兔主动脉平滑肌细胞低密度脂蛋白受体相关蛋白(LRP)的诱导表达调节

在兔主动脉平滑肌细胞 ( SMC)培养基中分别加入正常低密度脂蛋白 ( N- LDL)、氧化低密度脂蛋白 ( ox- LDL)、正常极低密度脂蛋白 ( N- VLDL)、氧化极低密度脂蛋白 ( ox- VLDL)和 β-极低密度脂蛋白 (β- VLDL )培养 2 4 h后 ,用定量 RT- PCR和配体结合实验检测平滑肌细胞 LRP的m RNA和蛋白质水平的表达 .结果表明 :五种脂蛋白均能在转录和翻译水平诱导兔主动脉平滑肌细胞的 LRP表达 ,尤以富含胆固醇的 N- LDL ,ox- LDL和β- VLDL的刺激作用更明显 .用胆固醇单独或与脂蛋白共同温育 SMC后 ,发现胆固醇本身可促进 SMC的 LRP蛋白水平的表达 ,脂蛋白与胆固醇的共同刺激作用更为显著 .结果提示 :上述五种脂蛋白对 SMC上 LRP的表达有上调作用 ,其机制可能主要是通过其中的胆固醇来实现的 .     

The effects of hyperlipidemia on lipoprotein metabolism in squirrel monkeys and rabbits.

We studied the metabolism of different classes of lipoprotein in squirrel monkeys and rabbits. Lipoproteins were labeled in vivo in donor animals with (3H)leucine and (3H)cholesterol. The rate of disappearance from plasma of recipient squirrel monkeys of the protein moiety of the very low density lipoproteins was rapid, that of high density lipoproteins slow, and the rate for low density lipoproteins was intermediate. The fractional turnover of the apoprotein of low density lipoproteins was slightly reduced in hyperlipidemic monkeys, but the absolute rates of synthesis and catabolism were increased. Hyperdipidemia in rabbits resulted in a dramatic reduction in the fractional catabolic rate of low density lipoprotein apoprotein. Hyperlipidemia in the donors of biosynthetic low density lipoproteins also influenced the rates of catabolism in rabbits. We showed the cycloheximide that although there was recycling of (3H)leucine into other proteins, the reutilization of leucine from low density lipoproteins for nascent low density lipoproteins was not significant. In most tissues the ratio of cholesterol:protein radioactivity was much greater than that for plasma 24 h after administration of labeled low density lipoproteins, but the ratios for aortic intima plus inner media and for plasma low density lipoproteins were similar. The presence of atherosclerosis resulted in a large increase in the apparent uptake of low density lipoproteins by the aortas of rabbits and monkeys.     

Identification of lipoprotein X-like particles in rat plasma following Intralipid infusion.

Fasting rats were infused with 10% Intralipid for 24 h (0.33 mL/h per 100 g body weight) and the plasma lipoproteins isolated and compared with those of fed animals and animals with bile duct ligatures as controls. There was a 6- to 10-fold increase in the free cholesterol and phospholipid content of total plasma in animals infused with Intralipid or with ligated bile ducts. The changes were largely restricted to the low density lipoproteins (d=1.019--1.063 g/mL) where free cholesterol and phospholipid increased 30- to 60-fold compared with fed control animals. Hydroxylapatite chromatography of the low density lipoprotein fractions of both Intralipid-infused and bile duct ligated animals yielded a subfraction which was rich in free cholesterol (27%), phosphatidylcholine (66%), and protein (6%); the latter was composed primarily of albumin and apo C proteins. The electrophoretic mobility and polyanionic precipitation properties of the abnormal lipoprotein were indistinguishable from those of lipoprotein X isolated from the animals with bile duct ligatures. The albumin in the abnormal lipoprotein from both groups of experimental animals was detected immunochemically only after delipidation of the lipoprotein. Twice as much of the lipoprotein X accumulated in Intralipid-infused than in the bile duct ligated animals. On rechromatography of the residual low density lipoprotein other subfractions could be isolated which possessed lipid and protein proportions intermediate between those of the lipoprotein X and of normal rat plasma low density lipoprotein. The activity of lecithin cholesterol acyl transferase was increased twofold in the Intralipid-infused animals when compared with control animals, but it decreased by 50% in the animals with bile duct ligatures. It is concluded that the unusual lipoprotein X accumulates in the plasma of Intralipid-infused animals owing to incomplete clearance of the exogenous phospholipid, which mobilized tissue cholesterol and in the form of vesicular particles serves as a lipid phase for apo C proteins. A comparable mechanism is suggested for the formation of lipoprotein X in the animals with bile duct ligature.     

Scavenger receptor class B, type I-mediated [3H]cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding to the receptor

The murine scavenger receptor class B, type I (mSR-BI) is a receptor for high density lipoprotein (HDL), low density lipoprotein (LDL), and acetylated LDL (AcLDL). It mediates selective uptake of lipoprotein lipid and stimulates efflux of [(3)H]cholesterol to lipoproteins. SR-BI-mediated [(3)H]cholesterol efflux was proposed to be independent of ligand binding. In this study, using anti-mSR-BI antibody KKB-1 and two mSR-BI mutants with altered ligand binding properties, we demonstrated that SR-BI-mediated [(3)H]cholesterol efflux to lipoproteins was correlated with ligand binding and lipid uptake activities of the receptor. The KKB-1 antibody, which blocked lipoprotein binding without substantially altering the cholesterol oxidase-accessible cellular [(3)H]cholesterol, also blocked [(3)H]cholesterol efflux to HDL and LDL. One of the SR-BI mutants, which has a double substitution of arginines for glutamines at positions 402 and 418 (Q402R/Q418R), exhibited a high level of LDL binding and lipid uptake from LDL, but lost most of the corresponding HDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to LDL, but not to HDL. Another mutant, M158R, with an arginine in place of methionine at position 158, exhibited reduced HDL and LDL receptor activities, but apparently normal AcLDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to AcLDL, but not to HDL or LDL. These results suggest that SR-BI-stimulated [(3)H]cholesterol efflux to lipoproteins critically depends on ligand binding to this receptor and raise the possibility that the mechanisms of selective lipid uptake and [(3)H]cholesterol efflux may be intimately related.     

Enhanced binding of phospholipase-A2-modified low density lipoprotein by human adipocytes

Recognition of low density lipoprotein (LDL) by human adipocytes is not dependent on the classical LDL (apoprotein B-E) receptor. To assess whether LDL phospholipids have a role in adipocyte-LDL interactions, binding studies were carried out with human LDL modified with cobra venom phospholipase A2 (PLA2) and freshly isolated adipocytes and purified adipocyte plasma membranes prepared from surgical biopsies. LDL incubated with PLA2 showed increased monoacylphospholipid content, decreased diacylphospholipid content, and increased anodic migration on agarose gel electrophoresis. LDL cholesterol, triglyceride, and protein content remained unchanged. Typically, modification of 16 and 47% of LDL phospholipids enhanced specific binding of 125I-labelled LDL to plasma membranes progressively from 3.1 micrograms LDL bound/mg membrane protein (control) to 5.8 and 28.2 micrograms LDL bound/mg membrane protein, respectively. Nonspecific binding was not altered significantly. Excess unlabelled native LDL and high density lipoprotein (HDL3) effectively inhibited binding of PLA2-modified LDL. Freshly isolated adipocytes also showed enhanced binding and uptake of PLA2-modified LDL (0.1 vs. 0.9 micrograms LDL/10(6) cells x 2 h), control vs. modified). The results demonstrate that alterations of LDL phospholipids significantly enhance LDL binding and suggest a regulatory role for phospholipids in lipoprotein-cell interaction. Furthermore, the results support the view that human adipose tissue may be involved in the metabolism of modified lipoproteins, in vivo.     

Intracellular trafficking of pigeon beta-very low density lipoprotein and low density lipoprotein at low and high concentrations in pigeon macrophages

Foam cell formation occurs in vitro at lipoprotein concentrations above 50 microgram/ml in pigeon macrophages. Hypothetically, intracellular trafficking of lipoproteins at higher concentrations may differ from uptake of lipoproteins associated with low concentrations, revealing a separate atherogenic endocytic pathway. Macrophage intracellular trafficking of pigeon beta-very low density lipoprotein (beta-VLDL) and low density lipoprotein (LDL) at low concentrations (12 microgram/ml) near the saturation of high affinity binding sites and high lipoprotein concentrations (50-150 microgram/ml) used to induce foam cell formation were examined. Pigeon beta-VLDL and LDL, differentially labeled with colloidal gold, were added simultaneously to contrast trafficking of beta-VLDL, which causes in vitro foam cell formation, with LDL, which does not. The binding of lipoproteins to cell surface structures, distribution of lipoproteins in endocytic organelles, and the extent of colabeling in the endocytic organelles were determined by thin-section transmission electron microscopy.At low concentrations, the intracellular trafficking of pigeon LDL and beta-VLDL was identical. At high concentrations, LDL was removed more rapidly from the plasma membrane and reached lysosomes more quickly than beta-VLDL. No separate endocytic route was present at high concentrations of beta-VLDL; rather, an increased residence on the plasma membrane, association with nonmicrovillar portions of the plasma membrane, and slower trafficking in organelles of coated-pit endocytosis reflected a more atherogenic trafficking pattern.     

Presence and isolation of 2 lipoproteins immunologically related to apolipoprotein A I in human serum

Very high density lipoproteins d : 1.23--1.25 g/ml (VHDL2) have been isolated from human serum by preparative ultracentrifugation. They contain 80 per cent proteins and 20 per cent lipids. Lipids are mainly phospholipids (80 per cent). The proportion of lysolecithin (50 per cent) is higher than that of lecithin (40 per cent). The quantity of cholesterol is low, the free cholesterol: total cholesterol ratio is 0.35. VHDL2 consisted principally in lipoprotein D and two lipoproteins immunologically apparented to apolipoprotein A I, called LP A I1 and LP A I2. The LP A I1 has a molecular weight slightly higher and a hydrated density lower than that of LP AI2. Our experiments suggest that LP A I1 exists in the serum before ultracentrifugation while LP A I 2 comes from HDL degradation during ultracentrifugation. The immunological heterogeneity of apo A I forming different protein-lipid complexes is discussed.     

Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice

The human cholesteryl ester transfer protein (CETP) facilitates the exchange of neutral lipids among lipoproteins. In order to evaluate the effects of increased plasma CETP on lipoprotein levels, a human CETP minigene was placed under the control of the mouse metallothionein-I promoter and used to develop transgenic mice. Integration of the human CETP transgene into the mouse genome resulted in the production of active plasma CETP. Zinc induction of CETP transgene expression caused depression of serum cholesterol due to a significant reduction of high density lipoprotein cholesterol. There was no change in total cholesterol content in very low and low density lipoproteins. However, there was a decrease in the free cholesterol/cholesteryl ester ratio in plasma and in all lipoprotein fractions of transgenic mouse plasma, suggesting stimulation of plasma cholesterol esterification. The results suggest that high levels of plasma CETP activity may be a cause of reduced high density lipoproteins in humans.     

Clearance of chylomicron remnants by the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

The involvement of the low density lipoprotein receptor-related protein (LRP) in chylomicron remnant (CR) catabolism was investigated. Ligand blot analyses demonstrated that beta-very low density lipoproteins (beta-VLDL) incubated with apolipoprotein E (beta-VLDL+E) bound to the LRP and low density lipoprotein receptors, whereas active (receptor-binding) alpha 2-macroglobulin (alpha 2M) bound only to LRP partially purified from rat liver membranes. Iodinated beta-VLDL+E and active alpha 2M showed high affinity binding to the LRP/alpha 2M receptor of low density lipoprotein receptor-negative fibroblasts. The binding and degradation of radiolabeled alpha 2M by these cells were partially inhibited by beta-VLDL+E. Furthermore, alpha 2M interfered with the internalization of beta-VLDL+E and subsequent induction in the cholesterol esterification by these cells. These studies suggested that remnant lipoproteins and active alpha 2M compete for binding to the LRP/alpha 2M receptor. Next, we examined whether the LRP/alpha 2M receptor plays a role, in the presence of low density lipoprotein receptors, in the in vivo catabolism of CR in mice. In vivo studies demonstrated that the unlabeled active, but not the native, alpha 2M partially inhibited the plasma clearance and hepatic uptake of radiolabeled CR or apoE-enriched radiolabled CR. Likewise, apoE-enriched CR retarded the plasma clearance and hepatic uptake of radiolabeled active alpha 2M. These studies provide physiological evidence that the LRP/alpha 2M receptor may function as a CR receptor that removes CR from the plasma.     

In vitro incorporation of cholesterol-14C into very low density lipoprotein cholesteryl esters

The cholesteryl esters of very low density lipoproteins become labeled when human plasma is incubated with cholesterol-(14)C. The relative order of magnitude of the specific activity of the cholesteryl esters of the major lipoprotein fractions is: high density lipoproteins > very low density lipoproteins > low density lipoproteins. This pattern of labeling is similar to that found by others in experiments performed in vivo. Very low density lipoprotein cholesteryl esters are probably not formed by direct action of the plasma lecithin:cholesteryl acyltransferase, since significant esterification of cholesterol does not occur when very low density lipoproteins are incubated separately with the enzyme. Instead, labeled cholesteryl esters formed in the other lipoprotein fractions transfer to the very low density lipoproteins, the relative amount of monounsaturated esters transferred being slightly greater than that of saturated and polyunsaturated esters. The results support the possibility that the acyltransferase indirectly increases the concentration of very low density lipoprotein cholesteryl esters in vivo.     

Rat high density lipoprotein subfraction (HDL3) uptake and catabolism by isolated rat liver parenchymal cells.

Rat liver parenchymal cell binding, uptake, and proteolytic degradation of rat 125I-labeled high density lipoprotein (HDL) subfraction, HDL3 (1.10 less than d less than 1.210 g/ml), in which apo-A-I is the major polypeptide, were investigated. Structural and metabolic integrity of the isolated cells was verified by trypan blue exclusion, low lactic dehydrogenase leakage, expected morphology, and gluconeogenesis from lactate and pyruvate. 125I-labeled HDL3 was incubated with 10 X 10(6) cells at 37 degrees and 4 degrees in albumin and Krebs-Henseleit bicarbonate buffer, pH 7.4. Binding and uptake were determined by radioactivity in washed cells. Proteolytic degradation was determined by trichloroacetic acid-soluble radioactivity in the incubation medium. At 37 degrees, maximum HDL3 binding (Bmax) and uptake occurred at 30 min with a Bmax of 31 ng/mg dry weight of cells. The apparent dissociation constant of the HDL3 receptor system (Kd) was 60 X 10(-8) M, based on Mr = 28,000 of apo-A-I, the predominant rat HDL3 protein. Proteolytic degradation showed a 15-min lag and then constant proteolysis. After 2 hours 5.8% of incubated 125I-labeled HDL3 was degraded. Sixty per cent of cell radioactivity at 37 degrees was trypsin-releasable. At 37 degrees, 125I-labeled HDL3 was incubated with cells in the presence of varying concentrations of native (cold) HDL3, very low density lipoproteins, and low density lipoproteins. Incubation with native HDL3 resulted in greatest inhibition of 125I-labeled HDL3 binding, uptake, and proteolytic degradation. When 125I-labeled HDL3 was preincubated with increasing amounts of HDL3 antiserum, binding and uptake by cells were decreased to complete inhibition. Cell binding, uptake, and proteolytic degradation of 125I-labeled HDL3 were markedly diminished at 4 degrees. Less than 1 mM chloroquine enhanced 125I-labeled HDL3 proteolysis but at 5 mM or greater, chloroquine inhibited proteolysis with 125I-labeled HDL3 accumulation in cells. L-[U-14C]Lysine-labeled HDL3 was bound, taken up, and degraded by cells as effectively as 125I-labeled HDL3. These data suggest that liver cell binding, uptake, and proteolytic degradation of rat HDL3 are actively performed and linked in the sequence:binding, then uptake, and finally proteolytic degradation. Furthermore, there may be a specific HDL3 (lipoprotein A) receptor of recognition site(s) on the plasma membrane. Finally, our data further support our previous reports of the important role of liver lysosomes in proteolytic degradation of HDL3.     

Investigations on the mechanism of hypercholesterolemia observed in copper deficiency in rats

The mechanism of hypercholesterolemia effect of Cu²⁺ deficiency was studied in rats. There was increased activity of hepatic hydroxymethylglutaryl-coenzyme A reductase and increased incorporation of labelled acetate into free cholesterol of liver in the Cu²⁺ deficient rats. Incorporation of label into ester cholesterol was however decreased in the liver. Concentration of bile acids in the liver was not significantly altered. Increase in the incorporation of labelled acetate into serum cholesterol and increase in the concentration of cholesterol and apo B in the low density lipoproteins + very low density lipoproteins fractions were observed. Activity of lipoprotein lipase of the extrahepatic tissues decreased in the Cu²⁺ deficient rats.     

Effects of cholestyramine on low density lipoprotein binding sites on liver membranes from rabbits with endogenous hypercholesterolemia induced by a wheat starch-casein diet

Rabbits fed a wheat starch-casein diet develop a marked hypercholesterolemia with a lipoprotein distribution similar to that of humans. Approximately 76% of the total cholesterol is carried in the low density lipoprotein (LDL) fraction (1.006 less than d less than 1.063 g/ml). Inclusion of 1% cholestyramine in the diet prevents the increase in plasma cholesterol. The cholestyramine effect is mediated through an increased fractional catabolic rate of 125I-LDL. In order to determine the potential role of hepatic LDL receptors in the removal of LDL from the plasma, binding of 125I-LDL and 125I-beta-VLDL (beta-migrating very low density lipoproteins) to hepatic membranes prepared from livers of rabbits fed the wheat starch-casein diet with or without cholestyramine supplementation was investigated. Membranes from livers of the cholestyramine-supplemented animals exhibit high levels of specific EDTA-sensitive binding of either of the 125I-labeled lipoproteins. Very little EDTA-sensitive binding occurs on liver membranes from wheat starch-casein-fed rabbits that have not been treated with cholestyramine. These results indicate that the hypercholesterolemia in rabbits associated with the wheat starch-casein diet is wholly or partially the result of a decreased number of specific hepatic LDL receptors and thus a decreased catabolism of plasma cholesterol. The response of the liver to the inclusion in the diet of the bile acid sequestrant, cholestyramine, is to maintain or increase the number of specific LDL binding sites, thus promoting catabolism of plasma cholesterol.     

Uptake and degradation of low density lipoprotein by swine arterial smoot muscle cells with inhibition of cholesterol biosynthesis.

We have previously proposed on the basis of studies in hepatectomized animals that low density lipoproteins are degraded at a significant rate by peripheral tissues. To test the capacity of one peripheral cell type to catabolize low density lipoprotein, cultures of swine aortic smooth muscle cells were incubated with homologous 125I-labeled low density lipoprotein and uptake and degradation measured. Degradation of 125I-labeled low density lipoprotein to products soluble in trichloroacetic acid showed an initial lag period of 1--2 h after which the rate increased and remained linear for the following 15 h. Rates of degradation increased sharply with low density lipoprotein concentration over the lower range (from 0--25 mug protein/ml) and then more slowly up to the highest concentration tested, 300 mug protein/ml. Even at very low concentrations, 1 mug low density lipoprotein protein/ml (less than 10% of the plasma low density lipoprotein concentration), the in vitro degradation rate (per kg of smooth muscle cells) exceeded the in vivo degradation rate (per kg of total body weight). To the extent that smooth muscle cells are representative of other peripheral cells, the results support the proposal that peripheral degradation of low density lipoprotein apoprotein may be quantitatively important. The rate of incorporation of labeled acetate into sterols was suppressed in cells incubated with whole serum, low density and very low density lipoproteins, or suspensions of free cholesterol. In this respect, the results were similar to those observed in human skin fibroblasts studied concurrently. However, high density lipoprotein inhibited sterol synthesis by about 25% in swine smooth muscle cells while it had no effect in human skin fibroblasts.     

Lipoprotein metabolism in the ovariectomized rat

The hyperlipoproteinemia observed after ovariectomy in rats was previously shown to be associated with increased concentrations of cholesterol, triglycerides, and apolipoproteins B, E, and C. In the present study, it was shown that increases in low density lipoproteins and high density lipoproteins were almost entirely responsible for the changes in plasma lipids and apolipoproteins after ovariectomy. The size of the low density lipoproteins and high density lipoproteins isolated from the plasma of ovariectomized rats as determined by agarose chromatography appeared to be somewhat different from that of control rats. Specifically, the apolipoprotein B appeared to be associated with somewhat smaller particles, whereas the apolipoprotein E from those rats appeared to be associated with larger particles than that of control rats. To determine the mechanism for the increased plasma low density lipoproteins, apolipoprotein B pool sizes and turnover rates were calculated and compared. In addition to an increased mass of low density lipoproteins in ovariectomized rats, the turnover rate of low density lipoproteins was increased almost twofold, indicating an increased low density lipoprotein synthesis and catabolism in those animals. We postulate that the increased low density lipoprotein levels of ovariectomized rats are due to an initial increased production of low density lipoproteins, followed by an enhanced catabolism of low density lipoproteins to establish a steady state at higher plasma low density lipoprotein concentrations.     

Hyperlipoproteinemia in Nagase analbuminemic rats: effects of pravastatin on plasma (apo)lipoproteins and lecithin:cholesterol acyltransferase activity.

The present study demonstrates very high levels of plasma lipids and high density lipoprotein (HDL) apolipoproteins (apoA-I and apoE) in female Nagase analbuminemic rats (NAR) fed a semi-synthetic diet in order to further increase the hyperlipidemia present in this strain. Plasma apoB-containing lipoproteins (very low, intermediate, and low density lipoproteins) were also elevated in NAR. Plasma cholesterol was mainly present in lipoprotein particles with a density between 1.02 and 1.12 g/ml. Separation of lipoprotein classes by gel filtration showed that the major cholesterol-carrying lipoprotein fractions in NAR plasma are apoE-rich HDL and apoA-I-rich HDL. The high HDL levels in NAR are explained, at least partly, by the two- to threefold elevated activity of plasma lecithin:cholesterol acyltransferase (LCAT). The lysophosphatidylcholine generated in the LCAT reaction, as well as plasma free fatty acids, are bound to lipoproteins in NAR plasma. A study was carried out to determine whether the elevated LDL and aopoE-rich HDL levels could be corrected by administration of the HMG-CoA reductase inhibitor pravastatin (at a dose of 1 mg/kg per day). Pravastatin treatment results in a 43% decrease in plasma triglycerides in NAR, but not in Sprague-Dawley (SDR) rats, and had no significant effect on plasma total cholesterol, phospholipids apolipoproteins A-I, A-IV, B, or E, as well as on plasma LCAT activity levels in NAR or SDR.     

Characterization of guinea pig plasma lipoproteins: the appearance of new lipoproteins in response to dietary cholesterol

Dietary cholesterol induces a hemolytic anemia in guinea pigs, accompanied by changes in the lipid composition of red cells and of plasma lipoproteins. This report presents a characterization of the lipoprotein species present in each main density class in both control and cholesterol-fed guinea pigs. Traces of a typical high density lipoprotein (HDL) were detected in control plasma. HDL from cholesterol-fed, anemic guinea pigs differed from control HDL in electron microscopic appearance and lipid and peptide composition. Long stacks of discs were observed in the electron microscope in addition to smaller, spherical particles characteristic of control HDL. Low density lipoproteins (LDL) from cholesterol-fed, anemic guinea pigs had two main populations, which were separated by gel chromatography. One population appeared in the electron microscope as large transparent discs and contained mainly unesterified cholesterol and phospholipids in a 2:1 molar ratio. The other population resembled control LDL in size and composition except for its high unesterified cholesterol content. Dietary cholesterol also altered the composition and decreased the electrophoretic mobility of very low density lipoproteins. Gel electrophoretic and immunochemical evidence indicates that a peptide (mol wt 35,000) appears in lipoproteins from cholesterol-fed, anemic guinea pigs that is undetectable in those of controls. Similarities between the cholesterol-induced lipoprotein abnormalities in guinea pigs and those reported in patients with obstructive jaundice, biliary cirrhosis, type III hyperlipoproteinemia, or familial lecithin:cholesterol acyltransferase deficiency are discussed.     

Differential scanning calorimetric studies of native and freeze-damaged very low density lipoproteins in hen’s egg yolk plasma

Lipid thermal transition patterns of the very low density lipoproteins in native and variously treated egg yolk plasma and extracted total very low density lipoproteins lipids have been recorded by differential scanning calorimetry in the temperature range 220–300 K, after lowering the freeze endotherm of free water in the sample with ethylene glycol. Three distinguishable patterns of lipid endotherms, designated types 1, 2 and 3 were obtained, respectively, from (i) native very low density lipoproteins in egg yolk plasma, (ii) freeze damaged very low density lipoproteins in gelled egg yolk plasma and (iii) extracted total lipids of very low density lipoproteins dispersed in water. Protein-depleted ‘lipid core’ particles of very low density lipoproteins obtained by exhaustive proteolysis of egg yolk plasma gave type 2 lipid transition pattern suggesting similarities in its lipid association with that of the freeze damaged very low density lipoproteins. Freezing the ‘lipid cores’ of very low density lipoproteins led to phase separation and gave type 3 lipid transition pattern of water-dispersed, phase-separated total very low density lipoprotein lipids. Relative heat uptake of native very low density lipoproteins in egg yolk plasma was about 15% lower than the freeze damaged sample or of the extracted total lipids. Treatments which prevented aggregation and gelation of very low density lipoproteins in egg yolk plasma during frozen storage, namely with additives such as glycerol or NaCl, gave subsequent lipid transition pattern intermediate between type 1 and 2, indicating that while very low density lipoprotein aggregation is prevented, additives do not altogether prevent changes in lipid association in these particles.     

Triglyceride-rich lipoprotein metabolism in unique VLDL receptor, LDL receptor, and LRP triple-deficient mice

The very low density lipoprotein receptor (VLDLR), low density lipoprotein receptor (LDLR), and low density lipoprotein receptor-related protein (LRP) are the three main apolipoprotein E-recognizing endocytic receptors involved in the clearance of triglyceride (TG)-rich lipoproteins from plasma. Whereas LDLR deficiency in mice results in the accumulation of plasma LDL-sized lipoproteins, VLDLR or LRP deficiency alone only minimally affects plasma lipoproteins. To investigate the combined effect of the absence of these receptors on TG-rich lipoprotein levels, we have generated unique VLDLR, LDLR, and LRP triple-deficient mice. Compared with wild-type mice, these mice markedly accumulated plasma lipids and lipases. These mice did not show aggravated hyperlipidemia compared with LDLR and LRP double-deficient mice, but plasma TG was increased after high-fat diet feeding. In addition, these mice showed a severely decreased postprandial TG clearance typical of VLDLR-deficient (VLDLR-/-) mice. Collectively, although VLDLR deficiency in LRP- and LDLR-/- mice does not aggravate hyperlipidemia, these triple-deficient mice represent a unique model of markedly delayed TG clearance on a hyperlipidemic background.     

Receptor-mediated uptake of lipoproteins by cultured porcine granulosa cells

Receptor-mediated uptake of low density lipoprotein (LDL) has been shown to provide a major source of cholesterol for a variety of cell types, particularly steroidogenic cells. In this study, the functional significance of lipoproteins in porcine ovarian granulosa cells and their mechanism of uptake by the cell was examined. Porcine LDL and high density lipoprotein (HDL) were isolated using a KBr density gradient, and the purity of both lipoproteins was confirmed by single corresponding bands on agarose gel stained for lipid and protein. Purified LDL and HDL were radioiodinated and labelled with colloidal gold for binding and tracer studies respectively. Both lipoproteins bind to cell surface and are internalized within 30 min at 37 degrees C. The cultured granulosa cells possess more HDL binding sites than LDL binding sites and are more responsive in progesterone production when supplemented with HDL. These results suggest that granulosa cells may preferentially utilize HDL over LDL as a source of cholesterol for steroidogenesis.