthesis genes (P < 0.01) and transporters mediating biliary sterol excretion (P < 0.01) was decreased by inflammation. In conclusion, our data demonstrate that acute inflammation impairs cholesterol efflux in patients and macrophage-to-feces RCT in vivo in mice. Myeloperoxidase and SAA contribute to a certain extent to reduced RCT during inflammation but not sPLA₂. However, reduced bile acid formation and decreased biliary sterol excretion might represent major contributing factors to decreased RCT in inflammation.

Baseline cholesterol absorption and the response to ezetimibe/simvastatin therapy: a post-hoc analysis of the ENHANCE trial [Research Articles]

Tue, 09 Mar 2010 11:16:08 PST

Subjects with increased cholesterol absorption might benefit more from statin therapy combined with a cholesterol absorption inhibitor. We assessed whether baseline cholesterol absorption markers were associated with response to ezetimibe/simvastatin therapy, in terms of LDL-cholesterol (LDL-C) lowering and cholesterol absorption inhibition, in patients with familial hypercholesterolemia (FH). In a posthoc analysis of the two-year ENHANCE trial, we assessed baseline cholesterol-adjusted campesterol (campesterol/TC) and sitosterol/TC ratios in 591 FH patients. Associations with LDL-C changes and changes in cholesterol absorption markers were evaluated by multiple regression analysis. No association was observed between baseline markers of cholesterol absorption and the extent of LDL-C response to ezetimibe/simvastatin therapy (β = 0.020, P = 0.587 for campesterol/TC and β<0.001, P = 0.992 for sitosterol/TC). Ezetimibe/simvastatin treatment reduced campesterol levels by 68% and sitosterol levels by 62%; reductions were most pronounced in subjects with the highest cholesterol absorption markers at baseline, the so-called high absorbers (P < 0.001). Baseline cholesterol absorption status does not determine LDL-C lowering response to ezetimibe/simvastatin therapy in FH, despite more pronounced cholesterol absorption inhibition in high absorbers. Hence, these data do not support the use of baseline absorption markers as a tool to determine optimal cholesterol lowering strategy in FH patients. However, due to the exploratory nature of any posthoc analysis, these results warrant further prospective evaluation in different populations.

Liver X receptor activation promotes macrophage-to-feces reverse cholesterol transport in a dyslipidemic hamster model [Research Articles]

Tue, 09 Mar 2010 11:16:08 PST

Liver X receptor (LXR) activation promotes reverse cholesterol transport (RCT) in rodents but has major side effects (increased triglycerides and LDL-cholesterol levels) in species expressing cholesteryl ester transfer protein (CETP). In the face of dyslipidemia, it remains unclear whether LXR activation stimulates RCT in CETP species. We therefore used a hamster model made dyslipidemic with a 0.3% cholesterol diet and treated with vehicle or LXR agonist GW3965 (30 mg/kg bid) over 10 days. To investigate RCT, radiolabeled ³H-cholesterol macrophages or ³H-cholesteryl oleate-HDL were then injected to measure plasma and feces radioactivity over 72 or 48 h, respectively. The cholesterol-enriched diet increased VLDL-triglycerides and total cholesterol levels in all lipoprotein fractions and strongly increased liver lipids. Overall, GW3965 failed to improve both dyslipidemia and liver steatosis. However, after ³H-cholesterol labeled macrophage injection, GW3965 treatment significantly increased the ³H-tracer appearance by 30% in plasma over 72 h, while fecal ³H-cholesterol excretion increased by 156% (P < 0.001). After ³H-cholesteryl oleate-HDL injection, GW3965 increased HDL-derived cholesterol fecal excretion by 64% (P < 0.01 vs. vehicle), while plasma fractional catabolic rate remained unchanged. Despite no beneficial effect on dyslipidemia, LXR activation promotes macrophage-to-feces RCT in dyslipidemic hamsters. These results emphasize the use of species with a more human-like lipoprotein metabolism for drug profiling.

FXR activation reverses insulin resistance and lipid abnormalities and protects against liver steatosis in Zucker (fa/fa) obese rats [Research Articles]

Cipriani, S., Mencarelli, A., Palladino, G., Fiorucci, S. — Tue, 09 Mar 2010 11:16:08 PST

The farnesoid X receptor (FXR) is a bile acid activated nuclear receptor. Zucker (fa/fa) rats, harboring a loss of function mutation of the leptin receptor, develop diabetes, insulin resistance, obesity, and liver steatosis. In this study, we investigated the effect of FXR activation by 6-ethyl-chenodeoxycholic acid, (6E-CDCA, 10 mg/kg) on insulin resistance and liver and muscle lipid metabolism in fa/fa rats and compared its activity with rosiglitazone (10 mg/kg) alone or in combination with 6E-CDCA (5 mg/kg each). In comparison to lean (fa/+), fa/fa rats on a normal diet developed insulin resistance and liver steatosis. FXR activation protected against body weight gain and liver and muscle fat deposition and reversed insulin resistance as assessed by insulin responsive substrate-1 phosphorylation on serine 312 in liver and muscles. Activation of FXR reduced liver expression of genes involved in fatty acid synthesis, lipogenesis, and gluconeogenesis. In the muscles, FXR treatment reduced free fatty acid synthesis. Rosiglitazone reduced blood insulin, glucose, triglyceride, free fatty acid, and cholesterol plasma levels but promoted body weight gain (20%) and liver fat deposition. FXR activation reduced high density lipoprotein plasma levels. In summary, FXR administration reversed insulin resistance and correct lipid metabolism abnormalities in an obesity animal model.

Analysis of lipid transfer activity between model nascent HDL particles and plasma lipoproteins: implications for current concepts of nascent HDL maturation and genesis [Research Articles]

Tue, 09 Mar 2010 11:16:08 PST

The specifics of nascent HDL remodeling within the plasma compartment remain poorly understood. We developed an in vitro assay to monitor the lipid transfer between model nascent HDL (LpA-I) and plasma lipoproteins. Incubation of -¹²⁵I-LpA-I with plasma resulted in association of LpA-I with existing plasma HDL, whereas incubation with TD plasma or LDL resulted in conversion of -¹²⁵I-LpA-I to preβ-HDL. To further investigate the dynamics of lipid transfer, nascent LpA-I were labeled with cell-derived [³H]cholesterol (UC) or [³H]phosphatidylcholine (PC) and incubated with plasma at 37°C. The majority of UC and PC were rapidly transferred to apolipoprotein B (apoB). Subsequently, UC was redistributed to HDL for esterification before being returned to apoB. The presence of a phospholipid transfer protein (PLTP) stimulator or purified PLTP promoted PC transfer to apoB. Conversely, PC transfer was abolished in plasma from PLTP^–/– mice. Injection of ¹²⁵I-LpA-I into rabbits resulted in a rapid size redistribution of ¹²⁵I-LpA-I. The majority of [³H]UC from labeled r(HDL) was esterified in vivo within HDL, whereas a minority was found in LDL. These data suggest that apoB plays a major role in nascent HDL remodeling by accepting their lipids and donating UC to the LCAT reaction. The finding that nascent particles were depleted of their lipids and remodeled in the presence of plasma lipoproteins raises questions about their stability and subsequent interaction with LCAT.

Photoactivable sphingosine as a tool to study membrane microenvironments in cultured cells [Research Articles]

Tue, 09 Mar 2010 11:16:08 PST

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [³H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([³H]SM-N₃). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [³H]SM-N₃. However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions.

Disruption of the C-terminal helix by single amino acid deletion is directly responsible for impaired cholesterol efflux ability of apolipoprotein A-I Nichinan [Research Articles]

Tue, 09 Mar 2010 11:16:08 PST

Apolipoprotein A-I (apoA-I) Nichinan, a naturally occurring variant with E235 in the C terminus, is associated with low plasma HDL levels. Here, we investigated the tertiary structure, lipid-binding properties, and ability to induce cellular cholesterol efflux of apoA-I Nichinan and its C-terminal peptide. Thermal and chemical denaturation experiments demonstrated that the E235 mutation decreased the protein stability compared with wild type (WT). ApoA-I Nichinan exhibited capabilities to bind to or solubilize lipid vesicles that are intermediate to that of WT and a L230P/L233P/Y236P variant in which the C-terminal -helix folding is completely disrupted and forms relatively larger and unstable discoidal complexes, indicating that perturbation of the C-terminal -helical structure by the E235 mutation leads to reduced lipid binding. Supporting this, apoA-I 209-241/E235 peptide showed significantly decreased ability to form -helix both in the lipid-free and lipid-bound states, and reduced efficiency to solubilize vesicles. In addition, both apoA-I Nichinan and its C-terminal peptide exhibited reduced activity in ABCA1-mediated cellular cholesterol efflux. Thus, the disruption of the ability of the C-terminal region to form -helix caused by the E235 deletion appears to be the important determinant of impaired lipid binding and cholesterol efflux ability and, consequently, the low plasma HDL levels of apoA-I Nichinan probands.

Marked accumulation of 27-hydroxycholesterol in SPG5 patients with hereditary spastic paresis [Research Articles]

Tue, 09 Mar 2010 11:16:08 PST

Patients with a recessively inherited "pure" hereditary spastic paresis (SPG5) have mutations in the gene coding for the oxysterol 7 hydroxylase (CYP7B1). One of the expected metabolic consequences of such mutations is accumulation of oxysterol substrates due to decreased enzyme activity. In accordance with this, we demonstrate here that four patients with the SPG5 disease have 6- to 9-fold increased plasma levels of 27-hydroxycholesterol. A much higher increase, 30- to 50-fold, was found in cerebrospinal fluid. The plasma levels of 25-hydroxycholesterol were increased about 100-fold. There were no measurable levels of this oxysterol in cerebrospinal fluid. The pattern of bile acids in serum was normal, suggesting a normal bile acid synthesis. The findings are discussed in relation to two transgenic mouse models with increased levels of 27-hydroxy cholesterol in the circulation but without neurological symptoms: the cyp27a1 transgenic mouse and the cyp7b1 knockout mouse. The absolute plasma levels of 27-hydroxycholesterol in the latter models are, however, only about 20% of those in the SPG5 patients. If the accumulation of 27-hydroxycholesterol is an important pathogenetic factor, a reduction of its levels may reduce or prevent the neurological symptoms. A possible strategy to achieve this is discussed.

Quantitation of serum angiopoietin-like proteins 3 and 4 in a Finnish population sample [Research Articles]

Robciuc, M. R., Tahvanainen, E., Jauhiainen, M., Ehnholm, C. — Tue, 09 Mar 2010 11:16:08 PST

We have developed and validated quantitative ELISAs for human angiopoietin-like (ANGPTL)3 and 4 and correlated their serum levels with parameters of lipid and carbohydrate metabolism. For this study, we used a random subsample of the Health 2000 Health Examination Survey consisting of 125 men and 125 women, aged 30–94 years. The anthropometric and biochemical parameters of subjects were characterized in detail. ANGPTL 3 and 4 levels were determined using the developed ELISAs. The intra- and inter-assay coefficients of variation for the assays were less than 15%. The average serum concentration of ANGPTL3 was 368 ± 168 ng/ml (mean ± SD) and for ANGPTL4 it was 18 ± 23 ng/ml (mean ± SD). ANGPTL4 serum levels displayed high variability between individuals ranging from 2 to 158 ng/ml. In post-heparin plasma, both ANGPTL 3 and 4 were increased. Low levels of ANGPTL3 were associated with decreased HDL-cholesterol and increased triglyceride levels. ANGPTL4 levels were positively correlated with FFAs (P = 0.044) and waist-hip ratio (P = 0.016). The developed ELISAs will be important tools to clarify the role of ANGPTL 3 and 4 in human energy metabolism and partitioning of triglycerides between sites of storage (adipose tissue) and oxidation (skeletal and cardiac muscle).

Glucose stimulates cholesterol 7{alpha}-hydroxylase gene transcription in human hepatocytes [Research Articles]

Li, T., Chanda, D., Zhang, Y., Choi, H.-S., Chiang, J. Y. L. — Tue, 09 Mar 2010 11:16:08 PST

Bile acids play important roles in the regulation of lipid, glucose, and energy homeostasis. Recent studies suggest that glucose regulates gene transcription in the liver. The aim of this study was to investigate the potential role of glucose in regulation of bile acid synthesis in human hepatocytes. High glucose stimulated bile acid synthesis and induced mRNA expression of cholesterol 7-hydroxylase (CYP7A1), the key regulatory gene in bile acid synthesis. Activation of an AMP-activated protein kinase (AMPK) decreased CYP7A1 mRNA, hepatocyte nuclear factor 4 (HNF4) protein, and binding to CYP7A1 chromatin. Glucose increased ATP levels to inhibit AMPK and induce HNF4 to stimulate CYP7A1 gene transcription. Furthermore, glucose increased histone acetylation and decreased H3K9 di- and tri-methylation in the CYP7A1 chromatin. Knockdown of ATP-citrate lyase, which converts citrate to acetyl-CoA, decreased histone acetylation and attenuated glucose induction of CYP7A1 mRNA expression. These results suggest that glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. This study uncovers a novel link between hepatic glucose metabolism and bile acid synthesis. Glucose induction of bile acid synthesis may have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions.

APOC1 T45S polymorphism is associated with reduced obesity indices and lower plasma concentrations of leptin and apolipoprotein C-I in aboriginal Canadians [Patient-Oriented and Epidemiological Research]

Tue, 09 Mar 2010 11:16:08 PST

Apolipoprotein (apo) C-I is a constituent of chylomicrons, very low density lipoprotein, and high density lipoprotein. The role of apo C-I in human metabolism is incompletely defined. We took advantage of a naturally occurring amino acid polymorphism that is present in aboriginal North Americans, namely apo C-I T45S. We assessed the hypothesis that metabolic traits, including obesity-related and lipoprotein-related traits, would differ between carriers and noncarriers of apo C-I T45S. A genotyping assay was developed for APOC1 T45S and genotypes were determined in a sample of 410 Canadian Oji-Cree subjects. The allele frequency of the apo C-I S45 allele was ~8% in this sample. We observed the apo C-I S45 allele was significantly associated with 1) lower percent body fat (P < 0.05), 2) lower waist circumference (P = 0.058), 3) lower serum leptin levels (P < 0.05), and 4) lower plasma apo C-I levels (P < 0.0001), using a newly developed ELISA-based method. Taken together, these results suggest that at the whole human phenotype level, apo C-I is associated with the complex metabolic trait of obesity as well as with serum leptin levels.

Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11 [Methods]

Cheng, D., MacArthur, P. S., Rong, S., Parks, J. S., Shelness, G. S. — Tue, 09 Mar 2010 11:16:08 PST

The plasmid vector pLIV11 is used commonly to achieve liver-specific expression of genes of interest in transgenic mice and rabbits. Expression is driven by the human apolipoprotein (apo)E 5' proximal promoter, which includes 5 kb of upstream sequence, exon 1, intron 1, and 5 bp of exon 2. A 3.8 kb 3' hepatic control region, derived from a region ~18 kb downstream of the apoE gene, enhances liver-specific expression. Here, we report that cDNA sequences inserted into the multiple cloning site (MCS) of pLIV11, which is positioned just downstream of truncated exon 2, can cause exon 2 skipping. Hence, splicing is displaced to downstream cryptic 3' splice acceptor sites causing deletion of cloned 5' untranslated mRNA sequences and, in some cases, deletion of the 5' end of an open reading frame. To prevent use of cryptic splice sites, the pLIV11 vector was modified with an engineered 3' splice acceptor site inserted immediately downstream of truncated apoE exon 2. Presence of this sequence fully shifted splicing of exon 1 from the native intron 1–exon 2 splice acceptor site to the engineered site. This finding confirmed that sequences inserted into the MCS of the vector pLIV11 can affect exon 2 recognition and provides a strategy to protect cloned sequences from alternative splicing and possible attenuation of transgenic expression.

Separation and characterization of cardiolipin molecular species by reverse-phase ion pair high-performance liquid chromatography-mass spectrometry [Methods]

Minkler, P. E., Hoppel, C. L. — Tue, 09 Mar 2010 11:16:08 PST

An improved high-performance liquid chromatography-mass spectrometry method for the separation and characterization of cardiolipin molecular species is presented. Reverse-phase ion pair chromatography with acidified triethylamine resulted in increased chromatographic retention and resolution when compared with chromatography without acidified triethylamine. Using a hybrid triple quadrupole linear ion trap mass spectrometer to generate MS/MS spectra revealed three regions within each spectrum that could be used to deduce the structure of the cardiolipin molecular species: the diacylglycerol phosphate region, the monoacylglycerol phosphate region, and the fatty acid region. Cardiolipin standards of known composition were analyzed and exhibited expected chromatographic and mass spectral results. Two minor components in commercial bovine heart cardiolipin, (with the same molecular weight but different chromatographic retention times), were shown to differ by fatty acid composition: (C18:2)₂(C18:1)₂ versus (C18:2)₃(C18:0)₁. These compounds were then analyzed by HPLC-MS³ to examine specific diac ylglycerol phosphate generated fatty acid fragmentation. Also, two commercial sources of bovine heart cardiolipin were shown to have minor differences in cardiolipin species content. Cardiolipin isolated from rat liver, mouse heart, and dog heart mitochondria were then characterized and the relative distributions of the major cardiolipin species were determined.

Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase [Methods]

Tue, 09 Mar 2010 11:16:08 PST

Glucosylceramide synthase (GCS or GlcT-1), converting ceramide to glucosylceramide, is a key enzyme for the synthesis of glycosphingolipids. Due to its diverse roles in physiology and diseases, GCS may be a disease marker and drug target. Current assays for enzymes including GCS are based on reactions conducted in a test tube using enzyme preparations. Measurement of enzyme activity in laboratory-made conditions cannot directly evaluate the role of GCS in cells. Here, we introduce a new approach to determine GCS cellular activity using fluorescent NBD C6-ceramide in vivo. Cellular GCS transfers UDP-glucose to NBD C6-ceramide and produces NBD C6-glucosylceramide. C6-glucosylceramide is then separated from C6-ceramide by thin-layer chromatography and both are then quantitated by spectrophotometer. This cell-based method is able to quantitate glucosylceramide in pmol range, produced by approximately 50,000 cells or 1.0 mg tissue. This method has been used successfully to evaluate the degrees of GCS enzyme in cells and in tumors subjected to gene manipulation and chemical inhibition. These data indicate that this cell-based fluorescent method is direct, reproducible, and simple for assessing ceramide glycosylation. It is applicable to validate GCS activity in drug-resistant cancers and in other disorders.

ERRATA [Errata]

Tue, 09 Mar 2010 11:16:08 PST

ERRATA [Errata]

Tue, 09 Mar 2010 11:16:08 PST