Very Low Density Lipoprotein Receptor (VLDLR) Expression Is a Determinant Factor in Adipose Tissue Inflammation and Adipocyte-Macrophage Interaction

Obesity is associated with adipose tissue remodeling, characterized by adipocyte hypertrophy and macrophage infiltration. Previously, we have shown that very low density lipoprotein receptor (VLDLR) is virtually absent in preadipocytes but is strongly induced during adipogenesis and actively participates in adipocyte hypertrophy. In this study, we investigated the role of VLDLR in adipose tissue inflammation and adipocyte-macrophage interactions in wild type and VLDLR-deficient mice fed a high fat diet. The results show that VLDLR deficiency reduced high fat diet-induced inflammation and endoplasmic reticulum (ER) stress in adipose tissue in conjunction with reduced macrophage infiltration, especially those expressing pro-inflammatory markers. In adipocyte culture, VLDLR deficiency prevented adipocyte hypertrophy and strongly reduced VLDL-induced ER stress and inflammation. Likewise, cultures of primary peritoneal macrophages show that VLDLR deficiency reduced lipid accumulation and inflammation but did not alter chemotactic response of macrophages to adipocyte signals. Moreover, VLDLR deficiency tempered the synergistic inflammatory interactions between adipocytes and macrophages in a co-culture system. Collectively, these results show that VLDLR contributes to adipose tissue inflammation and mediates VLDL-induced lipid accumulation and induction of inflammation and ER stress in adipocytes and macrophages.     

The conversion of apoB100 low density lipoprotein/high density lipoprotein particles to apoB100 very low density lipoproteins in response to oleic acid occurs in the endoplasmic reticulum and not in the Golgi in McA RH7777 cells

The site where bulk lipid is added to apoB100 low density lipoproteins (LDL)/high density lipoproteins (HDL) particles to form triglyceride-enriched very low density lipoproteins (VLDL) has not been identified definitively. We employed several strategies to address this question. First, McA RH7777 cells were pulse-labeled for 20 min with [35S]methionine/cysteine and chased for 1 h (Chase I) to allow study of newly synthesized apoB100 LDL/HDL remaining in the endoplasmic reticulum (ER). After Chase I, cells were incubated for another hour (C2) with/without brefeldin A (BFA) and nocodazole (Noc) (to block ER to Golgi trafficking) and with/without oleic acid (OA). OA treatment alone during C2 increased VLDL secretion. This was prevented by the addition of BFA/Noc in C2. When C2 media were replaced by control media for another 1-h chase (C3), VLDL formed during OA treatment in C2 were secreted into C3 medium. Thus, OA-induced conversion of apoB100 LDL/HDL to VLDL during C2 occurred in the ER. Next, newly synthesized apoB100 lipoproteins were trapped in the Golgi by treatment with Noc and monensin during Chase I (C1), and C2 was carried out in the presence of BFA/Noc with/without OA and without monensin. Under these conditions, OA treatment during C2 did not stimulate VLDL secretion. The same pulse/chase protocols were followed by iodixanol subcellular fractionation, extraction of lipoproteins from ER and Golgi, and sucrose gradient separation of extracted lipoproteins. Cells treated with BFA/Noc and OA in C2 had VLDL in the ER. In the absence of OA, only LDL/HDL were present in the ER. The density of Golgi lipoproteins in these cells was not affected by OA. Similar results were obtained when ER were immuno-isolated with anti-calnexin antibodies. In conclusion, apoB100 bulk lipidation, resulting in conversion of LDL/HDL to VLDL, can occur in the ER, but not in the Golgi, in McA RH7777 cells.     

AMPK activation prevents excess nutrient-induced hepatic lipid accumulation by inhibiting mTORC1 signaling and endoplasmic reticulum stress response

Lipid accumulation is a central event in the development of chronic metabolic diseases, including obesity and type 2 diabetes, but the mechanisms responsible for lipid accumulation are incompletely understood. This study was designed to investigate the mechanisms for excess nutrient-induced lipid accumulation and whether activation of AMP-activated protein kinase (AMPK) prevents the hepatic lipid accumulation in excess nutrient-treated HepG2 cells and high fat diet (HFD)-fed mice. Exposure of HepG2 cells to high levels of glucose or palmitate induced the endoplasmic reticulum (ER) stress response, activated sterol regulatory element-binding protein-1 (SREBP-1), and enhanced lipid accumulation, all of which were sensitive to ER stress inhibitor and gene silencing of eukaryotic initiation factor 2α. The increases in ER stress response and lipid accumulation were associated with activation of mammalian target of rapamycin complex 1 (mTORC1) signaling. Inhibition of mTORC1 signaling attenuated the ER stress response and lipid accumulation induced by high glucose or by deletion of tuberous sclerosis 2. In addition, AMPK activation prevented the mTORC1 activation, ER stress response, and lipid accumulation. This effect was mimicked or abrogated, respectively, by overexpression of constitutively active and dominant-negative AMPK mutants. Finally, treatment of HFD-fed mice with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside inhibited the mTORC1 pathway, suppressed the ER stress response, and prevented insulin resistance and hepatic lipid accumulation. We conclude that activation of AMPK prevents excess nutrient-induced hepatic lipid accumulation by inhibiting mTORC1 and ER stress response.     

Thermal transitions in human very-low-density lipoprotein: fusion, rupture, and dissociation of HDL-like particles

Very-low-density lipoproteins (VLDL) are metabolic precursors of low-density lipoproteins (LDL) and a risk factor for atherosclerosis. Human VLDL are heterogeneous complexes containing a triacylglycerol-rich apolar lipid core and polar surface composed of phospholipids, a nonexchangeable apolipoprotein B, and exchangeable apolipoproteins E and Cs. We report the first stability study of VLDL. Circular dichroism and turbidity data reveal an irreversible heat-induced VLDL transition that involves formation of larger particles and repacking of apolar lipids but no global protein unfolding. Heating rate effect on the melting temperature indicates a kinetically controlled reaction with high activation energy, Ea. Arrhenius analysis of the turbidity data reveals two kinetic phases with Ea = 53 +/- 7 kcal/mol that correspond to distinct morphological transitions observed by electron microscopy. One transition involves VLDL fusion, partial rupture, and dissociation of small spherical particles (d = 7-15 nm), and another involves complete lipoprotein disintegration and lipid coalescence into droplets accompanied by dissociation of apolipoprotein B. The small particles, which are unique to VLDL denaturation, are comparable in size and density to high-density lipoproteins (HDL); they have an apolar lipid core and polar surface composed of exchangeable apolipoproteins (E and possibly Cs) and phospholipids. We conclude that, similar to HDL and LDL, VLDL are stabilized by kinetic barriers that prevent particle fusion and rupture and decelerate spontaneous interconversion among lipoprotein classes and subclasses. In addition to fusion, VLDL disruption involves transient formation of HDL-like particles that may mimic protein exchange among VLDL and HDL pools in plasma.     

Regulation of plasma lecithin:cholesterol acyltransferase in man. III. Role of high density lipoprotein cholesteryl esters in the activating effect of a high-fat test meal.

Plasma lecithin:cholesterol acyltransferase (LCAT) activity is increased during the clearance phase of alimentary lipemia induced by a high-fat test meal in normal subjects. Ultracentrifugal fractionation of high density lipoproteins (HDL) into HDL(2), HDL(3), and very high density (VHD) subfractions followed by analyses of lipid and protein components has been accomplished at intervals during alimentary lipemia to seek associations with enzyme changes. HDL(2) lipids and protein increased substantially, characterized primarily by enrichment with lecithin. HDL(3), which contain the main LCAT substrates, revealed increased triglycerides and generally reduced cholesteryl esters which were reciprocally correlated, demonstrating a phenomenon previously observed in vitro by others. Both changes correlated with LCAT activation, but partial correlation analysis indicated that ester content is primarily related to triglycerides rather than LCAT activity. The VHD cholesteryl esters and lysolecithin were also reduced. Plasma incubation experiments with inactivated LCAT showed that alimentary lipemic very low density lipoproteins (VLDL) could reduce levels of cholesteryl esters in HDL by a nonenzymatic mechanism. In vitro substitution of lipemic VLDL for postabsorptive VLDL resulted in enhanced reduction of cholesteryl esters in HDL(3) and VDH, but not in HDL(2), during incubation. Nevertheless, augmentation of LCAT activity did not result, indicating that cholesteryl ester removal from substrate lipoproteins is an unlikely explanation for activation. Since VHD and HDL(3), which contain the most active LCAT substrates, were also most clearly involved in transfers of esters to VLDL and low density lipoproteins, the suggestion that LCAT product lipoproteins are preferentially involved in nonenzymatic transfer and exchange is made. The main determinant of ester transfer, however, appears to be the level of VLDL, both in vitro and in vivo. Rose, H. G., and J. Juliano. Regulation of plasma lecithin: cholesteryl acyltransferase in man. III. Role of high density lipoprotein cholesteryl esters in the activating effect of a high-fat test meal.     

Low density lipoproteins isolated from the blood of patients with ischemic heart disease induce accumulation of lipids in human aortic intima cells

Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were isolated from the blood of healthy subjects and CHD patients. LDL from the blood of healthy individuals did not raise the intracellular lipid values within 24 h of cultivation. During intracellular lipid values within 24 h of cultivation. During the same incubation period. LDL obtained from the blood of CHD patients caused a 2- to 5-fold rise in cholesterol esters as well as a 1.5- to 3-fold rise in free cholesterol and triglycerides, while the intracellular phospholipid levels remained unchanged. In one of the three cases, the ability to raise the intracellular level of cholesterol esters was demonstrated by VLDL (500 micrograms/ml) derived from CHD patients blood. HDL did not affect the lipid levels in smooth muscle cells cultured from unaffected intima. The obtained data suggests that circulating LDL and, possibly, VLDL in the blood of CHD patients are capable of inducing the accumulation of fat in vascular wall cells.     

The aggregation of isolated human platelets in the presence of lipoproteins and prostacyclin.

Addition of prostacyclin (PGI2) temporarily inhibits platelet aggregation and permits the isolation of platelets free from plasma proteins, which have the same sensitivity as those in plasma [Moncada, Radomski & Vargas (1982) Br. J. Pharmacol. 75, 165P]. By using a modification of this technique we have established that platelets isolated from normal subjects aggregate more readily in response to ADP and adrenaline when physiological concentrations of low-density lipoproteins (LDL) are present. At high LDL concentrations spontaneous aggregation occurs. High-density lipoproteins (HDL) and very-low-density lipoproteins (VLDL) had no effect on agonist-induced platelet aggregation at normal concentrations, but HDL sensitized at higher concentrations. These effects by lipoproteins are not accompanied by changes in platelet lipid content. Cyclohexanedione treatment of LDL to modify apolipoproteins appeared to abolish the sensitization effect, indicating that binding to receptors was essential for the effects of LDL. LDL, but not HDL, overcame the inhibitory effect of PGI2 on platelet aggregation, except at very high concentrations of PGI2. PGI2 raised the cyclic AMP content of isolated platelets, but LDL only partially prevented this rise. These results suggest that LDL may have a greater role in platelet aggregation than previously recognized and may also regulate effects of PGI2. These findings may be of relevance to an understanding of cardiovascular diseases.     

Free cholesterol is a potent regulator of lipid transfer protein function

This study investigates the effect of altered lipoprotein free cholesterol (FC) content on the transfer of cholesteryl ester (CE) and triglyceride (TG) from very low- (VLDL), low- (LDL), and high-(HDL) density lipoproteins by the plasma-derived lipid transfer protein (LTP). The FC content of VLDL and HDL was selectively altered by incubating these lipoproteins with FC/phospholipid dispersions of varying composition. FC-modified lipoproteins were then equilibrated with [3H] TG, [14C]CE-labeled lipoproteins of another class to facilitate the subsequent modification of the radiolabeled donor lipoproteins. LTP was added and the extent of radiolabeled TG and CE transfer determined after 1 h. With either LDL or VLDL as lipid donor, an increase in the FC content of these lipoproteins caused a concentration-dependent inhibition (up to 50%) of CE transfer from these particles, without any significant effect on TG transfer. In contrast, with HDL as donor, increasing the HDL FC content had little effect on CE transfer from HDL, but markedly stimulated (up to 2.5-fold) the transfer of TG. This differential effect of FC on the unidirectional transfer of radiolabeled lipids from VLDL and HDL led to marked effects on LTP-facilitated net mass transfer of lipids. During long-term incubation of a constant amount of LTP with FC-modified VLDL and HDL, the extent of net mass transfer was linearly related to lipoprotein FC content; a 4-fold increase in FC content resulted in a 3-fold stimulation of the CE mass transferred to VLDL, which was coupled to an equimolar, reciprocal transfer of TG mass to HDL. Since lipid transfer between lipoproteins is integral to the process of reverse cholesterol transport, we conclude that lipoprotein FC levels are a potent, positive regulator of the pathways involved in sterol clearance. FC may modulate lipid transfer by altering the availability of CE and TG to LTP at the lipoprotein surface.     

Mechanisms of enhancement of cholesteryl ester transfer protein activity by lipolysis

Lipoprotein lipase enhances the cholesteryl ester transfer protein (CETP)-mediated transfer of cholesteryl esters from plasma high density lipoproteins (HDL) to very low density lipoproteins (VLDL). In time course studies the stimulation of cholesteryl ester transfer by bovine milk lipase was correlated with accumulation of fatty acids in VLDL remnants. As the amount of fatty acid-poor albumin in the incubations was increased, there was decreased accumulation of fatty acids in VLDL remnants and a parallel decrease in the stimulation of cholesteryl ester transfer by lipolysis. Addition of sodium oleate to VLDL and albumin resulted in stimulation of the CETP-mediated transfer of cholesteryl esters from HDL to VLDL. The stimulation of transfer of cholesteryl esters into previously lipolyzed VLDL was abolished by lowering the pH from 7.5 to 6.0, consistent with a role of lipoprotein ionized fatty acids. CETP-mediated cholesteryl ester transfer from HDL to VLDL was also augmented by phosholipase A2 and by a bacterial lipase which lacked phospholipase activity. When VLDL and HDL were re-isolated after a lipolysis experiment, both lipoproteins stimulated CETP activity. Postlipolysis VLDL and HDL bound much more CETP than native VLDL or HDL. Lipolysis of apoprotein-free phospholipid/triglyceride emulsions also resulted in enhanced binding of CETP to the emulsion particles. Incubation conditions which abolished the enhanced cholesteryl ester transfer into VLDL remnants reduced binding of CETP to remnants, emulsions, and HDL. In conclusion, the enhanced CETP-mediated transfer of cholesteryl esters from HDL to VLDL during lipolysis is related to the accumulation of products of lipolysis, especially fatty acids, in the lipoproteins. Lipids accumulating in VLDL remnants and HDL as a result of lipolysis may augment binding of CETP to these lipoproteins, leading to more efficient transfer of cholesteryl esters from HDL to VLDL.     

Hypoxia-inducible factor-1 (HIF-1) promotes LDL and VLDL uptake through inducing VLDLR under hypoxia

Metabolism under hypoxia is significantly different from that under normoxia. It has been well elucidated that HIF-1 (hypoxia-inducible factor-1) plays a central role in regulating glucose metabolism under hypoxia; however, the role of HIF-1 in lipid metabolism has not yet been well addressed. In the present study we demonstrate that HIF-1 promotes LDL (low-density lipoprotein) and VLDL (very-LDL) uptake through regulation of VLDLR (VLDL receptor) gene expression under hypoxia. Increased VLDLR mRNA and protein levels were observed under hypoxic or DFO (deferoxamine mesylate salt) treatment in MCF7, HepG2 and HeLa cells. Using dual-luciferase reporter and ChIP (chromatin immunoprecipitation) assays we confirmed a functional HRE (hypoxia-response element) which is localized at +405 in exon 1 of the VLDLR gene. Knockdown of HIF1A (the α subunit of HIF-1) and VLDLR, but not HIF2A (the α subunit of HIF-2), attenuated hypoxia-induced lipid accumulation through affecting LDL and VLDL uptake. Additionally we also observed a correlation between HIF-1 activity and VLDLR expression in hepatocellular carcinoma specimens. The results of the present study suggest that HIF-1-mediated VLDLR induction influences intracellular lipid accumulation through regulating LDL and VLDL uptake under hypoxia.     

Dose and duration effects of estradiol valerate on serum and lipoprotein lipids

Non-alkylated estrogens, like estradiol valerate (F2V), are widely used in the treatment of the postmenopausal hormonal deficiency syndrome. Their effects on serum and lipoprotein lipids are characterized by an increase in the lipid constituents of high density lipoproteins (HDL) and, usually, a decrease in low density lipoproteins (LDL). These effects are considered beneficial as regards atherogenesis and the risk for cardiovascular diseases. Unlike the effects of alkylated estrogens, no concomitant increase in triglycerides (TG) in serum and very low density lipoproteins (VLDL) - adverse effects - are seen in doses of up to 2 mg E2V. In order to compare the effects of 2 and 4 mg of E2V on serum and lipoprotein lipids, 19 bilaterally oophorectomized women participated in a cross-over study after a 4 week long wash-out period. To evaluate the influence of the time factor, 10 of the women continued taking 2 mg and 9 taking 4 mg of E2V respectively for an additional period of 12 weeks, resulting in a total treatment period of 24 weeks per group. The serum lipoproteins were separated by preparative ultracentrifugation, the serum and lipoprotein lipids being assessed using commercially available kits. In the cross-over part of the study, total (TC) and free cholesterol (FC) and phospholipids (PL) increased in HDL and decreased in LDL. Neither dose increased TG in serum or VLDL. These changes in the lipoprotein pattern persisted at the end of the entire study. Consequently, within the range of commonly used doses (2 and 4 mg) E2V seems to have a constant and, in terms of cardiovascular disease, favourable influence on lipoprotein metabolism irrespective of doses and periods studied.     

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca(2+)-independent phospholipase A(2) (iPLA(2)β), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.     

Hepatic ABCA1 and VLDL triglyceride production

Elevated plasma triglyceride (TG) and reduced high density lipoprotein (HDL) concentrations are prominent features of metabolic syndrome (MS) and type 2 diabetes (T2D). Individuals with Tangier disease also have elevated plasma TG concentrations and a near absence of HDL, resulting from mutations in ATP binding cassette transporter A1 (ABCA1), which facilitates the efflux of cellular phospholipid and free cholesterol to assemble with apolipoprotein A-I (apoA-I), forming nascent HDL particles. In this review, we summarize studies focused on the regulation of hepatic very low density lipoprotein (VLDL) TG production, with particular attention on recent evidence connecting hepatic ABCA1 expression to VLDL, LDL, and HDL metabolism. Silencing ABCA1 in McArdle rat hepatoma cells results in diminished assembly of large (>10nm) nascent HDL particles, diminished PI3 kinase activation, and increased secretion of large, TG-enriched VLDL1 particles. Hepatocyte-specific ABCA1 knockout (HSKO) mice have a similar plasma lipid phenotype as Tangier disease subjects, with a two-fold elevation of plasma VLDL TG, 50% lower LDL, and 80% reduction in HDL concentrations. This lipid phenotype arises from increased hepatic secretion of VLDL1 particles, increased hepatic uptake of plasma LDL by the LDL receptor, elimination of nascent HDL particle assembly by the liver, and hypercatabolism of apoA-I by the kidney. These studies highlight a novel role for hepatic ABCA1 in the metabolism of all three major classes of plasma lipoproteins and provide a metabolic link between elevated TG and reduced HDL levels that are a common feature of Tangier disease, MS, and T2D. This article is part of a Special Issue entitled: Triglyceride Metabolism and Disease.     

Proteinuria increases oxylipid concentrations in VLDL and HDL but not LDL particles in the rat

We previously established that proteinuria alters the apolipoprotein content of lipoproteins. This study was conducted to establish whether proteinuria also alters the concentrations of oxidized lipids within lipoprotein density fractions. To this end, we induced passive Heymann nephritis in Sprague Dawley rats and measured an array of alkaline-stable oxylipids in VLDL, LDL, and HDL particles. Proteinuria increased the total oxylipid amounts in the HDL and VLDL fractions. More importantly, these levels were increased when expressed per unit lipoprotein protein, indicating that the oxidized lipid load per particle was increased. Epoxides and diols increased approximately 2-fold in HDL and approximately 5-fold in VLDL, whereas LDL showed approximately 2-fold decreases. The hydroxyeicosatetraenoic acids and hydroxyoctadecadienoic acids (HODEs) increased >4-fold in HDL and >20-fold in VLDL, whereas LDL showed approximately 2-fold decreases in the HODEs. Therefore, nephrotic syndrome alters the lipoprotein oxylipid composition independently of an increase in total lipoprotein levels. These proteinuria-induced changes may be associated with the cardiovascular risk of lipoprotein oxidation.     

Determinants of F2-isoprostane biosynthesis and inhibition in man

Several cardiovascular risk factors are characterized by the coexistence of low-grade inflammation, enhanced oxidative stress and lipid peroxidation. It has been hypothesized that F2-isoprostanes, a product of in vivo lipid peroxidation, may transduce the effects of metabolic and hemodynamic abnormalities into increased cardiovascular risk. Thus, the formation of these compounds, including urinary 8-iso-Prostaglandin (PG) F2alpha, has been investigated in clinical settings putatively associated with oxidant stress. Enhanced lipid peroxidation together with increased in vivo platelet activation have been found in association with the major cardiovascular risk factors. Thus, F2-isoprostanes may transduce the effects of oxidant stress associated with complex metabolic disorders into specialized forms of cellular activation. In particular, the low-grade inflammatory state characterizing metabolic disorders such as obesity, hypercholesterolemia, type 2 diabetes mellitus, and homozygous homocystinuria may be the primary trigger of thromboxane-dependent platelet activation mediated, at least in part, through enhanced lipid peroxidation. Moreover, oxidative stress may promote endothelial dysfunction through increased production of reactive oxygen species that inactivate nitric oxide. Accumulation and activation of leukocytes plays a key role in atherosclerosis and its complications. Interestingly, neutrophil adhesion induced by minimally modified low-density lipoproteins is mainly mediated by F2-isoprostanes. Although epidemiological studies suggest an inverse relationship between antioxidant vitamin intake and cardiovascular disease, several clinical trials have obtained conflicting results on the effects of vitamin E supplementation on the risk of cardiovascular events. On the other hand, the use of F2-isoprostane formation as a biochemical end-point for dose-finding studies of vitamin E supplementation has helped clarifying the unique features of its pharmacodynamic effects on lipid peroxidation. This information could be extremely valuable in the selection of the appropriate patient subgroups that may benefit from antioxidant interventions.     

Lipid class and molecular species interrelationships among plasma lipoproteins of type III and type IV hyperlipemic subjects

As a further appraisal of lipoprotein interconversion and equilibration of lipid components a detailed examination was made of the chemical class and molecular species interrelationships among the major fasting plasma lipoprotein fractions within each of six male Type III and Type IV hyperlipemic subjects subsisting on free choice diets. The lipoprotein fractions were prepared by conventional ultracentrifugation and the lipid class and molecular species composition of the corresponding lipoprotein fractions were determined by gas chromatography of the intact glycerol esters and ceramides. In general, each lipoprotein fraction possessed a well defined lipid class composition, which was characterized by a dramatically decreasing triacylglycerol and increasing phospholipid and cholesteryl ester content, when progressing from the very low (VLDL) to the low (LDL) and high (HDL) density lipoproteins, as already established for normolipemic subjects. Likewise, the LDL, and LDL₂ of the hyperlipemic subjects contained about two times higher proportion of total phospholipid as sphingomyelin than VLDL and HDL. Furthermore, the sphingomyelins of the HDL fraction contained about 30% more of the higher and 30% less of the lower molecular weight species than the sphingomyelins of the VLDL. Smaller differences were seen in the molecular species composition of the phosphatidylcholines, cholesteryl esters and triacylglycerols among the corresponding lipoproteins. In comparison to normolipemic subjects analyzed previously, the hyperlipemic subjects showed greater individual variability. Despite this variability the lipid class and molecular species composition in the hyperlipemic subjects was again incompatible with the hypothesis which postulates direct VLDL conversion into LDL and HDL under the influence of lipoprotein lipase and lecithin: cholesterol acyltransferase. The main differences between normolipemic and hyperlipemic plasma were found to reside in the number of the VLDL and LDL, lipoprotein particles and not in their chemical composition or physical structure, or in the apparent mechanism of their metabolic interconversion.     

Role of lipid transfers in the formation of a subpopulation of small high density lipoproteins

The effect of lipid transfers on the structure and composition of high density lipoproteins (HDL) has been studied in vitro in incubations that contained the lipoprotein-free fraction of human plasma as a source of lipid transfer protein. These incubations did not contain lecithin:cholesterol acyltransferase activity and were not supplemented with lipoprotein lipase. Incubations were performed at 37 degrees C for 6 hr in both the presence and absence of either added very low density lipoproteins (VLDL) or the artificial triglyceride emulsion, Intralipid. Incubation in the absence of added VLDL or Intralipid had little or no effect on the HDL. By contrast, incubation in the presence of either VLDL or Intralipid resulted in marked changes in the HDL. The effect of incubation with VLDL was qualitatively similar to that of Intralipid; both resulted in obvious transfers of lipid and changes in the density, particle size, and composition of HDL. Incubation of the plasma fraction of density 1.006-1.21 g/ml, total HDL, or HDL3 with either VLDL or Intralipid resulted in the following: 1) a depletion of the cholesteryl ester and free cholesterol content and an increase in the triglyceride content of both HDL2 and HDL3; 2) a decrease in density and an increase in particle size of the HDL3 to form a population of HDL2-like particles; and 3) the formation of a discrete population of very small lipoproteins with a density greater than that of the parent HDL3. The newly formed lipoproteins had a mean particle radius of 3.7-3.8 nm and consisted mainly of protein, predominantly apolipoprotein A-I and phospholipid.     

Concentration and composition of serum lipoproteins in the fetal rat]

1. Concentration and composition of the "very low density lipoproteins" (VLDL), "low density lipoproteins" (LDL) and "high density lipoproteins" (HDL) and of non-floatable lipids of fetal rat serum (day 22 of pregnancy) were determined by ultracentrifugation, thin-layer chromatographic separation of the floated lipids and quantitation of the lipid and protein moiety. 2. The concentration of VLDL is in the fetal rat by one order of magnitude lower, and that of LDL, 5fold higher than in the adult animal; the concentration of HDL in fetal serum amounts to 60% of the value of adult animals. 3. The composition of LDL and HDL of fetal serum does not differ from that in the serum of adult animals; in contrast, the fetal VLDL have a higher proportion of protein and cholesterol and a lower proportion of triglycerides than the VLDL of adult serum. The electrophoretic mobility of the fetal VLDL is lower than that of adult VLDL.     

Interactions of serum proteins with small unilamellar liposomes composed of dioleoylphosphatidylethanolamine and oleic acid: high-density lipoprotein, apolipoprotein A1, and amphipathic peptides stabilize liposomes

Small unilamellar liposomes composed to dioleoylphosphatidylethanolamine (DOPE) and oleic acid (OA) are stabilized by incubation with normal human serum or plasma [Liu, D., & Huang, L. (1989) Biochemistry 28, 7700-7707]. The present report describes a systematic study of interactions of purified serum proteins and lipoproteins with these liposomes. Albumin destabilized liposomes by extracting OA from the liposomes, whereas immunoglobulins and lipoproteins (HDL, LDL, and VLDL) had no effect. However, HDL and, to some extent, VLDL showed a rapid stabilization activity against the lytic effect of albumin. HDL added together with or shortly after the addition of albumin completely abolished the liposome leakage and aggregation effects induced by albumin. SDS-PAGE analysis of the HDL-stabilized liposomes revealed that apolipoprotein A1 was associated with liposomes. Purified apolipoprotein A1, but not a lipid mixture resembling the lipid composition of HDL, showed comparable liposome stabilization activity as HDL. Furthermore, synthetic peptides resembling the amphipathic helices found in apolipoprotein A1 also showed strong liposome stabilization activity. Peptides which were able to form amphipathic helixes of a wedge shape were more effective stabilizers than those which could not. These data indicate that HDL plays a major role in human serum or plasma for the liposome stabilization activity. HDL exerts its activity probably by the interactions of the amphipathic helices of apolipoprotein A1 with the hydrophobic voids found on the outer surface of the highly curved, small liposomes.     

Isolation and analysis of lipoproteins secreted by rat liver hepatocytes

A procedure has been developed for the small-scale isolation and characterization of lipoproteins secreted by cultured rat liver hepatocytes. The lipoproteins in the culture medium were separated into VLDL, LDL, HDL and a fraction with d greater than 1.21 on single-spin density-gradients. The lipoproteins were removed from the gradients by adsorption onto Cab-O-Sil, a hydrated colloidal silica. The lipid components were extracted from the silica with CHCl3/CH3OH and the apoproteins solubilized in a buffer that contained 2% sodium dodecyl sulfate and 6 M urea. The proteins were analyzed on 3-20% acrylamide electrophoresis gels that contained 1% sodium dodecyl sulfate. The two major rat-plasma lipoproteins, VLDL and HDL, were well separated by the gradients. The Cab-O-Sil was shown to bind 90-95% of the HDL and VLDL in the fractions from the gradient. The recovery of the lipid components was essentially quantitative. The recovery of the apolipoproteins was only about 60% but with very good precision. Over a 20 h period, the lipid phosphorus associated with secreted lipoproteins increased linearly. The secretion of apolipoprotein A1 and apolipoprotein E associated with HDL and apolipoprotein B associated with VLDL also increased as a nearly linear function with time. The secretion of apolipoprotein E associated with VLDL was linear only up to approx. 6 h. The availability of this procedure should greatly facilitate further studies on the characterization of lipoproteins secreted by hepatocytes and mechanisms that regulate lipoprotein synthesis and secretion.