Lipoprotein-deficient Serum Research Articles

Modulation of 3-hydroxy-3-methylglutaryl-CoA reductase by changes in microsomal cholesterol content or phospholipid composition.

Microsomal preparations from normal human skin fibroblasts were used to investigate the role of free cholesterol in the endoplasmic reticulum in the control of cholesterol biosynthesis by regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (NADPH) [(S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]. Controlled changes in the cholesterol/phospholipid ratio of microsomes were introduced either in intact cells by incubating fibroblast cultures with whole serum or lipoprotein-deficient serum or by enrichment or depletion of cholesterol in microsomal preparations by incubating microsomes with cholesterol-rich or cholesterol-poor egg phosphatidylcholine unilamellar vesicles. Cholesterol enrichment resulted in suppression of reductase activity and increased ESR order parameters for 12-nitroxystearate in the microsomal preparations. Conversely, cholesterol depletion caused an activation of reductase and a decrease in order parameter. Enrichment of microsomal preparations with a "nonfluid" lipid, dipalmitoyl phosphatidylcholine, also suppressed enzyme activity and increased membrane order. The effect was reversed by subsequent enrichment of the microsomes with fluid egg phosphatidylcholine. Our findings suggest that cholesterol may regulate its own biosynthesis, at least in part, by suppression of hydroxymethylglutaryl-CoA reductase mediated through changes in membrane fluidity as measured by ESR order parameter.

The role of lipoproteins in the degradation of platelet-activating factor

Measuring both platelet-stimulating activity and liberation of acetate, the capacity of serum and individual lipoproteins to degrade the platelet-activating factor (PAF) was studied. The highest degrading effect relative to the protein content was found in very low density lipoproteins (VLDL) and in low density lipoproteins (LDL). The effect is about 10- and 100-fold higher than that of high density lipoproteins (HDL) and serum, respectively. In lipoprotein deficient serum (LPDS) less than 5% of serum activity is detectable. Considerable individual variations are observed measuring the degradation of PAF under standard conditions in plasma from 37 healthy volunteers. Moreover, this activity is shown to correlate strongly with the plasma concentration of LDL. On the other hand, a significant negative correlation was found between the PAF-degrading capacity and the plasma concentration of HDL. In contrast, the PAF-degradation is unrelated to the concentration of plasma triglycerides. The results point to a possible role of plasma lipoproteins in regulating the degradation of PAF released into the circulation.

Role of lipoproteins in growth of human adult arterial endothelial and smooth muscle cells in low lipoprotein-deficient serum.

Recently improved culture conditions for human adult arterial endothelial and smooth muscle cells from a wide variety of donors have been used to study the effects of lipoproteins on proliferation of both cell types in low serum culture medium. Optimal growth of endothelial and smooth muscle cells in an optimal nutrient medium (MCDB 107) containing epidermal growth factor, a partially purified fraction from bovine brain, and 1% (v/v) lipoprotein-deficient serum was dependent on either high- or low-density lipoprotein. High- and low-density lipoprotein stimulated cell growth by three- and five-fold, respectively, over a 6-day period. Optimal stimulation of both endothelial and smooth muscle cell growth occurred between 20 and 60 micrograms/ml of high- and low-density lipoproteins, respectively. No correlation between the activation of 3-hydroxyl-3-methylglutaryl coenzyme. A reductase activity and lipoprotein-stimulated cell proliferation was observed. Lipid-free total apolipoproteins or apolipoprotein C peptides from high-density lipoprotein were partially effective and together with oleic acid effectively replaced native high-density lipoprotein for the support of endothelial cell growth. In contrast, apolipoproteins or apolipoprotein C peptides from high-density lipoprotein alone or with oleic acid had no effect on smooth muscle cell proliferation. The results suggest a functional role of high- and low-density lipoproteins and apolipoproteins in the proliferation of human adult endothelial and smooth muscle cells.

Mevinolin, an inhibitor of cholesterol biosynthesis, drastically depresses Ca2+ channel activity and uncouples excitation from contraction in cardiac cells in culture.

Mevinolin (MK803), a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) (Ki, 30 X 10(-9) M), depressed de novo synthesis of cholesterol in 11-day chicken embryonic cardiac cells cultured in lipoprotein-deficient serum (LPDS). Cardiac cells exposed to different concentrations of mevinolin for 1-3 days presented different electrophysiological and mechanical properties: The resting membrane potential, the rate of increase, and the shape of the action potential and contractile properties were changed at concentrations as low as 0.1 microM mevinolin. At a concentration of 1 microM mevinolin, the cardiac cells became quiescent and electrical stimulation induced action potentials of short duration without contraction. Isoproterenol and Bay K8644 were unable to restore excitability and contraction. Although the number of receptors for the tritiated Ca2+ channel blocker nitrendipine was the same in control and in mevinolin-treated cells, voltage-clamp data on isolated cardiac cells and 45Ca2+ flux experiments on monolayers showed that most of the slow Ca2+ channel activity was lost in mevinolin-treated cells. These results suggest that the disappearance of Ca2+ channel activity is most probably at the origin of the loss of cardiac contractility.

Regulation of synthesis of lactosylceramide and long chain bases in normal and familial hypercholesterolemic cultured proximal tubular cells.

We have investigated the effects of lipoproteins on sphingolipid metabolism in proximal renal tubular cells from normal subjects and low density lipoprotein (LDL) receptor-negative homozygous familial hypercholesterolemic subjects employing radioactive precursors, e.g. [3H]serine, [3H]glucose, and [14C]galactose. Compared to cells incubated with lipoprotein-deficient serum, maximum suppression (70-80%) of incorporation of [3H]glucose and [3H]serine into ceramide and LacCer occurred when the LDL concentration in the medium was 25 micrograms/ml medium, and addition of higher amounts of LDL (up to 500 micrograms/ml medium) to normal cells did not produce further suppression. In contrast, high density lipoproteins did not suppress the incorporation of [3H]glucose into lactosylceramide (LacCer) in normal cells. The incorporation of [14C] galactose into LacCer was also suppressed by LDL (50% suppression at a concentration of 100 micrograms/ml medium). In contrast, LDL modified by reductive methylation of lysine residues did not suppress the incorporation of [3H]glucose into LacCer and the incorporation of [3H]serine into ceramide, whereas, native LDL exerted a concentration-dependent suppression of [3H]serine incorporation into ceramide and sphingomyelin in normal cells. At high concentrations of LDL (50-500 micrograms/ml medium), the incorporation of [3H]glucose and [14C]galactose into LacCer in homozygous FH cells was stimulated approximately 2-fold. Maximum stimulation of [3H]serine incorporation into ceramides, LacCer, and sphingomyelin occurred at 100 micrograms LDL/ml medium. Our studies indicate that the endogenous synthesis of sphingolipids in normal renal cells is regulated by the LDL receptor. Modification of the lysine residues in LDL by reductive methylation results in the inability to suppress sphingolipid synthesis in normal cells. Lack of LDL receptors, as in the case of homozygous FH cells, results in the lack of suppression of endogenous sphingolipid synthesis.

62 MEVALONIC ACIDURIA. MEMBRANE COMPOSITION AND FLUIDITY IN AN INBORN ERROR OF CHOLESTEROL SYNTHESIS

Mevalonate aciduria is an inherited disorder of cholesterol synthesis. Mevalonate kinase activity in lysates of fibroblasts from a patient was <1% of control mean and cholesterol synthesis in intact fibroblasts with labeled acetate or mevalonolactone was 33-54% of parallel controls. However, fibroblast growth rates in media containing whole serum and lipoprotein-deficient serum (LPDS), respectively, were normal. Mutant fibroblasts grown in the presence of whole serum had a normal cholesterol content and a normal free to ester-ified cholesterol ratio. Lipid fluidity of plasma membranes of fibroblasts was measured by steady-state fluorescence polarization. Fluorophores used were diphenylhexatriene (DPH), a cationic analogue (TMA-DPH), and a set of anthroyloxy fatty acids. For all fluorophores used the results for the mutant cells were within the normal range. Even when grown in LPDS medium, membrane fluidity of mutant cells was not increased. Hence, mutant cells were able to preserve, a normal growth rate and membrane function despite their impaired cholesterol synthesis. We are now investigating the compensatory mechanisms developed by cells with this mutation. (Supported by DFG grant K0756.)

Growth and lipid composition of rat brain glial cells cultured in lipoprotein deficient serum.

The purpose of this study was to examine the effects of substituting lipoprotein deficient serum (LPDS) for complete fetal calf serum (FCS) in culture media on the growth and lipid composition of cells dissociated from 1 to 2-day-old rat brain. The results show that in FCS cultures DNA, protein and all lipids increase with an increase in the number of days in culture. Substitution of LPDS for FCS in the culture media caused a slower increase in each of these constituents. Esterified cholesterol remained unaltered with time in LPDS cultures but increased continuously in FCS cultures. Substitution of LPDS for FCS reduced the DNA:protein ratio, and unesterified cholesterol:phospholipid ratio but the protein:phospholipid ratio and the proportion of individual phospholipids were not affected. The data indicate that removal of low density lipoprotein (LDL) from serum used in culture media reduces cell proliferation and causes alterations in cellular lipid composition specifically ratio of cholesterol:phospholipids.

Induction of corticosteroid biosynthetic pathway by ACTH and high-density lipoprotein in newborn rat adrenocortical cells cultured in serum-free medium

In order to investigate the role of rat high-density lipoprotein (HDL) on adrenal cholesterol accumulation and steroidogenic pathways (corticosteroid, i.e., 21-hydroxysteroid biosynthesis and reductive metabolism of progesterone), newborn rat adrenal cells cultured in serum-free medium were used. Incubation of [4- 14C]cholesterol-HDL (i) in serum-free medium compared to those in medium with lipoprotein-deficient serum, (ii) in serum-free medium with ACTH compared to those without ACTH, both showed an increase of labelled cholesterol in cells and of labelled 21-hydroxysteroids excreted in medium. Substitution of serum-supplemented medium by serum-free and cholesterol-free medium led to a deep decrease of ACTH-induced steroid biosynthesis with a predominance of 20α-reduced steroids; addition of HDL restored the corticosteroid biosynthesis and decreased the reductive metabolism. Addition of increased concentrations of HDL (7–150 μg cholesterol/ml) enhanced, in a saturable fashion, the total cholesterol uptake and the corticosteroid biosynthesis. The total cholesterol accumulation in cells exceeded by 4-fold the steroid production at saturation. The ratio between the two steroidogenic pathways increased up to 40 at saturation in favor of corticosteroids. These results suggest that HDL is at least partly internalized and that probably its constituents contribute greatly to the control of the two different steroidogenic pathways.

Oxidized low density lipoprotein induces ceroid accumulation by murine peritoneal macrophages in vitro

The presence of ceroid pigment inclusions within macrophage foam cells is a well-recognized phenomenon in human atherosclerosis but its significance is unclear. Murine peritoneal macrophages, maintained in a medium containing 10% lipoprotein-deficient fetal calf serum to which oxidized low density lipoprotein (LDL) has been added, rapidly accumulate similar ceroid inclusions. However, cells exposed to LDL, acetyl-LDL or dextran sulphate-LDL complexes fail to develop ceroid under the same culture conditions. Oxidation of LDL results in considerable physico-chemical changes to both protein and lipid moieties, and these may be important in the uptake of the particles by macrophages and, subsequently, the accumulation of intracellular ceroid.

Low apparent affinity for low-density lipoprotein of receptors expressed by human macrophages maintained with whole serum.

Normal human monocyte-derived macrophages maintained in medium containing whole serum exhibited saturable degradation of low-density lipoprotein (LDL) that was mediated by LDL receptors. This degradation required a higher concentration of LDL to achieve one-half saturation than that in cells preincubated with lipoprotein-deficient serum (LPDS). Studies of short-term uptake and of heparin-releasable binding of LDL showed that binding to the surface receptors was the limiting factor for degradation under both conditions and that the LDL receptors expressed by cells in whole serum had a significantly lower affinity for LDL than those in cells pre-incubated in LPDS. LDL receptors in monocyte-macrophages could mediate the uptake and degradation of complexes between apolipoprotein E (apoE) and phospholipid. The receptors in cells pre-incubated in LPDS bound the complexes and LDL with apparently the same affinity and in approximately the same molar ratio. Receptors in cells maintained with whole serum did not have a lower affinity for the complexes than cells pre-incubated in LPDS, although the molar ratio of maximum degradation of LDL to that of complexes was greater.

The effects of phorbol ester on anabolism of lipids of cultured human skin fibroblasts

We have investigated the independent effects of phorbol ester (phorbol 12-myristate 13-acetate) on anabolism of the major lipid components in cultured diploid human skin fibroblasts. When we incubated these cells with [ 3H]acetate in serum-free medium for 18 h in the presence of 16 nM phorbol ester, [ 3H]acetate incorporation and the cellular content of cholesterol ester increased, and free cholesterol decreased. Enhancement of [ 3H]acetate incorporation into cholesterol ester was also observed when the cells were incubated with phorbol ester for 5 h in medium containing lipoprotein-deficient serum. Incorporation of [ 3H]galactose into glycosphingolipids increased many fold upon exposure of the cells either to fetal calf serum or separately to phorbol ester. Therefore, phorbol ester independently affects cholesterol and glycosphingolipid metabolism in a way that may be similar to that reported for serum low-density lipoproteins and unknown other factors in fetal calf serum. We have observed these effects of phorbol ester in the intact living cell. These findings should provide useful means for the study of metabolism of the plasma membrane lipid components in fibroblasts.

Effect of lipoprotein on LPL activity of guinea pig alveolar macrophages

Macrophages may play an important role in the atherogenic process because they appear to be precursors of some arterial wall foam cells. Recently, it has been reported that human peripheral monocytes in culture synthesize and secrete lipoprotein lipase (LPL). LPL catalyzes the hydrolysis of triglycerides which are transported in plasma by chylomicrons and very low density lipoprotein (VLDL). Consequently, it is possible that LPL secretion by human monocytes is related to atherogenesis. As shown below, human monocytes and guinea pig alveolar macrophages in culture synthesize and secrete LPL. The purpose of this research is to study the significance of this macrophage LPL.Therefore I investigated the effect of lipoprotein on LPL activity of guinea pig alveolar macrophages.Monocytes were obtained from a normal healthy donor and were separated from whole blood by Ficoll-Hypaque. The lungs of guinea pig were washed 10-12 times with 0.9% NaCl and the washings were cultured. The enzyme activity was determined by a modification of the method of Schotz et al. The separation of the lipoprotein fractions was performed in a type RP-40 rotor by ultracentrifugation by the method of Havel et al.The rate of LPL secretion from human monocytes was low initially but increased with time in culture. During 14 days in culture, maximal activity was attained on the 12th day.On the other hand, guinea pig alveolar macrophages increased from the beginning. During 8 days in culture, maximal activity was attained on the 4th day. Next, we investigated the effect of lipoprotein on LPL activity of guinea pig alveolar macrophages. Alveolar macrophages were cultured for 2 days with either RPMI-1640 medium containing 10% lipoprotein deficient serum (LPDS) or that medium with the addition of VLDL, LDL, and HDL respectively in the concentrations of 300μg protein/ml. When VLDL was added to LPDS after one day, LPL activity was significantly reduced to 48.3% within 2 days of exposure when compared with LPDS controls. (p<0.02). A possible explanation for this phenomenon, is that LPL activity may be inhibited by apoprotein CIII of VLDL.

LDLによる培養ヒト動脈壁平滑筋細胞のDNA合成能の検討

Many investigators have studied the effect of low density lipoprotein (LDL) on the proliferation of arterial smooth muscle cells (SMC). The methodology is not well studied. The purpose of this study was to investigate the method for analysis of the effect of LDL on DNA synthesis in cultured human arterial SMC.Human arterial SMC were cultured from splenic artery with MEM containing 5% of LPDS (lipoprotein deficient serum from normal subjects). LDLs were ultracentrifugally prepared from normal subjects (n=8) and diabetics (n=12). 0.2×105 cells/dish were cultured with 5% LPDS-MEM for 48 hours. This medium was replaced with fresh 5% LPDS-MEM containing LDL (25 microgr./ml) and 0.5 micro Ci of 3H-Thymidine. After 6 hours intracellular radioactivity was measured and DNA synthesis calculated as cpm/microgr, cell protein. This experiment was repeated for 6 times during 36 hours. In another experiment DNA synthesis in these SMC was studied during 36 hours after 3H-Thymidine addition.DNA synthesis in SMC cultured without LDL addition was almostly constant during 36 hours, however, the value of cpm/microgr, protein was variated in each 7-experiment. Therefore, we calculated the value of Index as shown below: Index (%)This Index was useful in comparison of data among 7-experiments. The time point of the maximum value of Index in the culture with each LDL was variated from 18 to 36 hours after LDL addition in the experiment repeatedly performed for 6 hours during 36 hours. The maximum Index was higher in the culture with diabetic LDL than in that with normal LDL. When DNA synthesis was studied during 36 hours after 3H-Thymidine addition, Index in the culture with diabetic LDL was significantly (p<0.05) higher than that in the culture with normal LDL.We concluded that (1) Index of DNA synthesis was useful for analysing the effect of LDL on SMC in vitro, (2) DNA synthesis should be studied for 36 hours after addition of LDL and 3H-Thymidine.

細胞内コレステロール処理機構に対する低酸素の影響

To elucidate the mechanism of cholesterol accumulation in cultured cells under hypoxic conditions which we previously reported, the effect of hypoxia on the efflux of cholesterol from cultured rabbit skin fibroblasts was tested.Cells were incubated with hyperlipemic rabbit serum (HRS) for 48 hours (load phase), then washed three times with phosphate buffered saline and incubated with normolipemic rabbit serum (NRS) for 24 hours (efflux phase). In the efflux phase cells were divided into two groups, one group was incubated under 95% N2 and 5% CO2 (hypoxic group), another was 95% room air and 5% CO2 (control group).At the end of load phase cellular content of esterified cholesterol was 39.0±1.5nmol/mg protein and after efflux phase it was remarkably decreased in control group (22.4±2.5nmol/mg protein) while it was almost unchanged in hypoxic group (38.2±1.7nmol/mg protein).When cells were incubated with lipoprotein deficient serum instead of NRS to exclude the uptake of extracellular cholesterol in efflux phase, the cellular content of esterified cholesterol was also significantly higher in hypoxic group than control group (53.1±1.5, 36.2±3.2nmol/mg protein respectively).These results suggested that the affected efflux may play a role in accumulation of esterified cholesterol in cells under hypoxic conditions.

Cholesteryl ester efflux from extracellular and cellular elements of the arterial wall. Model systems in culture with cholesteryl linoleyl ether.

Cholesteryl ester (CE) accretion in human atheroma is a slow process during which lipoproteins infiltrate the arterial extracellular space and then gain entry into the cellular components. The present aim was to simulate this process in model systems in culture to learn whether cholesteryl ester transfer protein (CETP) may promote CE efflux at different stages of atheroma formation. To simulate CE efflux from arterial interstitium, cationized LDL labeled with 3H-cholesteryl linoleyl ether (3H-CLE) was added to fixed aortic smooth muscle cells (SMC) or to extracellular matrix. To study efflux of 3H-CLE taken up by cells via receptor-mediated endocytosis of LDL, the SMC cultures were fixed and permeabilized prior to the determination of CE efflux. The cellular model included macrophages, which had ingested acetylated LDL labeled with 3H-CLE. Efflux of 3H-CLE and 14C-CE was studied during postincubation of the labeled cultures with human lipoprotein deficient serum (LPDS) or partially purified CETP. As controls, we used SMC cultures incubated with albumin. In all systems, a 3- to 12-fold increase of 3H-CLE or 14C-CE efflux was found in the postincubation medium containing human LPDS or partially purified CETP when compared to controls. Permeabilization of the cells with saponin enhanced cellular 3H-CLE and 14C-CE efflux in the presence of human LPDS. The findings indicate that CETP may promote CE efflux from aortic interstitium or disintegrating cells. We propose that CETP may play an important role in aortic CE homeostasis under physiological and pathological conditions.

家族性高コレステロール血症患者のLDLレセプター活性とそれに対する薬物療法の影響

We examined low density lipoprotein (LDL) receptor activities of 13 heterozygous patients with familial hypercholesterolemia (FH) using peripheral lymphocytes.The receptor activities were measured as the degradation of 125I-labeled LDL in lymphocytes which had been cultured for three days in 10% lipoprotein-deficient serum. After incubation, 10μg/ml of 125I-LDL were added to the medium in the presence or absence of 250μg/ml of nonlabeled LDL and then the lymphocytes were incubated for five hours further. The radioactivity of the trichloroacetic acid-soluble fraction of the medium was measured.As a result, LDL receptor activities of FH heterozygotes were revealed to correlate significantly and negatively with serum LDL-cholesterol concentration. If we combined them with those of two FH homozygotes and 18 hyperlipidemic patients other than FH and normolipidemic persons, the latter of which we reported previously (J. Jpn. Atheroscler. Soc. 12: 303-308, 1984), not only the grade of the negative correlation between LDL receptor activities and LDL-cholesterol became greater, but LDL receptor activities also correlated negatively with total cholesterol (TC) (p<0.01). Extrapolation from these data suggested that LDL receptor activity would disappear when TC concentration was higher than 718mg/dl. The value estimated agreed well with the clinical findings of FH.Then, probucol (750mg a day) was administered to eight heterozygous patients with FH for four to six months, by which TC concentration decreased significantly from 319.6±36.7 (mg/dl) to 282.3±17.4 (mg/dl). The result suggests that probucol is effective in the treatment of FH. The levels of LDL receptor activities in the pretreatment state was not correlated to the effectiveness of probucol treatment in each case.LDL receptor activities were also measured after probucol administration and were compared with those of the pretreatment state. There were a great variations of change of LDL receptor activity among the eight patients, and no definite trend of the activity could be found. But, it was suggested that the percentage change of TC concentration was correlated negatively with the difference in LDL receptor activity before and after probucol administration, that is, the more the TC concentration decreased, the more the LDL receptor activity increased.These results may suggest that the main action of probucol is not on LDL receptor and probucol indirectly influences LDL receptor activity by the change in serum cholesterol concentration.

Diabetic lipoprotein deficient serum: Its effect in low density lipoprotein (LDL) uptake and degradation by fibroblasts

Low density lipoproteins (LDL) isolated from poorly controlled diabetic patients are known to be taken up and degraded by fibroblasts at a lower rate than LDL isolated from normal subjects. This aberrant metabolic behavior has been attributed to a diabetic-related abnormality in LDL composition yet to be characterized. The studies reported in this article show that the decrease in uptake and intracellular degradation of LDL from diabetic patients is further enhanced when the cells are exposed to lipoprotein deficient serum (LPDS) isolated from the same poorly controlled diabetic patients. Comparative studies of the composition of LPDS obtained from normal donors and poorly controlled diabetic patients showed an increase in saturated and total unesterified fatty acids (UFA), lecithin, apolipoprotein A 1, and immunoreactive insulin in the LPDS from diabetic patients. We postulate that exposure of cells to LPDS obtained from poorly controlled diabetic patients may induce changes in the composition of the fibroblast membrane and alter its fluidity, leading to further decrease in the uptake and degradation of LDL. During poor diabetic control, cell membrane changes, and modification of LDL composition are likely to act either additively or synergistically to induce an abnormal LDL-cell interaction. This abnormal interaction may be a relevant factor to explain the greater incidence of arteriosclerosis in diabetes mellitus.

Deficiency of serum cholesteryl-ester transfer activity in patients with familial hyperalphalipoproteinaemia

Lipoprotein patterns and cholesteryl ester transfer activity (CETA) were examined in 2 patients with familial hyperalphalipoproteinaemia (FHALP). The proband was a healthy 58-year-old Japanese male who had an HDL cholesterol of 7.83 mmol/1 (301 mg/dl). His sister's HDL cholesterol was 4.52 mmol/l (174 mg/dl), which suggested that both were homozygous carriers of FHALP. In both subjects HDL showed a high cholesterol/apo A-I ratio and appeared to be a larger-sized particle than normal HDL on agarose gel chromatography. Two of the proband's children showed higher HDL cholesterol levels (1.74 mmol/l, 2.16 mmol/l) than normal, but another 2 children showed normal levels (1.48 mmol/l, 1.40 mmol/l). However, the ratios of HDL cholesterol to total cholesterol and to apo A-I in all children were higher than normal. These data suggest, but do not prove, that all his children were heterozygotes. Apo B levels in all of the family members studied were lower than normal (47 ∼ 80 mg/dl). Deceased members of the same family had not died from cardiovascular disease. Cholesteryl-ester transfer activity was studied in both patients. When serum or lipoprotein deficient serum ( d > 1.21) and [ 3H] cholesteryl ester labelled HDL 3 were incubated in the presence of an LCAT inhibitor, there was no evidence of cholesteryl ester transfer from HDL to VLDL and/or LDL, unlike normal subjects. The deficiency of CETA in these patients with FHALP presumably accounted for the increase in particle size and cholesterol enrichment of HDL.

The effect of non-receptor-mediated uptake of cholesterol on intracellular cholesterol metabolism in human skin fibroblasts.

Unilamellar lipid vesicles of various cholesterol:phosphatidylcholine molar ratios were used to alter, via passive exchange at the plasma membrane, the cellular free cholesterol content of cultured human skin fibroblasts which had been preincubated in lipoprotein-deficient serum. The effects of these net surface transfers of cholesterol on cellular cholesterol biosynthesis, cholesterol esterification and low density lipoprotein (LDL) binding were determined and were compared with the effects of cholesterol delivered to the cell interior via the receptor-mediated endocytosis of LDL. Both LDL and cholesterol-rich lipid vesicles increased cell cholesterol within 6 h. Cells exposed to LDL also showed, within 6 h, decreased cholesterol synthesis, decreased LDL binding and increased cholesterol esterification. Cells incubated with the cholesterol-rich vesicles showed similar changes but these were delayed and did not occur until 24 h. Fibroblasts incubated with cholesterol-free phosphatidylcholine vesicles had decreased cell cholesterol, increased cholesterol synthesis, increased LDL binding, and decreased esterification, but only after 24 h of incubation. These results suggest that passive net transfers of cholesterol occurring at the cell surface can with time modulate intracellular cholesterol metabolism. These findings are consistent with the idea that the movement of cholesterol from the cell surface to the cell interior is a limited and relatively slow process.

Oxidized low density lipoprotein can reduce the pinocytic activity in cultured human endothelial cells as measured by cellular uptake of [ 14C]sucrose

The effect of low density lipoprotein (LDL) on the pinocytic activity in human endothelial cells in culture, as measured by the uptake of [ 14C]sucrose, was studied. The cells were preincubated for 48 h with a low concentration of LDL (25 μg protein/ml) or a high concentration of LDL (150 μg protein/ml) in the presence of lipoprotein deficient serum. Then the pinocytic activity of the cells was measured in a subsequent incubation by measuring the uptake of [ 14C]sucrose. The higher LDL concentration resulted in a reduced cellular uptake of [ 14C]sucrose compared to the lower concentration of LDL When the additional LDL (the lower concentration subtracted from the higher) was replaced by high density lipoprotein, albumin or gamma globulin, no reduction of the uptake of [ 14C]sucrose was observed. The inhibitory effect of LDL on the uptake of [ 14C]sucrose was seen in endothelial cell cultures of different age and cell density. When the endothelial cell proliferation was arrested by gamma irradiation, the reducing effect of LDL on the pinocytic activity was unchanged as compared to nonirradiated controls. LDL prepared in the presence of EDTA and glutathione did not have the same reducing effect on the uptake of [ 14C]sucrose as control LDL prepared in the absence of antioxidants. Thus, oxidized LDL reduced the pinocytic activity in cultured endothelial cells. The inhibitory effect of LDL on the rate of pinocytosis was present before endothelial cell injury could be observed.