BBA - Molecular and Cell Biology of Lipids (v.1771, #10)

Metabolomics (liver and blood profiling) in a mouse model in response to fasting: A study of hepatic steatosis by V. van Ginneken; E. Verhey; R. Poelmann; R. Ramakers; K. Willems van Dijk; Lisanne Ham; P. Voshol; L. Havekes; M. Van Eck; J. van der Greef (1263-1270).
A metabolomic approach was applied to a mouse model of starvation-induced hepatic steatosis. After 24 h of fasting it appears that starvation reduced the phospholipids (PL), free cholesterol (FC), and cholesterol esters (CE) content of low-density lipoproteins (LDL). In liver lipid profiles major changes were observed using different techniques. High performance thin layer chromatography (HPTLC)-measurements of liver-homogenates indicated a significant rise of FC with 192%, triacylglycerols (TG) with 456% and cholesterol esters (CE) with 268% after 24 h of starvation in comparison with the control group. Reversed phase liquid chromatography coupled to mass spectrometry measurements (LC-MS) of liver homogenate indicated that the intensity of Phosphatidylcholine (PC) in the 24-h starvation group dropped to 90% of the value in the control group while the intensity of CE and TG increased to 157% and 331%, respectively, of the control group. Interestingly, a 49:4-TG with an odd number of C atoms appeared during starvation. This unique triacylglycerol has all characteristics of a biomarker for detection of hepatic steatosis. These observations indicate that in mammals liver lipid profiles are a dynamic system which are readily modulated by environmental factors like starvation.
Keywords: Metabolomics; Lipids; Liver; Starvation; Triacylglycerols; Hepatic steatosis;

The histone deacetylase inhibitor trichostatin A mediates upregulation of 5-lipoxygenase promoter activity by recruitment of Sp1 to distinct GC-boxes by Nicole Schnur; Sabine Seuter; Careen Katryniok; Olof Rådmark; Dieter Steinhilber (1271-1282).
The histone deacetylase inhibitor trichostatin A (TsA) potently induces 5-lipoxygenase (5-LO) promoter activity in reporter gene assays as well as 5-LO mRNA expression. We identified two proximal Sp1/Sp3 binding sites in the 5-LO gene promoter mediating the TsA effect in both 5-LO-negative HeLa cells and in 5-LO expressing Mono Mac 6 (MM6) cells, the tandem GC-boxes, by contrast, were not important for the TsA effect. TsA neither altered the protein expression levels of Sp1/Sp3 nor of the histone deacetylases HDAC1/2, nor did it apparently change the protein complex formation by these factors. Also, treatment of cells with TsA did not change the binding affinity of Sp1/Sp3 in cell extracts, as tested by DAPA analysis using probes containing the proximal GC boxes. However, in the living cell TsA induced Sp1, Sp3 and RNA polymerase II recruitment to the 5-LO promoter without changing the acetylation status of histone protein H4. Cotransfection studies suggest that both Sp1 and Sp3 can mediate the TsA effect. This is the first report demonstrating that Sp3 is involved in the regulation of 5-LO promoter activity. In summary, we show that TsA increases 5-LO promoter activity by the enhanced recruitment of Sp1 and Sp3 to the 5-LO promoter.
Keywords: 5-lipoxygenase; Histone deacetylase inhibitor; Trichostatin A; Sp1; Promoter activity;

Loss of intestinal fatty acid binding protein increases the susceptibility of male mice to high fat diet-induced fatty liver by Luis B. Agellon; Laurie Drozdowski; Lena Li; Claudiu Iordache; Le Luong; M. Tom Clandinin; Richard R.E. Uwiera; Matthew J. Toth; Alan B.R. Thomson (1283-1288).
Mice lacking I-FABP (encoded by the Fabp2 gene) exhibit a gender dimorphic response to a high fat/cholesterol diet challenge characterized by hepatomegaly in male I-FABP-deficient mice. In this study, we determined if this gender-specific modification of liver mass in mice lacking I-FABP is attributable to the high fat content of the diet alone and whether hepatic Fabp1 gene (encodes L-FABP) expression contributes to this difference. Wild-type and Fabp2 / mice of both genders were fed a diet enriched with either polyunsaturated or saturated fatty acids (PUFA or SFA, respectively) in the absence of cholesterol. Male Fabp2 / mice, but not female Fabp2 / mice, exhibited increased liver mass and hepatic triacylglycerol (TG) deposition as compared to corresponding wild-type mice. In wild-type mice that were fed the standard chow diet, there was no difference in the concentration of hepatic L-FABP protein between males and females although the loss of I-FABP did cause a slight reduction of hepatic L-FABP abundance in both genders. The hepatic L-FABP mRNA abundance in both male and female wild-type and Fabp2 / mice was higher in the PUFA-fed group than in the SFA-fed group, and was correlated with L-FABP protein abundance. No correlation between hepatic L-FABP protein abundance and hepatic TG concentration was found. The results obtained demonstrate that loss of I-FABP renders male mice sensitive to high fat diet-induced fatty liver, and this effect is independent of hepatic L-FABP.
Keywords: FABP; High fat diet; Fatty liver; Small intestine; Mouse; Gender effect;

A comparative study of microstructural development in paired human hepatic and gallbladder biles by Daphne Weihs; Judith Schmidt; Dganit Danino; Ilana Goldiner; Diana Leikin-Gobbi; Arieh Eitan; Moshe Rubin; Yeshayahu Talmon; Fred M. Konikoff (1289-1298).
Cholesterol gallstones usually develop in the gallbladder and rarely form in bile ducts even in patients with highly lithogenic bile. Bile concentration and proteins (e.g. mucin) may affect crystallization, but the exact nature of this effect, especially in relation to crystallization pathways and microstructural evolution remains unclear. We examined lipid microstructures in paired hepatic and gallbladder biles to reveal ones that are essential for crystallization. Combining digital light microscopy with cryogenic-temperature transmission electron microscopy we are able to directly visualize and compare the time evolution of lipid microstructures in paired hepatic, gallbladder and diluted gallbladder biles of gallstone patients and controls, without drying or separating. Gallbladder bile exhibited several multilamellar vesicles and spheroidal micelles preceding and throughout crystallization. Vesicle morphology changed before crystallization was observed. In contrast, hepatic bile revealed almost no crystallization and while a variety of unilamellar vesicles and spheroidal micelles existed throughout the examination, multilamellar vesicles were rare. Diluted gallbladder bile was different from native gallbladder bile, as well as the paired hepatic bile, yielding occasional crystallization. Our findings suggest that maturing multilamellar vesicles precede (and at least partially initiate) crystallization in gallbladder bile. Although microstructural development seems to be concentration dependent, dilution of gallbladder bile to hepatic bile concentrations neither makes it identical to hepatic bile, nor prevents crystallization.
Keywords: Cholesterol gallstones; Lipid microstructures; Cryogenic-temperature transmission electron microscopy; Digital light microscopy;

Metabolism and short-term metabolic effects of conjugated linoleic acids in rat hepatocytes by Paola Priore; Anna M. Giudetti; Francesco Natali; Gabriele V. Gnoni; Math J.H. Geelen (1299-1307).
Metabolic fate and short-term effects of a 1:1 mixture of cis-9,trans-11 and trans-10,cis-12-conjugated linoleic acids (CLA), compared to linoleic acid (LA), on lipid metabolism was investigated in rat liver. In isolated mitochondria CLA-CoA were poorer substrates than LA-CoA for carnitine palmitoyltransferase-I (CPT-I) activity. However, in digitonin-permeabilized hepatocytes, where interactions among different metabolic pathways can be simultaneously investigated, CLA induced a remarkable stimulatory effect on CPT-I activity. This stimulation can be ascribed to a reduced malonyl-CoA level in turn due to inhibition of acetyl-CoA carboxylase (ACC) activity. The ACC/malonyl-CoA/CPT-I system can therefore represent a coordinate control by which CLA may exert effects on the partitioning of fatty acids between esterification and oxidation. Moreover, the rate of oxidation to CO2 and ketone bodies was significantly higher from CLA; peroxisomes rather than mitochondria were responsible for this difference. Interestingly, peroxisomal acyl-CoA oxidase (AOX) activity strongly increased by CLA-CoA compared to LA-CoA. CLA, metabolized by hepatocytes at a higher rate than LA, were poorer substrates for cellular and VLDL-triacylglycerol (TAG) synthesis. Overall, our results suggest that increased fatty acid oxidation with consequent decreased fatty acid availability for TAG synthesis is a potential mechanism by which CLA reduce TAG level in rat liver.
Keywords: Acetyl-CoA carboxylase; Carnitine palmitoyltransferase-I; Conjugated linoleic acid; Linoleic acid; Mitochondria; Peroxisomes;

A role for diacylglycerol in annexin A7-mediated fusion of lung lamellar bodies by Avinash Chander; Xiao-Liang Chen; Devendra G. Naidu (1308-1318).
Lung surfactant secretion in alveolar type II cells occurs following lamellar body fusion with plasma membrane. Annexin A7 is a Ca2+-dependent membrane-binding protein that is postulated to promote membrane fusion during exocytosis in some cell types including type II cells. Since annexin A7 preferably binds to lamellar body membranes, we postulated that specific lipids could modify the mode of annexin A7 interaction with membranes and its membrane fusion activity. Initial studies with phospholipid vesicles containing phosphatidylserine and other lipids showed that certain lipids affected protein interaction with vesicle membranes as determined by change in protein tryptophan fluorescence, protein interaction with trans membranes, and by protein sensitivity to limited proteolysis. The presence of signaling lipids, diacylglycerol or phosphatidylinositol-4,5-bisphosphate, as minor components also modified the lipid vesicle effect on these characteristics and membrane fusion activity of annexin A7. In vitro incubation of lamellar bodies with diacylglycerol or phosphatidylinositol-4,5-bisphosphate caused their enrichment with either lipid, and increased the annexin A7 and Ca2+-mediated fusion of lamellar bodies. Treatment of isolated lung lamellar bodies with phosphatidylinositol- or phosphatidylcholine phospholipase C to increase diacylglycerol, without or with preincubation with phosphatidylinositol-4,5-bisphosphate, augmented the fusion activity of annexin A7. Thus, increased diacylglycerol in lamellar bodies following cell stimulation with secretagogues may enhance membrane fusion activity of annexin A7.
Keywords: Membrane fusion; Surfactant secretion; Protein fluorescence; Proteolysis; Membrane insertion; Membrane binding; Signaling lipids;

Docosahexaenoic acid (DHA; 22:6 n-3) is an essential fatty acid required for the normal function of several tissues, especially the brain. Previous studies suggested that lysophosphatidylcholine (lysoPC) is a preferred carrier of DHA to the brain, although the pathways of the formation of DHA-containing lysophospholipids in plasma have not been delineated. We propose that endothelial lipase (EL), a phospholipase A1 that plays an important role in the metabolism of high density lipoproteins, may be responsible for the generation of DHA lysophospholipids in plasma. Here we studied the substrate specificity of EL using deuterium-labeled phospholipids with different polar head groups, as well as DHA-enriched natural phospholipids to test this hypothesis. Glycerol-stabilized phospholipids were treated with recombinant EL, and the products were analyzed by liquid chromatography/electrospray ionization mass spectrometry. EL showed the polar head group specificity in the order of phosphatidylethanolamine > phosphatidylcholine > phosphatidylserine > phosphatidic acid. Within the same phospholipid class, the enzyme showed preference for the species containing DHA at the sn-2 position, and was inactive in the hydrolysis of phospholipids containing an ether linkage. Since EL is known to be secreted by the cells of blood–brain barrier, we suggest that it plays an important role in the delivery of DHA lysophospholipid carriers to the brain.
Keywords: Docosahexaenoic acid; Endothelial lipase; Phosphatidylethanolamine; Phosphatidylcholine; Docosahexaenoyl lysophosphatidylcholine; Docosahexaenoyl lysophosphatidylethanolamine;

17β-Estradiol (E2) fatty acyl esters naturally incorporate into high-density lipoprotein (HDL). The objective was to elucidate mechanisms involved in HDL-associated E2 cellular uptake and to determine the intracellular distribution of E2 and its fatty acyl esters (E2-FAE) after uptake. [3H]E2 or [3H] cholesterol was incubated with human serum for 24 h to allow for fatty acyl esterification. Total-HDL containing [3H]E2-FAE or [3H]cholesterol esters was isolated by sequential density ultracentrifugation and then incubated with Fu5AH rat hepatoma cells for various time points. Cellular uptake was determined by intracellular radioactivity as a percentage of total radioactivity. Chemical inhibition of scavenger receptor class B, type I and low-density lipoprotein (LDL) receptor competition assays were performed to determine cellular uptake mechanisms. Compared to HDL-[3H]cholesterol, cellular uptake of HDL-[3H]E2 occurred at an initially rapid rate. SR-BI inhibition resulted in a decrease in HDL-E2 uptake and LDL impaired this uptake in a concentration-dependent manner. Accordingly, pretreatment of cells with BLT-1 combined with LDL addition significantly attenuated HDL-E2 uptake. HDL-E2-FAE was hydrolyzed into free E2 with the maximum at 24 h. Fu5AH cells facilitate HDL-E2 uptake by at least SR-BI and LDL receptor pathways and intracellular hydrolysis of E2-FAE into free E2 ensues.
Keywords: Fu5AH hepatoma cell; BLT-1; apoE;

β-Amyloid (Aβ40, Aβ42) binding to modified LDL accelerates macrophage foam cell formation by Berta Schulz; Gerhard Liebisch; Margot Grandl; Tobias Werner; Stefan Barlage; Gerd Schmitz (1335-1344).
Apart from its role as a risk factor in arteriosclerosis, plasma cholesterol is increasingly recognized to play a major role in the pathogenesis of Alzheimer's disease (AD). Moreover, alterations of intracellular cholesterol metabolism in neuronal and vascular cells are of considerable importance for the understanding of AD. Cellular cholesterol accumulation enhances the deposition of insoluble β-amyloid peptides, which is considered a hallmark in the pathogenesis of AD. In order to test the hypothesis, whether exogenous β-amyloid peptides (Aβ42, Aβ40) might contribute to cellular cholesterol accumulation by opsonization of lipoproteins, we compared the binding and uptake of native LDL, enzymatically modified LDL (E-LDL), copper oxidized LDL (Ox-LDL) and HDL as control, preincubated either in the absence or presence of Aβ42 or Aβ40, by human monocytes or monocyte-derived macrophages. Incubation of monocytes and macrophages with Aβ–lipoprotein-complexes lead to increased cellular free and esterified cholesterol when compared to non-opsonized lipoproteins, except for HDL. Furthermore, the cellular uptake of these complexes regulated Aβ-receptors such as FPRL-1 or LRP/CD91. In summary, our results suggest that Aβ42 and Aβ40 act as potent opsonins for LDL, E-LDL and Ox-LDL and enhance cellular cholesterol accumulation as well as Aβ-deposition in vessel wall macrophages.
Keywords: β-Amyloid peptide; Lipoprotein; Macrophage; Alzheimer disease; Arteriosclerosis;