Byung Hong Chung

[8] Single vertical spin density gradient ultracentrifugation

Publisher Summary Density gradient ultracentrifugation in the swing out rotor has been successfully applied to fractionation of the major lipoprotein species and their subspecies; however, it requires a minimum of 24–36 h to complete the separation of the major lipoprotein fractions in plasma. This chapter describes 13 separate vertical spin procedures for the preparative separation of a number of different plasma lipoproteins and a method for the quantitative analysis of lipoprotein cholesterol from plasma separated by single vertical spin (SVS) density gradient ultracentrifugation. The vertical rotor works on the principle that compression of the gradient geometry by the vertical rotor when at speed shortens spin time over more conventional ultracentrifugation techniques without loss of resolution. The single vertical spin procedure has a number of advantages over the more conventional methods including decreased spin time, decreased lipoprotein degradation, and good resolution. But there are certain disadvantages of this method also, such as wall adherence of VLDL and albumin, resolution, and plasma volume limit per rotor. However, the chapter addresses solutions to the problems of single vertical spin ultracentrifugation such as angled-head rotors.

Dietary fructose induces a wide range of genes with distinct shift in carbohydrate and lipid metabolism in fed and fasted rat liver

Dietary fructose has been suspected to contribute to development of metabolic syndrome. However, underlying mechanisms of fructose effects are not well characterized. We investigated metabolic outcomes and hepatic expression of key regulatory genes upon fructose feeding under well defined conditions. Rats were fed a 63% (w/w) glucose or fructose diet for 4 h/day for 2 weeks, and were killed after feeding or 24-hour fasting. Liver glycogen was higher in the fructose-fed rats, indicating robust conversion of fructose to glycogen through gluconeogenesis despite simultaneous induction of genes for de novo lipogenesis and increased liver triglycerides. Fructose feeding increased mRNA of previously unidentified genes involved in macronutrient metabolism including fructokinase, aldolase B, phosphofructokinase-1, fructose-1,6-bisphosphatase and carbohydrate response element binding protein (ChREBP). Activity of glucose-6-phosphate dehydrogenase, a key enzyme for ChREBP activation, remained elevated in both fed and fasted fructose groups. In the fasted liver, the fructose group showed lower non-esterified fatty acids, triglycerides and microsomal triglyceride transfer protein mRNA, suggesting low VLDL synthesis even though plasma VLDL triglycerides were higher. In conclusion, fructose feeding induced a broader range of genes than previously identified with simultaneous increase in glycogen and triglycerides in liver. The induction may be in part mediated by ChREBP.

Lipoprotein Particles of Intraocular Origin in Human Bruch Membrane : An Unusual Lipid Profile

PURPOSE Throughout adulthood, Bruch membrane (BrM) accumulates esterified cholesterol (EC) associated with abundant 60- to 80-nm-diameter lipoprotein-like particles (LLP), putative apolipoprotein B (apoB) lipoproteins secreted by the retinal pigment epithelium (RPE). In the present study, neutral lipid, phospholipids, and retinoid components of human BrM-LLP were assayed. METHODS Particles isolated from paired choroids of human donors were subjected to comprehensive lipid profiling (preparative liquid chromatography [LC] gas chromatography [GC]), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), Western blot analysis, and negative stain electron microscopy. Results were compared to plasma lipoproteins isolated from normolipemic volunteers and to conditioned medium from RPE-J cells supplemented with palmitate to induce particle synthesis and secretion. RESULTS EC was the largest component (32.4+/-7.9 mol%) of BrM-LLP lipids. EC was 11.3-fold more abundant than triglyceride (TG), unlike large apoB lipoproteins in plasma. Of the fatty acids (FA) esterified to cholesterol, linoleate (18:2n6) was the most abundant (41.7+/-4.7 mol%). Retinyl ester (RE) was detectable at picomolar levels in BrM-LLP. Notably scarce in any BrM-LLP lipid class was the photoreceptor-abundant FA docosahexaenoate (DHA, 22:6n3). RPE-J cells synthesized apoB and numerous EC-rich spherical particles. CONCLUSIONS BrM-LLP composition resembles plasma LDL more than it does photoreceptors. An EC-rich core is possible for newly synthesized lipoproteins as well as those processed in plasma. Abundant EC could contribute to a transport barrier in aging and lesion formation in age-related maculopathy (ARM). Analysis of BrM-LLP composition has revealed new aspects of retinal cholesterol and retinoid homeostasis.

Disruption of endothelial barrier function by lipolytic remnants of triglyceride-rich lipoproteins.

Remnants, resulting from the lipolysis of triglyceride-rich lipoproteins, injured cultured endothelial cells and resulted in decreased barrier function of the vascular endothelium. Endothelial cells were cultured on micropore filters. Albumin transfer across endothelial cell monolayers was measured after a 24-h exposure to media enriched with control or in vitro-lipolyzed samples of various hypertriglyceridemic (HTG) sera and its isolated lipoprotein (VLDL, LDL and HDL) and serum free protein (d greater than 1.21 g/ml) fractions. Compared with control cultures, neither control HTG serum nor its isolated lipoprotein and serum-free protein fractions had any effect on albumin transfer. In contrast, lipolyzed HTG (L-HTG) serum and all of its isolated lipoprotein fractions (L-VLDL, L-IDL, L-LDL and L-HDL) caused a marked decrease in endothelial barrier function, evidenced by a significant increase in albumin transfer across endothelial monolayers. The L-IDL and L-HDL fractions were more effective in increasing albumin transfer than the L-VLDL and L-LDL fractions. The extent of the L-IDL and L-HDL mediated increases in albumin transfer was concentration dependent. An exposure of 12 h was required for L-HDL to increase albumin transfer. The L-HDL mediated increase in albumin transfer was reversible only after a 12-h exposure at low concentrations. The free protein fraction from L-HTG serum had no significant effect on the barrier function of endothelial cells. The presence of normolipidemic HDL in culture medium prevented disruption of the endothelial barrier induced by L-IDL but not by L-HDL. The decrease in endothelial barrier function induced by lipolyzed samples of HTG serum or lipoproteins appeared to be correlated with the level of free fatty acids contained in lipolytic remnants. Enrichment of LDL, and in particular HDL, with fatty acid significantly increased albumin transfer. Compared with lipolyzed samples, sera/lipoproteins oxidized in vitro by Cu2+ ions had little effect on endothelial barrier function, which did not correlate with their respective thiobarbituric acid-reacting substance (TBARS) values. TBARS remained within normal range after L-HDL incubation with endothelial cells for up to 48 h. At most concentrations tested, exposure to lipolyzed but not oxidized lipoproteins resulted in morphological perturbations of cell monolayers. These data suggest that lipolytic remnants of triglyceride-rich lipoproteins may play an important role in the development of atherosclerosis by decreasing the barrier function of the vascular endothelium. The remnant-induced injury of the arterial wall may permit the entry of cholesterol-rich lipolytic remnants as well as LDL into the arterial wall.

High-density lipoprotein recombinants: evidence for a bicycle tire micelle structure obtained by neutron scattering and electron microscopy.

Apo A-I, the major protein of HDL, interacts spontaneously with hydrated dimyristoyl phosphatidylcholine (DMPC) to form complexes smaller than 200 A in greatest dimension [ 1,2]. These A-I : DMPC recombinants have been suggested on the basis of negative stain electron microscopy [3] and low angle X-ray scattering [4] to be discoid in shape. Nascent HDL synthesized in the liver [5] and gut [6], and also the mature HDL of patients with the heritable disorder lecithin : cholesterol acyl transferase deficiency [7 1, all are discs of similar size and shape. These observations suggest that such a particle may be an intermediate in the formation of mature, spherical HDL. Based on thermodynamic considerations and differential scanning calorimetry, Segrest [8] and Tall et al. [9], respectively, have suggested that the A-I : DMPC recombinants represent unilamellar bilayer discs whose otherwise thermodynamically-unstable edges are lined by amphipathic helical domains [ 18 ] of apo A-I. In this study we analyze the size, shape and structure of the recombinants by a combination of Guinier region neutron scattering, electron microscopy and column chromatography. In the last five years, low-angle neutron scattering , has been used to study a number of macromolecules in solution. In almost all cases, including the studies of low density lipoprotein [lo], chromatin [ 111, ferritin [12] and ribosomes [13], contrast variation was achieved by measurement of scattering in solutions containing variable ratios of Hz0 to DzO. These measurements have two major drawbacks. First, the degree of hydrogen exchange differs with the ratio; second, incoherent scattering by hydrogen atoms contributes high background, thus decreasing the effective sensitivity of measurement. To circumvent these problems, we changed contrast by using deuterated (d-DMPC) and hydrogenated (l-r-DMPC) lipids to form recombinants, and did all of the scattering experiments in DzO.

The receptor binding domain of apolipoprotein E, linked to a model class A amphipathic helix, enhances internalization and degradation of LDL by fibroblasts.

Human apolipoprotein E (apo E) consists of two distinct domains, the lipid-associating domain (residues 192-299) and the globular domain (residues 1-191) which contains the LDL receptor (LDLR) binding site (residues 129-169). To test the hypothesis that an arginine-rich apo E receptor binding domain (residues 141-150) is sufficient to enhance low-density lipoprotein (LDL) uptake and clearance when covalently linked to a class A amphipathic helix, a peptide in which the receptor binding domain of human apo E, LRKLRKRLLR (hApoE[141-150]), is linked to 18A, a well-characterized high-affinity lipid-associating peptide (DWLKAFYDKVAEKLKEAF), we synthesized the peptide hApoE[141-150]-18A (hE18A) and its end-protected analogue, Ac-hE18A-NH(2). The importance of positively charged residues and the role of the hydrophobic residues in the receptor binding domain were also studied using four analogues. Ac-LRRLRRRLLR-18A-NH(2) [Ac-hE(R)18A-NH(2)] and Ac-LRKMRKRLMR-18A-NH(2) (Ac-mE18A-NH(2)) contained an extended hydrophobic face, including the receptor binding region. Control peptides, Ac-LRLLRKLKRR-18A-NH(2) [Ac-hE(Sc)18A-NH(2)], had the amino acid residues of the apo E receptor binding domain scrambled to disrupt the extended hydrophobic face, and Ac-RRRRRRRRRR-18A-NH(2) (Ac-R(10)18A-NH(2)) had only positively charged Arg residues as the receptor binding domain. The effect of the dual-domain peptides on the uptake and degradation of human LDL by fibroblasts was determined in murine embryonic fibroblasts (MEF1). LDL internalization was enhanced 3-, 5-, and 7-fold by Ac-mE18A-NH(2), Ac-hE18A-NH(2), and Ac-hE(R)18A-NH(2), respectively, whereas the control peptides had no significant biological activity. All three active peptides increased the level of degradation of LDL by 100%. The LDL binding and internalization to MEF1 cells in the presence of these peptides was not saturable over the LDL concentration range that was studied (1-10 microgram/mL). Furthermore, a similar enhancement of LDL internalization was observed independent of the presence of the LDL receptor-related protein (LRP), LDLR, or both. Pretreatment of cells with heparinase and heparitinase abolished more than 80% of the enhanced peptide-mediated LDL uptake and degradation by cells. We conclude that the dual-domain peptides enhanced LDL uptake and degradation by fibroblasts via a heparan sulfate proteoglycan (HSPG)-mediated pathway.

Effect of the fat composition of a single meal on the composition and cytotoxic potencies of lipolytically-releasable free fatty acids in postprandial plasma

Ingestion of a meal increases plasma levels of triglyceride (TG)-rich lipoproteins through the secretion of intestine-derived chylomcirons and liver-derived very low density lipoproteins (VLDL). We have determined the effects of the fat composition of a single meal on the composition of TG in TG-rich lipoproteins (VLDL + chylomicrons) and circulating and lipolytically-releasable free fatty acids (FFA) in postprandial (PP) plasma and on the cytotoxic potencies of the lipolytically-released FFA to cultured arterial wall cells. PP lipemia was induced by feeding fasted normolipidemic human subjects with a meal rich in saturated fat (SF) and another meal rich in polyunsaturated fat (PUF), or vice versa; each meal provided 65% of energy as fat, and polyunsaturated to saturated fatty acid ratios (P/S) of the SF and PUF in the meals were 0.40 and 2.49, respectively. The mean P/S of TG in TG-rich lipoproteins (1.43) and circulating FFA (1.46) in 4 h PP plasma of PUF were significantly higher than those in PP plasma of SF (0.44 and 0.59, respectively) in fasting plasma (0.52 and 0.53, respectively). In vitro lipolysis of fasting and PP serum by purified bovine milk lipoprotein lipase (LpL) resulted in a marked (8.8-12.3-fold) increase in the serum FFA level. The P/S of serum FFA in postlipolysis fasting and PP serum were consistently higher than that of FFA or that of TG associated with TG-rich lipoproteins in prelipolysis fasting and PP serum, indicating that polyunsaturated TG in VLDL and/or chylomicrons is more susceptible than saturated TG to lipolysis. When postlipolysis serum was interacted with cultured endothelial cells and mouse peritoneal macrophages (MPM), the lipolytically-released FFA in PP serum of SF and PUF disrupted the barrier function of endothelial cells and were cytotoxic to cultured MPM; FFA in postlipolysis fasting serum was not cytotoxic. FFA in postlipolysis PP serum of PUF were consistently more potent than that in postlipolysis PP serum of SF. Further study showed that all long-chain monounsaturated FFA and polyunsaturated FFA, but not saturated FFA, incorporated into lipoproteins (LDL) were cytotoxic to cultured MPM. In conclusion, despite the generally well-accepted belief that SF is more atherogenic than PUF, the present study provides in vitro evidence that the lipolytic remnant products of TG-rich lipoproteins produced after a meal rich in PUF are more injurious to arterial wall cells than those produced after a meal rich in SF.

Characterization of liver disease and lipid metabolism in the Niemann-Pick C1 mouse.

Niemann‐Pick type C1 (NPC1) disease is an autosomal‐recessive cholesterol‐storage disorder characterized by liver dysfunction, hepatosplenomegaly, and progressive neurodegeneration. The NPC1 gene is expressed in every tissue of the body, with liver expressing the highest amounts of NPC1 mRNA and protein. A number of studies have now indicated that the NPC1 protein regulates the transport of cholesterol from late endosomes/lysosomes to other cellular compartments involved in maintaining intracellular cholesterol homeostasis. The present study characterizes liver disease and lipid metabolism in NPC1 mice at 35 days of age before the development of weight loss and neurological symptoms. At this age, homozygous affected (NPC1−/−) mice were characterized with mild hepatomegaly, an elevation of liver enzymes, and an accumulation of liver cholesterol approximately four times that measured in normal (NPC1+/+) mice. In contrast, heterozygous (NPC1+/−) mice were without hepatomegaly and an elevation of liver enzymes, but the livers had a significant accumulation of triacylglycerol. With respect to apolipoprotein and lipoprotein metabolism, the results indicated only minor alterations in NPC1−/− mouse serum. Finally, compared to NPC1+/+ mouse livers, the amount and processing of SREBP‐1 and ‐2 proteins were significantly increased in NPC1−/− mouse livers, suggesting a relative deficiency of cholesterol at the metabolically active pool of cholesterol located at the endoplasmic reticulum. The results from this study further support the hypothesis that an accumulation of lipoprotein‐derived cholesterol within late endosomes/lysosomes, in addition to altered intracellular cholesterol homeostasis, has a key role in the biochemical and cellular pathophysiology associated with NPC1 liver disease. J. Cell. Biochem. 101: 498–516, 2007.

In vitro production of β-very low density lipoproteins and small, dense low density lipoproteins in mildly hypertriglyceridemic plasma : role of activities of lecithin : cholester acyltransferase, cholesterylester transfer proteins and lipoprotein lipase

As a model for the formation of beta-very low density lipoproteins (VLDL) and small, dense LDL by the intraplasma metabolic activities in vivo, lipoproteins in fresh plasma were interacted in vitro with endogenous lecithin:cholesterol acyltransferase (LCAT) and cholesterylester transfer proteins (CETP) and subsequently with purified lipoprotein lipase (LpL). The LCAT and CETP reactions in a mildly hypertriglyceridemic (HTG) plasma at 37 degrees C for 18 h resulted in (1) esterification of about 45% plasma unesterified cholesterol (UC), (2) a marked increase in cholesterylester (CE) (+129%) and a decrease in triglyceride (TG) (-45%) in VLDL, and (3) a marked increase of TG (+ 341%) with a small net decrease of CE (-3.6%) in LDL, causing a significant alteration in the TG/CE of VLDL (from 8.0 to 1.9) and of LDL (from 0.20 to 0.93). The LDL in LCAT and CETP-reacted plasma is larger and more buoyant than that in control plasma. In vitro lipolysis of control and LCAT and CETP-reacted plasma by LpL, which hydrolyzed >90% of VLDL-TG and about 50-60% of LDL-TG, converted most of VLDL in control plasma (>85%) but less than half (40%) of VLDL in LCAT and CETP-reacted plasma into the IDL-LDL density fraction and transformed the large, buoyant LDL in the LCAT and CETP-reacted plasma into particles smaller and denser than those in the control plasma. The remnants that accumulated in the VLDL density region of the postlipolysis LCAT and CETP-reacted plasma contained apo B-100 and E but little or no detectable apo Cs and consisted of particles having pre-beta and beta-electrophoretic mobilities. The inhibition of LCAT during incubation of plasma, which lessened the extent of alteration in VLDL and LDL core lipids, increased the extent of lipolytic removal of VLDL from the VLDL density region but lowered the extent of alteration in the size and density of LDL. The LCAT, CETP and/or LpL-mediated alterations in the density of LDL in normolipidemic fasting plasma were less pronounced than that in mildly HTG plasma, but they became highly pronounced upon increase of its TG-rich lipoprotein level by the addition of preisolated VLDL or by the induction of postprandial lipemia. Although the effect of LCAT, CETP and LpL reactions in non-circulating plasma in vitro may be different from that in vivo, the above data suggests that the plasma TG-rich lipoprotein level and the extent of intraplasma LCAT, CETP, LpL and likely hepatic lipase (HL) reactions in vivo may play a role in determining the LDL phenotype.

Electron spin resonance studies of fatty acid-induced alterations in membrane fluidity in cultured endothelial cells

Endothelial cell dysfunction has been implicated in the development of atherosclerosis. Of vital importance to the maintenance of endothelial cell integrity is the preservation of membrane functional and structural properties, such as membrane fluidity. The aim of this study was to develop a model for studying the relationship between endothelial cell integrity and membrane fluidity alterations in a well-defined cell culture setting. Alterations in membrane fluidity were assessed using electron spin resonance after labeling endothelial cells with the lipid-specific spin labels, CAT-16 and 12-nitroxide stearic acid. Endothelial cells were exposed to various 18-carbon fatty acids, i.e. stearic (18:0), oleic (18:1), linoleic (18:2), or linolenic (18:3), in addition to lipolyzed HDL (L-HDL) and benzyl alcohol. Membrane phospholipid fatty acid composition of endothelial cells supplemented with these fatty acids was analyzed using gas chromatography. All fatty acids, except 18:0, decreased membrane fluidity. A relationship between membrane fluidity and fatty acid compositional alterations in cellular phospholipids was observed. In particular, the arachidonic acid content decreased following exposure to 18:1, 18:2, or 18:3. Exposure of endothelial cells to L-HDL, lipoprotein particles which contain high levels of 18:1 and 18:2, also decreased membrane fluidity. The stabilization of cytoskeletal actin filaments by phalloidin partially prevented 18:2-induced increases in albumin transfer, thus implicating a cytoskeletal involvement in the 18:2-induced membrane fluidity changes involved in endothelial cell dysfunction. The present study shows that the exposure of endothelial cells to various lipids causes membrane fluidity alterations which may contribute to endothelial cell dysfunction and atherosclerosis.