The Journal of Lipid Research recent issues

[Thematic Reviews] Thematic Review Series: Glycerolipids. Acyltransferases in bacterial glycerophospholipid synthesis

Zhang, Y.-M., Rock, C. O. — 2008-08-15

Phospholipid biosynthesis is a vital facet of bacterial physiology that begins with the synthesis of the fatty acids by a soluble type II fatty acid synthase. The bacterial glycerol-phosphate acyltransferases utilize the completed fatty acid chains to form the first membrane phospholipid and thus play a critical role in the regulation of membrane biogenesis. The first bacterial acyltransferase described was PlsB, a glycerol-phosphate acyltransferase. PlsB is a key regulatory point that coordinates membrane phospholipid formation with cell growth and macromolecular synthesis. Phosphatidic acid is then produced by PlsC, a 1-acylglycerol-phosphate acyltransferase. These two acyltransferases use thioesters of either CoA or acyl carrier protein (ACP) as the acyl donors and have homologs that perform the same reactions in higher organisms. However, the most prevalent glycerol-phosphate acyltransferase in the bacterial world is PlsY, which uses a recently discovered acyl-phosphate fatty acid intermediate as an acyl donor. This unique activated fatty acid is formed from the acyl-ACP end products of the fatty acid biosynthetic pathway by PlsX, an acyl-ACP:phosphate transacylase.

[Reviews] Three-dimensional models of HDL apoA-I: implications for its assembly and function

Thomas, M. J., Bhat, S., Sorci-Thomas, M. G. — 2008-08-15

The purpose of this review is to highlight recent advances toward the refinement of a three-dimensional structure for lipid-bound apolipoprotein A-I (apoA-I) on recombinant HDL. Recently, X-ray crystallography has yielded a new structure for full-length, lipid-free apoA-I. Although this approach has not yet been successful in solving the three-dimensional structure of lipid-bound apoA-I, analysis of the X-ray structures has been of immense help in the interpretation of structural data obtained from other methods that yield structural information. Recent studies emphasize the use of mass spectrometry to unambiguously identify cross-linked peptides or to quantify solvent accessibility using hydrogen-deuterium exchange. The combination of mass spectrometry, molecular modeling, molecular dynamic analysis, and small-angle X-ray diffraction has provided additional structural information on apoA-I folding that complements previous approaches.

[Research Articles] {beta}-Glycosphingolipids-mediated lipid raft alteration is associated with redistribution of NKT cells and increased intrahepatic CD8+ T lymphocyte trapping

2008-08-15

The aim of this study was to determine the effect of β-glycosphingolipids on intra-hepatic natural killer T (NKT) lymphocyte regulatory function and on lymphocyte trapping via alteration of cell membrane lipid rafts. Immune-mediated colitis was induced by intracolonic instillation of trinitrobenzene sulfonic acid. Mice were treated with β-lactosylceramide (LC), β-glucosylceramide (GC), β-galactosylceramide, ceramide, or a combination of both GC and LC (IGL), or solvent alone. Lipid rafts were investigated by fluorescence-activated cell sorting analysis of ganglioside-GM1 and fluorescence microscopy of structure. Administration of β-glycosphingolipids resulted in an increased intrahepatic/peripheral NKT ratio, increased intrahepatic CD8+ lymphocyte trapping, decreased serum interferon- (IFN-) levels and decreased serum IFN-/interleukin-10 ratio. Administration of GC, LC, or IGL significantly altered the levels of GM1, a key marker of lipid rafts, on NKT regulatory lymphocytes. The immune modulatory effect of β-glycosphingolipids was associated with increased survival and significant alleviation of colitis as determined by improvement in both the macroscopic and microscopic scores. In conclusion, administration of β-glycosphingolipids increased NKT regulatory lymphocyte redistribution and intrahepatic CD8⁺ T lymphocyte trapping, resulting in alleviation of immune-mediated colitis. The effects of these naturally occurring compounds were associated with modification of the T lymphocyte lipid raft structure, which is a site for immune modulation.

[Research Articles] Neutrophils transiently infiltrate intra-abdominal fat early in the course of high-fat feeding

Elgazar-Carmon, V., Rudich, A., Hadad, N., Levy, R. — 2008-08-15

Chronic inflammation of adipose tissue in obesity is by now an established phenomenon, but the initiating event(s) of the inflammatory cascade are still unknown. We hypothesized that neutrophil infiltration into adipose tissue may precede macrophage infiltration as in classical immune responses. Here we demonstrate that early (3 and 7 days) after initiating high-fat feeding of C57BL/6J mice, neutrophils transiently infiltrate the parenchyma of intra-abdominal adipose tissue. Mean periepdidymal fat myeloperoxidase expression (representing neutrophils) was significantly increased 3.5-fold (P < 0.01) and 2.9-fold (P < 0.03), at days 3 and 7 compared with day 0. Immunohystochemistry analysis demonstrated a physical binding between neutrophils and adipocytes, which was supported by in vitro adherence assay: mouse peritoneal neutrophils adhered to a monolayer of 3T3-L1 mouse adipocytes, in a manner dependent on their activation state, 41.9 ± 3.7% or 29.5 ± 2%, by PMA or the IL-8 analog CXCL1 (KC), respectively, compared with 24.8 ± 1.5% in unstimulated neutrophils, respectively. The degree of surface exposure of CD11b (Mac-1) corresponded to the percentage of adhered neutrophils. The adherence was prevented by preincubating neutrophils or adipocytes with anti-CD11b or anti-ICAM-1 antibodies. Furthermore, immunoprecipitation of CD11b from lysates of a mixed neutrophil-adipocyte cell population resulted in coimmunoprecipitation of ICAM-1, indicating that the interaction is mediated by neutrophil CD11b and adipocyte ICAM-1.

[Research Articles] Liver X receptor-mediated activation of reverse cholesterol transport from macrophages to feces in vivo requires ABCG5/G8

2008-08-15

Liver X receptor (LXR) agonists increase both total fecal sterol excretion and macrophage-specific reverse cholesterol transport (RCT) in vivo. In this study, we assessed the effects of ABCG5/G8 deficiency as well as those of LXR agonist-induction of RCT from macrophages to feces in vivo. A [³H]cholesterol-labeled macrophage cell line was injected intraperitoneally into ABCG5/G8-deficient (G5/G8^–/–), heterozygous (G5G8^+/–), and wild-type G5/G8^+/+ mice. G5/G8^–/–mice presented increased radiolabeled HDL-bound [³H]cholesterol 24 h after the label injection. However, the magnitude of macrophage-derived [³H]cholesterol in liver and feces did not differ between groups. A separate experiment was conducted in G5G8^+/+ and G5G8^–/– mice treated with or without the LXR agonist T0901317. Treatment with T0901317 increased liver ABCG5/G8 expression, which was associated with a 2-fold increase in macrophage-derived [³H]cholesterol in feces of G5/G8^+/+ mice. However, T0901317 treatment had no effect on fecal [³H]cholesterol excretion in G5G8^–/– mice. Additionally, LXR activation stimulated the fecal excretion of labeled cholesterol after an intravenous injection of HDL-[³H]cholesteryl oleate in G5/G8^+/+ mice, but failed to enhance fecal [³H]cholesterol in G5/G8^–/– mice. Our data provide direct in vivo evidence of the crucial role of ABCG5 and ABCG8 in LXR-mediated induction of macrophage-specific RCT.

[Research Articles] Levels of apolipoprotein M are not associated with the risk of coronary heart disease in two independent case-control studies

2008-08-15

Apolipoprotein M (apoM), a 25 kDa plasma protein belonging to the lipocalin protein family, is predominantly associated with HDL. Studies in mice have suggested apoM to be important for the formation of pre-β-HDL and to increase cholesterol efflux from macrophage foam cells. Overexpression of human apoM in LDL receptor-deficient mice reduced the atherogenic effect of a cholesterol-rich diet. The aim of the present study was to investigate whether the apoM levels in man predict the risk for coronary heart disease (CHD). ApoM was measured in samples from two separate case-control studies. FINRISK '92 consisted of 255 individuals, of whom 80 developed CHD during follow-up and 175 were controls. The Copenhagen City Heart Study included 1,865 individuals, of whom 921 developed CHD during follow-up and 944 were controls. Correlation studies of apoM concentration with several analytes showed a marked positive correlation with HDL and total cholesterol as well as with apoA-I and apoB. There was no significant difference in mean apoM level between CHD and control subjects in either study. In conditional logistic regression analyses, apoM was not a predictor of CHD events, [odds ratio (95% CI) 0.97 (0.74–1.27) and 0.92 (0.84–1.02), respectively]. In conclusion, no association between apoM and CHD could be found in this study.

[Research Articles] Do unsaturated phosphoinositides mix with ordered phosphadidylcholine model membranes?

Hermelink, A., Brezesinski, G. — 2008-08-15

Phosphoinositides have been shown to control membrane trafficking events by targeting proteins to specific cellular sites, which requires a tight regulation of phosphoinositide generation and turnover as well as a high degree of compartmentalization. To shed light on the processes that lead to the formation of phosphoinositide-enriched microdomains, mixed monolayers of phosphatidylcholine and dioleoyl-phosphatidylinositol (DOPtdIns) or dioleoyl-phosphatidylinositol-bisphosphate [DOPtdIns(4,5)P₂] were investigated by isothermal area/pressure measurements, Brewster angle microscopy, and grazing incidence X-ray diffraction. The results are consistent with a charge-dependent formation of phosphatidylinositol-containing tightly packed phases. DOPtdIns is capable of mixing partially with condensed 1,2-distearoyl-phosphatidylcholine (DSPC) and of forming mixed crystals that differ significantly from those formed by pure DSPC. DOPtdIns(4,5)P₂ in mixtures with DSPC is, to a much larger extent, phase separated. The observed phase separation of the highly charged DOPtdIns(4,5)P₂ is presumably water stabilized by electrostatic interactions and hydrogen bonding. In biological systems, an enzymatic phosphorylation of phosphatidylinositol in mixed domains may cause their insolubility in ordered phosphatidylcholine areas and lead to a cooperative reorganization of the host lipid membrane. This strong cooperative effect underlines the important role of PtdIns(4,5)P₂ in signal transduction processes and suggests that the ability of phosphoinositides to induce or reduce long-range interactions in phospholipid mixtures is crucial.

[Research Articles] C-reactive protein enhances macrophage lipoprotein lipase expression

Maingrette, F., Li, L., Renier, G. — 2008-08-15

High serum levels of C-reactive protein (CRP), a strong predictor of cardiovascular events, are documented in patients with type 2 diabetes. Accumulating evidence suggests that CRP could directly promote arterial damage. To determine the role of CRP in diabetic atherosclerosis, we examined the effect of CRP on the expression of macrophage lipoprotein lipase (LPL), a proatherogenic molecule upregulated in type 2 diabetes. Treatment of human macrophages with native CRP increased, in a dose- and time-dependent manner, LPL protein expression and secretion. Modified CRP reproduced these effects. Preincubation of human macrophages with antioxidants, protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) inhibitors prevented CRP-induced LPL expression. Exposure of human macrophages to CRP further increased intracellular reactive oxygen species generation, classic PKC isozymes expression, and extracellular signal-regulated protein kinase 1/2 phosphorylation. In CRP-treated J774 macrophages, increased macrophage LPL mRNA levels and enhanced binding of nuclear proteins to the activated protein-1 (AP-1)-enhancing element were observed. These effects were prevented by antioxidants, as well as by PKC, MAPK, and AP-1 inhibitors. These data show for the first time that CRP directly increases macrophage LPL expression and secretion. Given the predominant role of macrophage LPL in atherogenesis, LPL might represent a novel factor underlying the adverse effect of CRP on the diabetic vasculature.

[Research Articles] Effects of high-fat diet exposure during fetal life on type 2 diabetes development in the progeny

2008-08-15

Nutrition during fetal life is a critical factor contributing to diabetes development in adulthood. The aim of our study was to verify: 1) whether a high-fat (HF) diet in young adult mice induces alterations in β-cell mass, proliferation, neogenesis, and apoptosis, as well as insulin sensitivity and secretion; 2) whether these alterations may be reversible after HF diet suspension; 3) the effects in a first (F1) and second generation (F2) of mice without direct exposure to a HF diet after birth. Type 2 diabetes developed in adult mice on a HF diet, in F1 mice that were HF diet-exposed during fetal or neonatal life, and in F2 mice whose mothers were HF diet-exposed during their fetal life. β-cell mass, replication, and neogenesis were high in HF diet-exposed mice and decreased after diet suspension. β-cell mass and replication remained high in F1 mice and decreased in F2 mice whose mothers were exposed to a HF diet. β-cell neogenesis was present in adult mice on a HF diet and in F1 mice that were HF diet-exposed during fetal and/or neonatal life. We conclude that a HF diet during fetal life, particularly if combined with the same insult during the suckling period, can induce the type 2 diabetes phenotype, which can be directly transmitted to the progeny even in the absence of additional dietary insults.

[Research Articles] A mechanism accounting for the low cellular level of linoleic acid in cystic fibrosis and its reversal by DHA

2008-08-15

Specific fatty acid alterations have been described in the blood and tissues of cystic fibrosis (CF) patients. The principal alterations include decreased levels of linoleic acid (LA) and docosahexaenoic acid (DHA). We investigated the potential mechanisms of these alterations by studying the cellular uptake of LA and DHA, their distribution among lipid classes, and the metabolism of LA in a human bronchial epithelial cell model of CF. CF (antisense) cells demonstrated decreased levels of LA and DHA compared with wild type (WT, sense) cells expressing normal CFTR. Cellular uptake of LA and DHA was higher in CF cells compared with WT cells at 1 h and 4 h. Subsequent incorporation of LA and DHA into most lipid classes and individual phospholipids was also increased in CF cells. The metabolic conversion of LA to n-6 metabolites, including 18:3n-6 and arachidonic acid, was upregulated in CF cells, indicating increased flux through the n-6 pathway. Supplementing CF cells with DHA inhibited the production of LA metabolites and corrected the n-6 fatty acid defect. In conclusion, the evidence suggests that low LA level in cultured CF cells is due to its increased metabolism, and this increased LA metabolism is corrected by DHA supplementation.

[Research Articles] Expression of CETP and of splice variants induces the same level of ER stress despite secretion efficiency differences

Lira, M. E., Loomis, A. K., Paciga, S. A., Lloyd, D. B., Thompson, J. F. — 2008-08-15

The cholesteryl ester transfer protein (CETP) gene has been associated with a variety of phenotypes, including HDL-cholesterol levels and, more sporadically, with cardiovascular disease, obesity, and extreme longevity. Alterations of CETP activity levels can be caused by single-base polymorphisms as well as by alternative splicing. In addition to the previously characterized alternative splicing that skips exon 9, we found additional minor variants and characterized the activity of the resultant proteins. The novel variants skipped exon 9 sequences and inserted one of two in-frame exons from Alu-derived intronic sequences. None of the alternatively spliced variants are efficiently secreted, and coexpression of them inhibits wild-type CETP secretion. Expression of the alternative spliced variants causes an induction of genes linked to the endoplasmic reticulum (ER) stress response, including the neighboring HERPUD1 (homocysteine- and ER stress-inducible protein, ubiquitin-like domain-containing) gene. Unexpectedly, even though wild-type CETP is secreted much more efficiently than spliced variants, it induces the same degree of stress response as spliced variants, whereas a control secreted protein does not. CETP plays a complex role in modulating ER stress, with its expression inducing the response and its cholesteryl ester transfer activity and differential splicing modulating the response in other ways.

[Research Articles] Effect of dietary docosahexaenoic acid on biosynthesis of docosahexaenoic acid from alpha-linolenic acid in young rats

DeMar, J. C., DiMartino, C., Baca, A. W., Lefkowitz, W., Salem, N. — 2008-08-15

Docosahexaenoic acid (DHA), a crucial nervous system n-3 PUFA, may be obtained in the diet or synthesized in vivo from dietary -linolenic acid (LNA). We addressed whether DHA synthesis is regulated by the availability of dietary DHA in artificially reared rat pups, during p8 to p28 development. Over 20 days, one group of rat pups was continuously fed deuterium-labeled LNA (d5-LNA) and no other n-3 PUFA (d5-LNA diet), and a second group of rat pups was fed a d5-LNA diet with unlabeled DHA (d5-LNA + DHA diet). The rat pups were then euthanized, and the total amount of deuterium-labeled docosahexaenoic acid (d5-DHA) (synthesized DHA) as well as other n-3 fatty acids present in various body tissues, was quantified. In the d5-LNA + DHA group, the presence of dietary DHA led to a marked decrease (3- to 5-fold) in the total amount of d5-DHA that accumulated in all tissues that we examined, except in adipose. Overall, DHA accretion from d5-DHA was generally diminished by availability of dietary preformed DHA, inasmuch as this was found to be the predominant source of tissue DHA. When preformed DHA was unavailable, d5-DHA and unlabeled DHA were preferentially accreted in some tissues along with a net loss of unlabeled DHA from other organs.

[Research Articles] TGF{beta}1, TNF{alpha}, and insulin signaling crosstalk in regulation of the rat cholesterol 7{alpha}-hydroxylase gene expression

Li, T., Ma, H., Chiang, J. Y. L. — 2008-08-15

The TGFβ1/Smad pathway plays a critical role in cholestasis and liver fibrosis. Previous studies show that TGFβ1, TNF, and insulin inhibit cholesterol 7-hydroxylase (CYP7A1) gene transcription and bile acid synthesis in human hepatocytes. In this study, we investigated insulin, TGFβ1, and TNF regulation of rat Cyp7a1 gene transcription. In contrast to inhibition of human CYP7A1 gene transcription, TGFβ1 stimulates rat Cyp7a1 reporter activity. Smad3, FoxO1, and HNF4 synergistically stimulated rat Cyp7a1 gene transcription. Mutations of the Smad3, FoxO1, or HNF4 binding site attenuated the rat Cyp7a1 promoter activity. Furthermore, TNF and cJun attenuated TGFβ1 stimulation of rat Cyp7a1. Insulin or adenovirus-mediated expression of constitutively active AKT1 inhibited FoxO1 and Smad3 synergy. In streptozotocin-induced diabetic rats, Cyp7a1 mRNA expression levels were induced and insulin attenuated CYP7A1 mRNA levels. Chromatin immunoprecipitation assay showed that FoxO1 binding to Cyp7a1 chromatin was increased in diabetic rat livers and insulin reduced FoxO1 binding. These results suggest a mechanistic basis for induction of Cyp7a1 activity and bile acid synthesis in cholestatic rats and in diabetic rats. The crosstalk of insulin, TGFβ and TNF signaling pathways may regulate bile acid synthesis and lipid homeostasis in diabetes, fatty liver disease, and liver fibrosis.

[Research Articles] Formation of eicosanoids, E2/D2 isoprostanes, and docosanoids following decapitation-induced ischemia, measured in high-energy-microwaved rat brain

2008-08-15

Inflammatory lipid mediators derived from arachidonic acid (AA) and docosahexaenoic acid (DHA) modify the pathophysiology of brain ischemia. The goal of this work was to investigate the formation of eicosanoids and docosanoids generated from AA and DHA, respectively, during no-flow cerebral ischemia. Rats were subjected to head-focused microwave irradiation 5 min following decapitation (complete ischemia) or prior to decapitation (controls). Brain lipids were extracted and analyzed by reverse-phase liquid chromatography-tandem mass spectrometry. After complete ischemia, brain AA, DHA, and docosapentaenoic acid concentrations increased 18-, 5- and 4-fold compared with controls, respectively. Prostaglandin E₂ (PGE₂) and PGD₂ could not be detected in control microwaved rat brain, suggesting little endogenous PGE₂/D₂ production in the brain in the absence of experimental manipulation. Concentrations of thromboxane B₂, E₂/D₂-isoprostanes, 5-hydroxyeicosatetraenoic acid (5-HETE), 5-oxo-eicosatetraenoic acid, and 12-HETE were significantly elevated in ischemic brains. In addition, DHA products such as mono-, di- and trihydroxy-DHA were detected in control and ischemic brains. Monohydroxy-DHA, identified as 17-hydroxy-DHA and thought to be the immediate precursor of neuroprotectin D₁, was 6.5-fold higher in ischemic than in control brain. The present study demonstrated increased formation of eicosanoids, E₂/D₂-IsoPs, and docosanoids following cerebral ischemia. A balance of these lipid mediators may mediate immediate events of ischemic injury and recovery.

[Research Articles] Identification of twenty-three mutations in fission yeast Scap that constitutively activate SREBP

Hughes, A. L., Stewart, E. V., Espenshade, P. J. — 2008-08-15

The endoplasmic reticulum membrane protein SREBP cleavage-activating protein (Scap) senses sterols and regulates activation of sterol-regulatory element binding proteins (SREBPs), membrane-bound transcription factors that control lipid homeostasis in fission yeast and mammals. Transmembrane segments 2–6 of Scap function as a sterol-sensing domain (SSD) that recognizes changes in cellular sterols and facilitates activation of SREBP. Previous studies identified conserved mutations Y298C, L315F, and D443N in the SSD of mammalian Scap and fission yeast Scap (Scp1) that render cells insensitive to sterols and cause constitutive SREBP activation. In this study, we utilized fission yeast genetics to identify additional functionally important residues in the SSD of Scp1 and Scap. Using a site-directed mutagenesis selection, we sampled all possible amino acid substitutions at 50 conserved residues in the SSD of Scp1 for their effects on yeast SREBP (Sre1) activation. We found mutations at 23 different amino acids in Scp1 that rendered Scp1 insensitive to sterols and caused constitutive activation of Sre1. To our surprise, the majority of the homologous Scap mutants displayed wild-type function, and only one mutation, V439G, caused constitutive activation of SREBP in mammals. These results suggest that the sterol-sensing mechanism of Scap and the functional requirements for SREBP activation are different between fission yeast and mammals.

[Research Articles] ApoB100 is required for increased VLDL-triglyceride secretion by microsomal triglyceride transfer protein in ob/ob mice

2008-08-15

Microsomal triglyceride transfer protein (Mttp) is a key player in the assembly and secretion of hepatic very low density lipoproteins (VLDL). Here we determined the effects of Mttp overexpression on hepatic triglyceride (TG) and VLDL secretion in leptin-deficient (ob/ob) mice, specifically in relation to apolipoproteinB (apoB) isoforms. We crossed Apobec1^–/– mice with congenic ob/ob mice to generate apoB100-only ob/ob mice (A-ob/ob). The obesity phenotype in both genotypes was similar, but A-ob/ob mice had greater hepatic TG content. Administration of recombinant adenovirus expressing murine Mttp cDNA (Ad-mMTP) increased hepatic Mttp content and activity and increased hepatic VLDL-TG secretion in A-ob/ob mice. However, despite equivalent overexpression of Mttp, there was no change in VLDL-TG secretion in ob/ob mice in a wild-type Apobec1 background. Metabolic labeling studies in primary hepatocytes from A-ob/ob mice demonstrated that Ad-mMTP increased triglyceride secretion without changing the synthesis and secretion of apoB100, suggesting greater incorporation of TG into existing VLDL particles rather than increased particle number. Ad-mMTP administration failed to increase hepatic VLDL secretion in lean Apobec1^–/– mice or controls. By contrast, VLDL secretion increased and hepatic TG content decreased following Ad-mMTP administration to human APOB transgenic mice crossed into the Apobec1^–/– line. These findings demonstrate that Ad-mMTP increases murine hepatic VLDL-TG secretion only in the apoB100 background, and even then only in situations with either increased hepatic TG accumulation or increased apoB100 expression.

[Research Articles] Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

Petersen, N. H., Faegeman, N. J., Yu, L., Wustner, D. — 2008-08-15

Niemann-Pick C1-like 1 (NPC1L1) is a recently identified protein that mediates intestinal cholesterol absorption and regulates biliary cholesterol excretion. The itineraries and kinetics of NPC1L1 trafficking remain uncertain. In this study, we have visualized movement of NPC1L1-enhanced green fluorescent protein (NPC1L1-EGFP) and cholesterol analogs in hepatoma cells. At steady state, about 42% of NPC1L1 resided in the transferrin (Tf)-positive, sterol-enriched endocytic recycling compartment (ERC), whereas time-lapse microscopy demonstrated NPC1L1 traffic between the plasma membrane and the ERC. Fluorescence recovery after photobleaching revealed rapid recovery (half-time ~2.5 min) of about 35% of NPC1L1 in the ERC, probably replenished from peripheral sorting endosomes. Acute cholesterol depletion blocked internalization of NPC1L1-EGFP and Tf and stimulated recycling of NPC1L1-EGFP from the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells, NPC1L1 resided almost exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between the cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1-mediated cellular sterol uptake.

[Research Articles] Blocking VLDL secretion causes hepatic steatosis but does not affect peripheral lipid stores or insulin sensitivity in mice

2008-08-15

The liver secretes triglyceride-rich VLDLs, and the triglycerides in these particles are taken up by peripheral tissues, mainly heart, skeletal muscle, and adipose tissue. Blocking hepatic VLDL secretion interferes with the delivery of liver-derived triglycerides to peripheral tissues and results in an accumulation of triglycerides in the liver. However, it is unclear how interfering with hepatic triglyceride secretion affects adiposity, muscle triglyceride stores, and insulin sensitivity. To explore these issues, we examined mice that cannot secrete VLDL [due to the absence of microsomal triglyceride transfer protein (Mttp) in the liver]. These mice exhibit markedly reduced levels of apolipoprotein B-100 in the plasma, along with reduced levels of triglycerides in the plasma. Despite the low plasma triglyceride levels, triglyceride levels in skeletal muscle were unaffected. Adiposity and adipose tissue triglyceride synthesis rates were also normal, and body weight curves were unaffected. Even though the blockade of VLDL secretion caused hepatic steatosis accompanied by increased ceramides and diacylglycerols in the liver, the mice exhibited normal glucose tolerance and were sensitive to insulin at the whole-body level, as judged by hyperinsulinemic euglycemic clamp studies. Normal hepatic glucose production and insulin signaling were also maintained in the fatty liver induced by Mttp deletion. Thus, blocking VLDL secretion causes hepatic steatosis without insulin resistance, and there is little effect on muscle triglyceride stores or adiposity.

[Patient-Oriented and Epidemiological Research] Reduced ileal expression of OST{alpha}-OST{beta} in non-obese gallstone disease

Renner, O., Harsch, S., Strohmeyer, A., Schimmel, S., Stange, E. F. — 2008-08-15

Cholelithiasis is a multifactorial process, and several mechanisms have been postulated. A decreased expression of the ileal apical sodium-dependent bile acid transporter (ASBT) and of the cytosolic ileal lipid binding protein (ILBP) was recently described in female non-obese patients. The role of the recently identified organic solute transporters and β (OST, OSTβ) in gallstone pathogenesis remains unclear. Therefore, we performed analysis of OST-OSTβ in gallstone patients according to body weight. Ileal mucosal biopsies were collected during routine colonoscopy from female gallstone carriers (n = 19) and controls (n = 34). OST-OSTβ mRNA expression was measured using the LightCycler sequence detection system; protein was analyzed by immunohistochemistry and Western blot. The mRNA expression of OST-OSTβ was significantly reduced (OST: 3.3-fold, P = 0.006; OSTβ: 2.6-fold, P = 0.03) in normal-weight but not overweight gallstone carriers compared with controls. OST-OSTβ protein levels also showed a reduction by 40–67%. The expression of OST-OSTβ correlated positively with ASBT (r = 0.65, 0.58, respectively), ILBP (r = 0.77, 0.67), and the farnesoid X receptor (r = 0.58, 0.50). Fibroblast growth factor-19 showed a 2.8-fold reduction (P = 0.06), and liver receptor homolog-1 showed a 2-fold reduction (P = 0.04) in non-obese patients. In conclusion, an impaired function of all three ileal bile acid transporters may lead to low ileal bile acid reabsorption and an altered bile acid pool composition and therefore may contribute to the formation of gallstones in non-obese patients.

[Methods] Composition of adipose tissue and marrow fat in humans by 1H NMR at 7 Tesla

Ren, J., Dimitrov, I., Sherry, A. D., Malloy, C. R. — 2008-08-15

Proton NMR spectroscopy at 7 Tesla (7T) was evaluated as a new method to quantify human fat composition noninvasively. In validation experiments, the composition of a known mixture of triolein, tristearin, and trilinolein agreed well with measurements by ¹H NMR spectroscopy. Triglycerides in calf subcutaneous tissue and tibial bone marrow were examined in 20 healthy subjects by ¹H spectroscopy. Ten well-resolved proton resonances from triglycerides were detected using stimulated echo acquisition mode sequence and small voxel (~0.1 ml), and T₁ and T₂ were measured. Triglyceride composition was not different between calf subcutaneous adipose tissue and tibial marrow for a given subject, and its variation among subjects, as a result of diet and genetic differences, fell in a narrow range. After correction for differential relaxation effects, the marrow fat composition was 29.1 ± 3.5% saturated, 46.4 ± 4.8% monounsaturated, and 24.5 ± 3.1% diunsaturated, compared with adipose fat composition, 27.1 ± 4.2% saturated, 49.6 ± 5.7% monounsaturated, and 23.4 ± 3.9% diunsaturated. Proton spectroscopy at 7T offers a simple, fast, noninvasive, and painless method for obtaining detailed information about lipid composition in humans, and the sensitivity and resolution of the method may facilitate longitudinal monitoring of changes in lipid composition in response to diet, exercise, and disease.

[Methods] Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS

2008-08-15

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 µl of dried serum were derivatized into picolinyl esters (3β-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 µl aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.

[Calendar] Calendar

2008-08-15

[Commentary] HDL and innate immunity: a tale of two apolipoproteins

Grunfeld, C., Feingold, K. R. — 2008-07-11

[Thematic Reviews] Thematic Review Series: Glycerolipids. Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes

Schlame, M. — 2008-07-11

In this article, the formation of prokaryotic and eukaryotic cardiolipin is reviewed in light of its biological function. I begin with a detailed account of the structure of cardiolipin, its stereochemistry, and the resulting physical properties, and I present structural analogs of cardiolipin that occur in some organisms. Then I continue to discuss i) the de novo formation of cardiolipin, ii) its acyl remodeling, iii) the assembly of cardiolipin into biological membranes, and iv) the degradation of cardiolipin, which may be involved in apoptosis and mitochondrial fusion. Thus, this article covers the entire metabolic cycle of this unique phospholipid. It is shown that mitochondria produce cardiolipin species with a high degree of structural uniformity and molecular symmetry, among which there is often a dominant form with four identical acyl chains. The subsequent assembly of cardiolipin into functional membranes is largely unknown, but the analysis of crystal structures of membrane proteins has revealed a first glimpse into the underlying principles of cardiolipin-protein interactions. Disturbances of cardiolipin metabolism are crucial in the pathophysiology of human Barth syndrome and perhaps also play a role in diabetes and ischemic heart disease.

[Thematic Reviews] Thematic Review Series: Sphingolipids. Biodiversity of sphingoid bases ("sphingosines") and related amino alcohols

2008-07-11

"Sphingosin" was first described by J. L. W. Thudichum in 1884 and structurally characterized as 2S,3R,4E-2-aminooctadec-4-ene-1,3-diol in 1947 by Herb Carter, who also proposed the designation of "lipides derived from sphingosine as sphingolipides." This category of amino alcohols is now known to encompass hundreds of compounds that are referred to as sphingoid bases and sphingoid base-like compounds, which vary in chain length, number, position, and stereochemistry of double bonds, hydroxyl groups, and other functionalities. Some have especially intriguing features, such as the tail-to-tail combination of two sphingoid bases in the ,-sphingoids produced by sponges. Most of these compounds participate in cell structure and regulation, and some (such as the fumonisins) disrupt normal sphingolipid metabolism and cause plant and animal disease. Many of the naturally occurring and synthetic sphingoid bases are cytotoxic for cancer cells and pathogenic microorganisms or have other potentially useful bioactivities; hence, they offer promise as pharmaceutical leads. This thematic review gives an overview of the biodiversity of the backbones of sphingolipids and the broader field of naturally occurring and synthetic sphingoid base-like compounds.

[Research Articles] Effect of lipid-bound apoA-I cysteine mutants on lipopolysaccharide-induced endotoxemia in mice

Wang, Y., Zhu, X., Wu, G., Shen, L., Chen, B. — 2008-07-11

HDL has been shown to be able to neutralize the toxicity of lipopolysaccharide (LPS). Our previous study (J. Lipid Res. 2005. 46: 1303–1311) characterized the properties of secondary structure and in vitro functions of different cysteine mutants of apolipoprotein A-I. Here, we reconstituted recombinant HDLs (named rHDLwt, rHDL52, rHDL74, rHDL107, rHDL129, rHDL173, rHDL195, and rHDL228) by mixing wild type or those mutants with dipalmitoyl phosphatidylcholine and examined their in vivo effects on LPS-induced endotoxemia in mice. Our results showed that 24 h after injection, mice receiving rHDL74 or rHDL52 had a significant decrease of plasma tumor necrosis factor (TNF-) and interleukin-1β (IL-1β), compared with control mice receiving either saline or rHDLwt (P < 0.05). Administration of rHDL74 to mice injected with LPS also led to a decrease of plasma IL-6, protection of lung against acute injury, and attenuation of endotoxin-induced clinical symptoms in mice, compared with controls injected with LPS only. However, injection of rHDL228 significantly increased plasma concentration of TNF- and exacerbated LPS-induced lung injury. In summary, compared with rHDLwt, rHDL74 and rHDL52 exhibit higher anti-inflammation capabilities, whereas rHDL228 shows hyper-proinflammation by exacerbating LPS-induced endotoxemia in mice.

[Research Articles] Wolman disease/cholesteryl ester storage disease: efficacy of plant-produced human lysosomal acid lipase in mice

2008-07-11

Lysosomal acid lipase (LAL) is an essential enzyme that hydrolyzes triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. Genetic LAL mutations lead to Wolman disease (WD) and cholesteryl ester storage disease (CESD). An LAL-null (lal^–/–) mouse model resembles human WD/CESD with storage of CEs and TGs in multiple organs. Human LAL (hLAL) was expressed in Nicotiana benthamiana using the GENEWARE® expression system (G-hLAL). Purified G-hLAL showed mannose receptor-dependent uptake into macrophage cell lines (J774E). Intraperitoneal injection of G-hLAL produced peak activities in plasma at 60 min and in the liver and spleen at 240 min. The t_1/2 values were: ~90 min (plasma), ~14 h (liver), and ~32 h (spleen), with return to baseline by ~150 h in liver and ~200 h in spleen. Ten injections of G-hLAL (every 3 days) into lal^–/– mice produced normalization of hepatic color, decreases in hepatic cholesterol and TG contents, and diminished foamy macrophages in liver, spleen, and intestinal villi. All injected lal^–/– mice developed anti-hLAL protein antibodies, but suffered no adverse events. These studies demonstrate the feasibility of using plant-expressed, recombinant hLAL for the enzyme therapy of human WD/CESD with general implications for other lysosomal storage diseases.

[Research Articles] L-4F treatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity in obese mice

2008-07-11

We hypothesized that the apolipoprotein mimetic peptide L-4F, which induces arterial anti-oxidative enzymes and is vasoprotective in a rat model of diabetes, would ameliorate insulin resistance and diabetes in obese mice. L-4F (2 mg/kg/d) administered to ob/ob mice for 6 weeks limited weight gain without altering food intake, decreased visceral (P < 0.02) and subcutaneous (P < 0.045) fat content, decreased plasma IL-1β and IL-6 levels (P < 0.05) and increased insulin sensitivity, resulting in decreased glucose (P < 0.001) and insulin (P < 0.036) levels. In addition, L-4F treatment increased aortic and bone marrow heme oxygenase (HO) activity and decreased aortic and bone marrow superoxide production (P < 0.001). L-4F treatment increased serum adiponectin levels (P < 0.037) and decreased adipogenesis in mouse bone marrow (P < 0.039) and in cultures of human bone marrow-derived mesenchymal stem cells (P < 0.022). This was manifested by reduced adiposity, improved insulin sensitivity, improved glucose tolerance, increased plasma adiponectin levels, and reduced IL-1β and IL-6 levels in obese mice. This study highlights the existence of a temporal relationship between HO-1 and adiponectin that is positively affected by L-4F in the ob/ob mouse model of diabetes, resulting in the amelioration of the deleterious effects of diabetes.

[Research Articles] Intracellular lipid droplet targeting by apolipoprotein A-V requires the carboxyl-terminal segment

Shu, X., Ryan, R. O., Forte, T. M. — 2008-07-11

The expression of apolipoprotein A-V (apoA-V) in hepatoma cells results in homing of this protein to intracellular lipid droplets. When hepatoma cells transfected with a full-length apoA-V-green fluorescent protein fusion protein were cultured in medium that was not supplemented with oleic acid (OA), intracellular lipid droplet size and number were reduced compared with those of cells supplemented with OA. Confocal microscopy studies revealed that apoA-V associates with lipid droplets under both conditions. To define the structural requirements for apoA-V lipid droplet association, hepatoma cells were transfected with a series of C-terminal truncated apoA-V variants. Confocal microscopy analysis revealed that, in a manner similar to mature full-length apoA-V (343 amino acids), truncation variants apoA-V(1-292), apoA-V(1-237), and apoA-V(1-191) associated with lipid droplets, while apoA-V(1-146) did not. Western blot analysis of the relative abundance of apoA-V in cell lysates versus conditioned medium indicated that apoA-V variants associated with lipid droplets were poorly secreted while apoA-V(1-146) was efficiently secreted. Ultracentrifugation of conditioned medium revealed that, unlike full-length apoA-V, which associates with lipoproteins, apoA-V(1-146) was present solely in the lipoprotein-deficient fraction. Deletion of the N-terminal signal peptide from apoA-V resulted in an inability of the protein to be secreted into the medium, although it associated with lipid droplets. Taken together, these data suggest that the C terminus of apoA-V is essential for lipid droplet association in transfected hepatoma cells and lipoprotein association in conditioned medium while the signal peptide is required for extracellular trafficking of this protein.

[Research Articles] Reduction of plasma glycosphingolipid levels has no impact on atherosclerosis in apolipoprotein E-null mice

Glaros, E. N., Kim, W. S., Rye, K.-A., Shayman, J. A., Garner, B. — 2008-07-11

Glycosphingolipids (GSLs) have been implicated as potential atherogenic lipids. Studies in apolipoprotein E-null (apoE^–/–) mice indicate that exacerbated tissue GSL accumulation resulting from -galactosidase deficiency promotes atherosclerosis, whereas the serine palmitoyl transferase inhibitor myriocin (which reduces plasma and tissue levels of several sphingolipids, including sphingomyelin, ceramide, sphingosine-1-phosphate, and GSLs) inhibits atherosclerosis. It is not clear whether GSL synthesis inhibition per se has an impact on atherosclerosis. To address this issue, apoE^–/– mice maintained on a high-fat diet were treated with a potent glucosylceramide synthesis inhibitor, d-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4), 10 mg/kg/day for 94 days, and lesion development was compared in mice that were treated with vehicle only. EtDO-P4 reduced plasma GSL concentration by approximately 50% but did not affect cholesterol or triglyceride levels. Assessment of atherosclerotic lesions at four different sites indicated that EtDO-P4 had no significant impact on lesion area. Thus, despite the previously observed positive correlations between plasma and aortic GSL concentrations and the development of atherosclerosis, and the in vitro evidence implying that GSLs may be pro-atherogenic, our current data indicate that inhibition of GSL synthesis does not inhibit atherosclerosis in vivo.

[Research Articles] Activation of the constitutive androstane receptor decreases HDL in wild-type and human apoA-I transgenic mice

2008-07-11

The nuclear hormone receptor constitutive androstane receptor (CAR, NR1I3) regulates detoxification of xenobiotics and endogenous molecules, and has been shown to be involved in the metabolism of hepatic bile acids and cholesterol. The goal of this study was to address potential effects of CAR on the metabolism of HDL particles, key components in the reverse transport of cholesterol to the liver. Wild-type (WT) mice, transgenic mice expressing human apolipoprotein A-I (HuAITg), and CAR-deficient (CAR^–/–) mice were treated with the specific CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). CAR activation decreased HDL cholesterol and plasma apolipoprotein A-I (apoA-I) levels in both WT and HuAITg mice, but not CAR^–/– mice. Both mouse apoA-I and human apoA-I were decreased by more than 40% after TCPOBOP treatment, and kinetic studies revealed that the production rate of HDL is reduced in TCPOBOP-treated WT mice. In transient transfections, TCPOBOP-activated CAR decreased the activity of the human apoA-I promoter. Although loss of CAR function did not alter HDL levels in normal chow-fed mice, HDL cholesterol, apoA-I concentration, and apoA-I mRNA levels were increased in CAR^–/– mice relative to WT mice when both were fed a high-fat diet. We conclude that CAR activation in mice induces a pronounced decrease in circulating levels of plasma HDL, at least in part through downregulation of apoA-I gene expression.

[Research Articles] Cell culture models demonstrate that CFTR dysfunction leads to defective fatty acid composition and metabolism

2008-07-11

Cystic fibrosis (CF) is associated with fatty acid alterations characterized by low linoleic and docosahexaenoic acid. It is not clear whether these fatty acid alterations are directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction or result from nutrient malabsorption. We hypothesized that if fatty acid alterations are a result of CFTR dysfunction, those alterations should be demonstrable in CF cell culture models. Two CF airway epithelial cell lines were used: 16HBE, sense and antisense CFTR cells, and C38/IB3-1 cells. Wild-type (WT) and CF cells were cultured in 10% fetal bovine serum (FBS) or 10% horse serum. Fatty acid levels were analyzed by GC-MS. Culture of both WT and CF cells in FBS resulted in very low linoleic acid levels. When cells were cultured in horse serum containing concentrations of linoleic acid matching those found in human plasma, physiological levels of linoleic acid were obtained and fatty acid alterations characteristic of CF tissues were then evident in CF compared with WT cells. Kinetic studies with radiolabeled linoleic acid demonstrated in CF cells increased conversion to longer and more-desaturated fatty acids such as arachidonic acid. In conclusion, these data demonstrate that CFTR dysfunction is associated with altered fatty acid metabolism in cultured airway epithelial cells.

[Research Articles] ESR1 polymorphism is associated with plasma lipid and apolipoprotein levels in Caucasians of the Rochester Family Heart Study

Klos, K. L. E., Boerwinkle, E., Ferrell, R. E., Turner, S. T., Morrison, A. C. — 2008-07-11

We evaluated six estrogen receptor 1 (ESR1) polymorphisms for association with ten plasma lipid and apolipoprotein traits in 1,847 individuals (941 females and 906 males) in the multi-generation Rochester Family Heart Study using a generalized estimating equation approach. Apolipoprotein A-I (apoA-I), apoA-II, and HDL-cholesterol (HDL-C) were associated with exon 4 rs1801132 (Pro325Pro) genotype (P = 0.0044, P = 0.0048, and P = 0.0035, respectively). Positive correlation between levels of apoA-I, apoA-II, and HDL-C and the number of G alleles was observed in females (P = 0.0120, P = 0.0032, and P = 0.0030), but not males (P > 0.05). Because few studies have evaluated the effect of ESR1 gene polymorphisms on lipid traits in children, we also stratified our sample at the age of 15 years. There was evidence of association between intron 1 single-nucleotide polymorphisms rs9322331 and rs9340799 and apoC-II, and triglycerides (TGs) in youths 15 years and younger. In youths, evidence of association between rs9322331 and rs9340799 and apoC-II was stronger in males (P = 0.0036 and P = 0.0124) than in females (P > 0.05), whereas evidence of association with TG was stronger in females (P = 0.0030 and P = 0.0024) than in males (P > 0.05). These findings suggest that ESR1 variation plays an age- and sex-dependent role in determining plasma lipid and apolipoprotein levels.

[Research Articles] Retinoic acid induces PGI synthase expression in human endothelial cells

2008-07-11

Retinoic acid (RA) exhibits anti-inflammatory, anti-tumor, and immuno-modulatory actions, and affects angiogenesis and thrombosis. Arachidonic acid (AA) metabolites are involved in all these processes. We explored the effect of RA on AA metabolism in human umbilical vein endothelial cells (HUVECs). 13-cis-RA increased the release of prostaglandin I₂ (PGI₂₎, both spontaneous and thrombin-induced, in terms of 6-oxo-PGF₁ analyzed by enzyme-immunoassay. Coincubation with 13-cis-RA and interleukin-1β resulted in a synergic increase in the release of PGI₂. Consistently, 13-cis-RA increased the ability of HUVECs to inhibit AA-induced platelet aggregation. 13-cis-RA did not induce cyclooxygenase-isoenzyme expression, determined by immunoblotting, or activity, evaluated by analyzing eicosanoids formed from exogenous labeled AA by HPLC. In contrast, RA induced PGI synthase (PGIS) activity and expression in terms of mRNA and protein determined by real-time PCR and Western blotting, respectively. Results from experiments with several species of RA and with retinoic acid receptor (RAR) and retinoid X receptor (RXR) antagonists showed that the effect of RA on PGIS expression was mediated by RAR. Actinomycin D and cycloheximide both inhibited RA-induced PGIS expression. Furthermore, RA increased PGIS transcriptional activity in transient transfection assays, an effect that was prevented by an RAR antagonist. These results reinforce the concept that RA could be beneficial for patients with cardiovascular risk.

[Research Articles] Xanthophylls are preferentially taken up compared with {beta}-carotene by retinal cells via a SRBI-dependent mechanism

During, A., Doraiswamy, S., Harrison, E. H. — 2008-07-11

The purpose of this study was to investigate the mechanisms by which carotenoids [xanthophylls vs. β-carotene(β-C)] are taken up by retinal pigment epithelial (RPE) cells. The human RPE cell line, ARPE-19, was used. When ARPE-19 cells were fully differentiated (7–9 weeks), the xanthophylls lutein (LUT) and zeaxanthin (ZEA) were taken up by cells to an extent 2-fold higher than β-C (P < 0.05). At 9 weeks, cellular uptakes were 1.6, 2.5, and 3.2%, respectively, for β-C, LUT, and ZEA. Similar extents were observed when carotenoids were delivered in either Tween 40 or "chylomicrons" produced by Caco-2 cells. Differentiated ARPE-19 cells did not exhibit any detectable β-C 15,15'-oxygenase activity or convert exogenous β-C into vitamin A. When using specific antibodies against the lipid transporters cluster determinant 36 (CD36) and scavenger receptor class B type I (SR-BI), cellular uptake of β-C and ZEA were significantly decreased (40–60%) with anti-SR-BI but not with anti-CD36. Small interfering RNA transfection for SR-BI led to marked knockdown of SR-BI protein expression (~90%), which resulted in decreased β-C and ZEA uptakes by 51% and 87%, respectively. Thus, the present data show that RPE cells preferentially take up xanthophylls versus the carotene by a process that appears to be entirely SR-BI-dependent for ZEA and partly so for β-C. This mechanism may explain, in part, the preferential accumulation of xanthophylls in the macula of the retina.

[Research Articles] Lipid composition of microdomains is altered in a cell model of Gaucher disease

Hein, L. K., Duplock, S., Hopwood, J. J., Fuller, M. — 2008-07-11

The formation of cholesterol and sphingolipids into specialized liquid-ordered membrane microdomains (rafts) has been proposed to function in the intracellular sorting and transport of proteins and lipids. Defined by biochemical criteria, rafts resist solubilization in nonionic detergents, enabling them to be isolated as detergent-resistant membranes (DRM). In this study, we characterized the lipid composition of DRM from a cell model of the sphingolipid storage disorder, Gaucher disease, in which the catabolism of the sphingolipid glucosylceramide (GC) is impaired. In this cell model, we showed that GC accumulated primarily in the DRM, with smaller secondary increases in ceramide, dihexosylceramide, trihexosylceramide, and phosphatidylglycerol. This suggested that not only was lipid metabolism altered as a consequence of the cells' inability to degrade GC, but this affected the DRM rather than other regions of the membrane. This increase in lipids in the DRM may be responsible for the altered lipid and protein sorting seen in Gaucher disease. Analysis of individual lipid species revealed preservation of the shorter and fully saturated fatty acid species in the DRM, suggesting that the highly ordered and tightly packed nature of the DRM is maintained.

[Research Articles] Rat heart cannot synthesize docosahexaenoic acid from circulating {alpha}-linolenic acid because it lacks elongase-2

Igarashi, M., Ma, K., Chang, L., Bell, J. M., Rapoport, S. I. — 2008-07-11

The extent to which the heart can convert -linolenic acid (-LNA, 18:3n-3) to longer chain n-3 PUFAs is not known. Conversion rates can be measured in vivo using radiolabeled -LNA and a kinetic fatty acid model. [1-¹⁴C]-LNA was infused intravenously for 5 min in unanesthetized rats that had been fed an n-3 PUFA-adequate [4.6% -LNA, no docosahexaenoic acid (DHA, 22:6n-3)] or n-3 PUFA-deficient diet (0.2% -LNA, nor DHA) for 15 weeks after weaning. Arterial plasma was sampled, as was the heart after high-energy microwaving. Rates of conversion of -LNA to longer chain n-3 PUFAs were low, and DHA was not synthesized at all in the heart. Most -LNA within the heart had been β-oxidized. In deprived compared with adequate rats, DHA concentrations in plasma and heart were both reduced by >90%, whereas heart and plasma levels of docosapentaenoic acid (DPAn-6, 22:5n-6) were elevated. Dietary deprivation did not affect cardiac mRNA levels of elongase-5 or desaturases 6 and 5, but elongase-2 mRNA could not be detected. In summary, the rat heart does not synthesize DHA from -LNA, owing to the absence of elongase-2, but must obtain its DHA entirely from plasma. Dietary n-3 PUFA deprivation markedly reduces heart DHA and increases heart DPAn-6, which may make the heart vulnerable to different insults.

[Research Articles] Plasma fatty acid binding protein 4 is associated with atherogenic dyslipidemia in diabetes

2008-07-11

The aim of this study was to evaluate the impact of adipocyte fatty acid binding protein 4 (FABP4) on the lipid profile in type 2 diabetic subjects. Plasma levels of FABP4 and adiponectin and an extensive lipid profile were analyzed in 169 type 2 diabetic subjects and 105 controls. Type 2 diabetic subjects were categorized according the presence of atherogenic dyslipidemia. Univariate statistical analyses, partial correlation tests, and binary logistic regression models were applied. In type 2 diabetic subjects, FABP4 was positively correlated with plasma triglycerides (P = 0.007), apolipoprotein C-III (apoC-III) (P = 0.009), and all the components of triglyceride-rich lipoproteins, including VLDL triglycerides (P = 0.002), VLDL-cholesterol (P = 0.001), and VLDL apoB (P = 0.001). FABP4 was inversely correlated with apoA-I (P = 0.038), HDL-cholesterol (P = 0.002), and HDL apoA-I (P = 0.010) in type 2 diabetic subjects. These correlations are not significantly affected by age, gender, body mass index, adiponectin, insulin, or any pharmacological treatment. The associations are even stronger when the FABP4/adiponectin ratio is considered. None of these associations were observed in controls. High FABP4 and low adiponectin levels are independent predictors of atherogenic dyslipidemia. In conclusion, FABP4 plasma concentrations hold strong potential for development as a clinical biomarker for atherogenic dyslipidemia, independent of obesity and insulin resistance, in type 2 diabetic subjects.

[Research Articles] Effects of acyl chain length, unsaturation, and pH on thermal stability of model discoidal HDLs

Guha, M., Gantz, D. L., Gursky, O. — 2008-07-11

HDLs prevent atherosclerosis by removing excess cell cholesterol. Lipid composition affects HDL functions in cholesterol removal, yet its effects on the disk stability remain unclear. We hypothesize that reduced length or increased cis-unsaturation of phosphatidylcholine acyl chains destabilize discoidal HDL and promote protein dissociation and lipoprotein fusion. To test this hypothesis, we determined thermal stability of binary complexes reconstituted from apoC-I and diacyl PCs containing 12–18 carbons with 0–2 cis-double bonds. Kinetic analysis using circular dichroism shows that, for fully saturated PCs, chain length increase by two carbons stabilizes lipoprotein by G* (37°C) 1.4 kcal/mol, suggesting that hydrophobic interactions dominate the disk stability; distinct effects of pH and salt indicate contribution of electrostatic interactions. Similarly, apoA-I-containing disks show increased stability with increasing chain length. Acyl chain unsaturation reduces disk stability. In summary, stability of discoidal HDL correlates directly with fatty acyl chain length and saturation: the longer and more fully saturated are the chains, the more extensive are the stabilizing lipid-protein and lipid-lipid interactions and the higher is the free energy barrier for protein dissociation and lipoprotein fusion. This sheds new light on the existing data of cholesterol efflux to discoidal HDL and suggests that moderate lipoprotein destabilization facilitates cholesterol insertion.

[Research Articles] Metabolism of apical versus basolateral sn-2-monoacylglycerol and fatty acids in rodent small intestine

Storch, J., Zhou, Y. X., Lagakos, W. S. — 2008-07-11

The metabolic fates of radiolabeled sn-2-monoacylglycerol (MG) and oleate (FA) in rat and mouse intestine, added in vivo to the apical (AP) surface in bile salt micelles, or to the basolateral (BL) surface via albumin-bound solution, were examined. Mucosal lipid products were quantified, and the results demonstrate a dramatic difference in the esterification patterns for both MG and FA, depending upon their site of entry into the enterocyte. For both lipids, the ratio of triacylglycerol to phospholipid (TG:PL) formed was approximately 10-fold higher for delivery at the AP relative to the BL surface. Further, a 3-fold higher level of FA oxidation was found for BL compared with AP substrate delivery. Incorporation of FA into individual PL species was also significantly different, with >2-fold greater incorporation into phosphatidylethanolamine (PE) and a 3-fold decrease in the phosphatidylcholine:PE ratio for AP- compared with BL-added lipid. Overnight fasting increased the TG:PL incorporation ratio for both AP and BL lipid addition, suggesting that metabolic compartmentation is a physiologically regulated phenomenon. These results support the existence of separate pools of TG and glycerolipid intermediates in the intestinal epithelial cell, and underscore the importance of substrate trafficking in the regulation of enterocyte lipid metabolism.

[Research Articles] Identification of putative active site residues of ACAT enzymes

Das, A., Davis, M. A., Rudel, L. L. — 2008-07-11

In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

[Research Articles] Infection induces a positive acute phase apolipoprotein E response from a negative acute phase gene: role of hepatic LDL receptors

Li, L., Thompson, P. A., Kitchens, R. L. — 2008-07-11

Apolipoprotein E (apoE) plays important roles in lipid homeostasis, anti-inflammation, and host defense. Since tissue apoE mRNA levels have been reported to decrease during inflammatory responses, we were surprised to find that plasma apoE levels were significantly elevated during septic infections in both humans and mice. This apparent paradox was also observed during lipopolysaccharide-induced acute inflammation in mice: plasma levels of apoE increased up to 4-fold despite sharply decreased apoE gene expression in the liver, macrophages, and extrahepatic tissues. We hypothesized that apoE levels were augmented by decreased plasma clearance. Our analysis revealed that apoE associated principally with HDL in mice and that apoE was cleared from the circulation principally via LDL receptors. The acute inflammatory response decreased LDL receptor expression in the liver and significantly reduced the rate of apoE clearance. In contrast, the same inflammatory stimuli increased LDL receptor expression in macrophages. Our results define a novel acute phase mechanism that increases circulating apoE levels as apoE production decreases. Diminished hepatic LDL receptor expression may thus cooperate with elevated LDL receptor expression in macrophages to facilitate the forward transport of apoE and its associated lipids to these key defense cells.

[Research Articles] Identification of a novel GPCAT activity and a new pathway for phosphatidylcholine biosynthesis in S. cerevisiae

Stalberg, K., Neal, A. C., Ronne, H., Stahl, U. — 2008-07-11

Turnover of phospholipids in the yeast Saccharomyces cerevisiae generates intracellular glycerophosphocholine (GPC). Here we show that GPC can be reacylated in an acyl-CoA-dependent reaction by yeast microsomal membranes. The lysophosphatidylcholine that is formed in this reaction is efficiently further acylated to phosphatidylcholine (PC) by yeast microsomes, thus providing a new pathway for PC biosynthesis that can either recycle endogenously generated GPC or utilize externally provided GPC. Genetic and biochemical evidence suggests that this new enzymatic activity, which we call GPC acyltransferase (GPCAT), is not mediated by any of the previously known acyltransferases in yeast. The GPCAT activity has an apparent V_max of 8.7 nmol/min/mg protein and an apparent K_m of 2.5 mM. It has a neutral pH optimum, similar to yeast glycerol-3-phosphate acyltransferase, but differs from the latter in being more heat stable. The GPCAT activity is sensitive to N-ethylmaleimide, phenanthroline, and Zn²⁺ ions. In vivo experiments showed that PC is efficiently labeled when yeast cells are fed with [³H]choline-GPC, and that this reaction occurs also in pct1 knockout strains, where de novo synthesis of PC by the CDP-choline pathway is blocked. This suggests that GPCAT can provide an alternative pathway for PC biosynthesis in vivo.

[Research Articles] Molecular mechanism of membrane targeting by the GRP1 PH domain,boxs

2008-07-11

The general receptor for phosphoinositides isoform 1 (GRP1) is recruited to the plasma membrane in response to activation of phosphoinositide 3-kinases and accumulation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃]. GRP1's pleckstrin homology (PH) domain recognizes PtdIns(3,4,5)P₃ with high specificity and affinity, however, the precise mechanism of its association with membranes remains unclear. Here, we detail the molecular basis of membrane anchoring by the GRP1 PH domain. Our data reveal a multivalent membrane docking involving PtdIns(3,4,5)P₃ binding, regulated by pH and facilitated by electrostatic interactions with other anionic lipids. The specific recognition of PtdIns(3,4,5)P₃ triggers insertion of the GRP1 PH domain into membranes. An acidic environment enhances PtdIns(3,4,5)P₃ binding and increases membrane penetration as demonstrated by NMR and monolayer surface tension and surface plasmon resonance experiments. The GRP1 PH domain displays a 28 nM affinity for POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/PtdIns(3,4,5)P₃ vesicles at pH 6.0, but binds 22-fold weaker at pH 8.0. The pH sensitivity is attributed in part to the His355 residue, protonation of which is required for the robust interaction with PtdIns(3,4,5)P₃ and significant membrane penetration, as illustrated by mutagenesis data. The binding affinity of the GRP1 PH domain for PtdIns(3,4,5)P₃-containing vesicles is further amplified (by ~6-fold) by nonspecific electrostatic interactions with phosphatidylserine/phosphatidylinositol. Together, our results provide new insight into the multivalent mechanism of the membrane targeting and regulation of the GRP1 PH domain.

[Research Articles] GM2/GD2 and GM3 gangliosides have no effect on cellular cholesterol pools or turnover in normal or NPC1 mice

Li, H., Turley, S. D., Liu, B., Repa, J. J., Dietschy, J. M. — 2008-07-11

These studies investigated the role of gangliosides in governing the steady-state concentration and turnover of unesterified cholesterol in normal tissues and in those of mice carrying the NPC1 mutation. In animals lacking either GM2/GD2 or GM3 synthase, tissue cholesterol concentrations and synthesis rates were normal in nearly all organs, and whole-animal sterol pools and turnover also were not different from control animals. Mice lacking both synthases, however, had small elevations in cholesterol concentrations in several organs, and the whole-animal cholesterol pool was marginally elevated. None of these three groups, however, had changes in any parameter of cholesterol homeostasis in the major regions of the central nervous system. When either the GM2/GD2 or GM3 synthase activity was deleted in mice lacking NPC1 function, the clinical phenotype was not changed, but lifespan was shortened. However, the abnormal cholesterol accumulation seen in the tissues of the NPC1 mouse was unaffected by loss of either synthase, and clinical and molecular markers of hepatic and cerebellar disease also were unchanged. These studies demonstrate that hydrophobic interactions between cholesterol and various gangliosides do not play an important role in determining cellular cholesterol concentrations in the normal animal or in the mouse with the NPC1 mutation.

[Research Articles] Hormone-sensitive lipase is involved in hepatic cholesteryl ester hydrolysis

2008-07-11

Hormone-sensitive lipase (HSL) regulates the hydrolysis of acylglycerol and cholesteryl ester (CE) in various organs, including adipose tissues. However, the hepatic expression level of HSL has been reported to be almost negligible. In the present study, we found that mice lacking both leptin and HSL (Lep^ob/ob/HSL^–/–) showed massive accumulation of CE in the liver compared with Lep^ob/ob/HSL^+/+ mice, while triacylglycerol (TG) accumulation was modest. Similarly, feeding with a high-cholesterol diet induced hepatic CE accumulation in HSL^–/– mice. Supporting these observations, we detected significant expression of protein as well as mRNA of HSL in the liver. HSL^–/– mice showed reduced activity of CE hydrolase, but not of TG lipase, in the liver compared with wild-type mice. Furthermore, we confirmed the expression of HSL in viable parenchymal cells isolated from wild-type mice. The hepatocytes from HSL^–/– mice showed reduced activity of CE hydrolase and contained more CE than those from HSL^+/+ mice even without the incubation with lipoproteins. Incubation with LDL further augmented the accumulation of CE in the HSL-deficient hepatocytes. From these results, we conclude that HSL is involved in the hydrolysis of CE in hepatocyes.

[Patient-Oriented and Epidemiological Research] The effect of IL6-174C/G polymorphism on postprandial triglyceride metabolism in the GOLDN study,boxs

2008-07-11

Chronically elevated interleukin-6 (IL-6) affects lipid and lipoprotein metabolism. Individuals genetically predisposed to higher IL-6 secretion may be at risk of dyslipidemia, especially during the postprandial phase. We investigated the effect of genetic variants at the IL6 locus on postprandial lipemia in US Whites participating in the Genetics of Lipid Lowering Drugs and Diet Network study. Subjects were given a single fat load composed of 3% of calories as protein, 14% as carbohydrate, and 83% as fat. Blood was drawn at 0 h, 3.5 h, and 6 h to determine plasma triglyceride (TG), TG-rich lipoprotein (TRL) and lipoprotein particle size. Homozygotes (GG) and heterozygotes (CG) of the -174C/G variant displayed higher plasma IL-6 concentrations compared with major allele homozygotes (CC) (P = 0.029). GG and CG subjects showed higher fasting plasma TG (P = 0.025), VLDL (P = 0.04), and large VLDL (P = 0.02) concentrations than did CC subjects. Moreover, GG and CG subjects experienced greater postprandial response of TG (P = 0.006) and TRL, including chylomicrons (P = 0.005), total VLDL (P = 0.029), and large VLDL (P = 0.017) than did CC subjects. These results suggest that the functional polymorphism -174C>G at the IL6 locus determines the difference in both fasting and postprandial TG metabolism. This phenomenon could be responsible for the observed association of this genetic variant with cardiovascular disease risk.

[Patient-Oriented and Epidemiological Research] An apolipoprotein A-V gene SNP is associated with marked hypertriglyceridemia among Asian-American patients

2008-07-11

Apolipoprotein A-V (apoA-V) is an important regulator of plasma levels of triglyceride (TG) in mice. In humans, APOA5 genetic variation is associated with TG in several populations. In this study, we determined the effects of the p.185Gly>Cys (c.553G>T; rs2075291) polymorphism on plasma TG levels in subjects of Chinese ancestry living in the United States and in a group of non-Chinese Asian ancestry. The frequency of the less common cysteine allele was 4-fold higher (15.1% vs. 3.7%) in Chinese high-TG subjects compared with a low-TG group (Chi-square = 20.2; P < 0.0001), corresponding with a 4.45 times higher risk of hypertriglyceridemia (95% confidence interval, 2.18–9.07; P < 0.001). These results were replicated in the non-Chinese Asians. Heterozygosity was associated, in the high-TG group, with a doubling of TG (P < 0.001), mainly VLDL TG (P = 0.014). All eleven TT homozygotes had severe hypertriglyceridemia, with mean TG of 2,292 ± 447 mg/dl. Compared with controls, carriers of the T allele had lower postheparin lipoprotein lipase activity but not hepatic lipase activity. In Asian populations, this common polymorphism can lead to profound adverse effects on lipoprotein profiles, with homozygosity accounting for a significant number of cases of severe hypertriglyceridemia. This specific apoA-V variant has a pronounced effect on TG metabolism, the mechanism of which remains to be elucidated.

[Methods] Rapid UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain fatty acid markers of peroxisomal disorders

2008-07-11

Quantification of pristanic acid, phytanic acid, and very long chain fatty acids (i.e., hexacosanoic, tetracosanoic, and docosanoic acids) in plasma is the primary method for investigateing a multitude of peroxisomal disorders (PDs). Typically based on GC-MS, existing methods are time-consuming and laborious. In this paper, we present a rapid and specific liquid chromatography tandem mass spectrometric method based on derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). Derivatization was undertaken to improve the poor mass spectrometric properties of these fatty acids. Analytes in plasma (20 µl) were hydrolyzed, extracted, and derivatized with DAABD-AE in ~2 h. Derivatives were separated on a reverse-phase column and detected by positive-ion electrospray ionization tandem mass spectrometry with a 5 min injection-to-injection time. Calibration plots were linear over ranges that cover physiological and pathological concentrations. Intraday (n = 12) and interday (n = 10) variations at low and high concentrations were less than 9.2%. Reference intervals in normal plasma (n = 250) were established for each compound and were in agreement with the literature. Using specimens from patients with established diagnosis (n = 20), various PDs were reliably detected. In conclusion, this method allows for the detection of at least nine PDs in a 5 min analytical run. Furthermore, this derivatization approach is potentially applicable to other disease markers carrying the carboxylic group.

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2008-07-11