Calorie restriction in mice does not affect LDL reverse cholesterol transport in vivo.

Calorie restriction (CR) prolongs life in animals, but may reduce plasma HDL, important in reverse cholesterol transport (RCT). The effect of CR, 60% of an ad libitum (AL) diet, on cholesterol removal from rectus femoris muscle injected with cationized LDL, was studied in C57BL male mice. RCT in vivo, on CR and AL diet, and cholesterol efflux from macrophages exposed to CR or AL sera, was similar, despite a 22% reduction in plasma HDL-cholesterol (HDL-C). In CR fed mice total cholesterol (TC) and phospholipid (T-PL) decreased by 32% and 38%, while HDL-C and HDL-PL decreased by 22% and 16% only, resulting in increased HDL-PL/T-PL ratio, which enhanced RCT. Partial re-feeding (CR-RF, 70% of AL) induced normalization of plasma lipids (excluding triglycerides), while HDL-PL/T-PL remained elevated. Thus, as CR did not interfere with RCT in vivo, it could possibly be beneficial to patients at risk for coronary heart disease.

Reverse cholesterol transport in mice expressing simian cholesteryl ester transfer protein.

The role of cholesteryl ester transfer protein (CETP) in atherogenesis remains ambiguous, as both pro and antiatherogenic effects have been described. Expression of CETP increases HDL-cholesteryl ester turnover, but there is no direct evidence whether CETP mobilizes cholesterol in vivo. The rate of cholesterol removal injected into a leg muscle as cationized low density lipoprotein (cat-LDL) was compared in CETP transgenic and control mice. Four days after injection the exogenous cholesterol mass retained in muscle was 65% in CETP transgenic and 70% of injected dose in controls; it decreased to 52-54% by day 8 and negligible amounts remained on day 28. The cat-LDL was labeled with either 3H-cholesterol oleate (3H-CE) or 3H-cholesteryl oleoyl ether (3H-COE), a nonhydrolyzable analog of 3H-CE. After injection of 3H-CE cat-LDL, clearance of 3H-cholesterol had a t(1/2) of 4 days between day 4 and 8 but there was little loss of 3H-COE between day 4 and 51. Liver radioactivity on day 4 was 1.7% in controls and 3.4% in CETP transgenics; it was 2.8 and 4.6%, respectively, on day 8. 3H-COE in liver accounted for 60% of label in CETP transgenics. In conclusion, high levels of plasma CETP in mice do not enhance reverse cholesterol transport in vivo but may act on extracellularly located cholesteryl ester.

Effect of atherogenic diet on reverse cholesterol transport in vivo in atherosclerosis susceptible (C57BL/6) and resistant (C3H) mice.

Mice susceptible (C57BL/6) or resistant (C3H) to atherosclerosis induced by a high cholesterol-cholate containing diet (A-diet) were used to study reverse cholesterol transport (RCT) in vivo as measured by loss of cholesterol from a depot created by injection of cationized LDL into the rectus femoris muscle. Plasma total and HDL-cholesterol (HDL-C), total and HDL phospholipid (HDL-PL) levels in chow fed C3H male and female mice were higher than in C57BL/6 mice. After one month on A-diet, plasma cholesterol more than doubled in both strains and genders. The decrease in HDL-C and HDL-PL was twice as great in C57BL/6 as in C3H female mice, while in male C3H mice there was no decrease. The loss of exogenous cholesterol mass (ECM) after injection of cationized LDL was more rapid in C3H than in C57BL/6 mice. In chow fed mice, ECM retained in muscle on day 12 was 37% in C57BL/6 and 20% in C3H females; in males it was 39% and 18% in C57BL/6 and C3H, respectively. On A-diet, 76% were retained in C57BL/6 and 28% in C3H females; these values were 59% and 28% in C57BL/6 and C3H males. Thus, the slow clearance of ECM (which represents RCT) in C57BL/6 mice on A-diet, that could be related to a marked decrease of HDL-PL, might contribute towards their susceptibility to atherosclerosis.

Macrophage cholesterol metabolism, apolipoprotein E, and scavenger receptor AI/II mRNA in atherosclerosis-susceptible and -resistant mice.

Female mice known to be susceptible (C57BL) and resistant (C3H and BALB/c) to diet-induced atherosclerosis were studied. Feeding of a cholate-containing atherogenic diet for 1 month resulted in an increase in plasma total cholesterol, little or no change in total phospholipids and high density lipoprotein (HDL) cholesterol, and a fall in HDL phospholipid, which was most pronounced in the C57BL strain. In elicited macrophages, cholesterol esterification was lower with acetylated low density lipoprotein (acLDL) and higher with beta-very low density lipoprotein (beta-VLDL) in C57BL than in C3H or BALB/C strains. In resident macrophages, acLDL enhanced cholesterol esterification more than did rabbit beta-VLDL. With acLDL, more apolipoprotein E (apoE) was recovered in all macrophage cultures. In macrophages from chow-fed mice, most apoE was in the medium, whereas in mice fed an atherogenic diet, half of the apoE was in the cells. ApoE protein was highest in macrophages from BALB/c mice fed an atherogenic diet; an increase in apoE mRNA occurred in BALB/c and C3H macrophages. Scavenger receptor AI/II mRNA was significantly higher in macrophages from atherosclerosis-resistant mice. Thus, higher HDL phospholipid and plasma apoE levels (reported by others), together with high macrophage scavenger receptor AI/II mRNA, could inhibit accretion of cholesterol in the vessel wall in the 2 resistant strains.

Clearance of cationized LDL cholesterol from a muscle depot is not enhanced in human apolipoprotein A-IV transgenic mice.

Human apolipoprotein A-IV (apoA-IV) transgenic mice fed an atherogenic diet were shown previously to develop less atherosclerosis than control mice. The question arose whether the antiatherogenic effect of human apoA-IV is due to enhancement of reverse cholesterol transport despite no increase in plasma high-density lipoprotein (HDL) cholesterol. We studied male and female mice overexpressing human apoA-IV and their wild-type (WT) controls, all of which were fed a chow diet. Plasma total and HDL cholesterol and total phospholipids were not increased in the transgenic mice, and regression analysis showed no correlation between plasma levels of cholesterol or phospholipids and plasma human apoA-IV. To study reverse cholesterol transport in vivo, the disappearance of cholesterol from a depot of [(3)H]cholesterol-labeled cationized low-density lipoprotein injected into the rectus femoris muscle was compared in high expressers of human apoA-IV and WT controls. The loss of radioactivity and the diminution of the exogenous cholesterol mass were determined on days 8 and 12 after injection. No enhanced loss of radioactivity or cholesterol mass was seen in the transgenic mice even at levels of 2500 mg/dL of human apoA-IV. In some instances, there was even slower loss of exogenous cholesterol (radioactivity and mass) in the transgenic mice. Although [(3)H]cholesterol efflux from cultured human skin fibroblasts and mouse peritoneal macrophages was only approximately 30% higher in the presence of sera from high expressers of human apoA-IV, addition of phosphatidylcholine liposomes enhanced the efflux in both groups to the same extent. Another paradoxical finding was that the cholesterol esterification rate in plasma was 34% to 36% lower in human apoA-IV mice than in WT controls. In conclusion, even though apoA-IV was found previously to be atheroprotective under hypercholesterolemic conditions, high plasma levels of human apoA-IV did not enhance cholesterol mobilization in vivo in normocholesterolemic mice.

High levels of human apolipoprotein A-I and high density lipoproteins in transgenic mice do not enhance efflux of cholesterol from a depot of injected lipoproteins. Relevance to regression of atherosclerosis?

The role of high density lipoprotein (HDL) and apolipoprotein A-I (apo A-I)in promoting cholesterol efflux from cultured cells and attenuation of development of atherosclerosis in transgenic (tg) animals has been well documented. The aim of the present study was to determine whether high levels of human (h) apo A-I will enhance cholesterol removal in vivo. h apo A-I in sera of tg mice was 429 +/- 18 and 308 +/- 10 mg/dl in male and female mice, the ratio of phospholipid (PL) to apo A-I was 0.94 in tg and 2.4 and 1.9 in male and female controls, taking mouse apo A-I as 100 mg/dl. The removal of lipoprotein cholesterol injected in the form of cationized low density lipoprotein (cat-LDL) into the rectus femoris muscle of h apo A-I tg is compared with control mice. After injection of cat-LDL labeled with [3H]cholesterol, the labeled cholesterol was cleared from the depot with a t 1/2 of about 4 days in both control and tg mice. The clearance of the exogenous cholesterol mass was initially much slower, it approached the t 1/2 of about 4 days between day 8 and 14 but there was no difference between tg and control mice. Cholesterol efflux from cultured macrophages exposed to media containing up to 10% serum was 56% higher with serum from tg mice than controls. In conclusion, the efflux of cholesterol from a localized depot of cat-LDL was not enhanced in h apo A-I tg mice. It appears, therefore, that while an increase above physiological levels of apo A-I or plasma HDL does play a pivotal role in the prevention of initiation and progression of early stages of atherosclerosis, the effectiveness of such an increase for the regression stage remains still to be demonstrated.

Dexamethasone impairs cholesterol egress from a localized lipoprotein depot in vivo.

Plasma high density lipoproteins play a central role in the prevention and regression of atherosclerosis, as they are known to promote egress of cholesterol from cells. Glucocorticoids increase plasma HDL, but enhance esterification of cholesterol in macrophages in vitro. A novel model to measure cholesterol egress from a well defined depot in vivo was used currently to study the effect of dexamethasone on reverse cholesterol transport. Cationized LDL (cat LDL) (200 microg cholesterol) was injected into the rectus femoris muscle of mice and the egress of cholesterol was studied as a function of time. Daily subcutaneous injection of dexamethasone (1.25 microg) raised plasma HDL levels by 40-80%. In mice injected with cat LDL labeled with 3H-cholesterol, daily treatment with dexamethasone slowed the loss of labeled cholesterol from the depot. With dexamethasone, there was no removal of the mass of lipoprotein cholesterol up to 14 days after injection of cat LDL, while in the controls 75% of the exogenous cholesterol mass had been cleared from the depot. When the cat LDL had been labeled with 3H-cholesteryl ester (3H-CE), apparent hydrolysis of 3H-CE amounted to 46, 75 and 97% in controls, but only to 20, 48 and 65% in dexamethasone treated mice on days 4, 8 and 14, respectively. In addition, dexamethasone stimulated cholesterol re-esterification as evidenced by recovery of 80% of the retained cholesterol mass as CE. In experiments with cultured macrophages exposed to modified LDL, dexamethasone increased the amount of labeled cholesteryl ester by 50-75% as compared to controls. Histological examination of the rectus femoris muscle after injection of cat LDL showed that in dexamethasone treated mice cellular infiltration was sparser on day 4, but not on day 8, and persisted longer than in controls. In conclusion, dexamethasone treatment impeded cholesterol egress from a lipoprotein depot by: a) reduction of early inflow of mononuclear cells; b) partial inhibition of cholesteryl ester hydrolysis, and c) enhancement of cholesterol esterification. The latter effect did not permit cholesterol egress from the injected site even in the presence of high plasma HDL in dexamethasone treated mice.

Delayed loss of cholesterol from a localized lipoprotein depot in apolipoprotein A-I-deficient mice.

The anti-atherogenic role of high density lipoprotein is well known even though the mechanism has not been established. In this study, we have used a novel model system to test whether removal of lipoprotein cholesterol from a localized depot will be affected by apolipoprotein A-I (apo A-I) deficiency. We compared the egress of cholesterol injected in the form of cationized low density lipoprotein into the rectus femoris muscle of apo A-I K-O and control mice. When the injected lipoprotein had been labeled with [3H]cholesterol, the t1/2 of labeled cholesterol loss from the muscle was about 4 days in controls and more than 7 days in apo A-I K-O mice. The loss of cholesterol mass had an initial slow (about 4 days) and a later more rapid component; after day 4, the disappearance curves for apo A-I K-O and controls began to diverge, and by day 7, the loss of injected cholesterol was significantly slower in apo A-I K-O than in controls. The injected lipoprotein cholesterol is about 70% in esterified form and undergoes hydrolysis, which by day 4 was similar in control and apo A-I K-O mice. The efflux potential of serum from control and apo A-I K-O mice was studied using media containing 2% native or delipidated serum. A significantly lower efflux of [3H]cholesterol from macrophages was found with native and delipidated serum from apo A-I K-O mice. In conclusion, these findings show that lack of apo A-I results in a delay in cholesterol loss from a localized depot in vivo and from macrophages in culture. These results provide support for the thesis that anti-atherogenicity of high density lipoprotein is related in part to its role in cholesterol removal.

Cholesterol efflux in vivo from a depot of cationized LDL injected into a thigh muscle of small rodents.

We have developed a model system to measure quantitatively removal of cholesterol from a well-defined depot in vivo. To that end, lipoproteins were injected into the rectus femoris muscle of small rodents, using a 25 microliters Hamilton syringe and a 27-gauge needle. In most experiments, the injected volume was 10 microliters containing 200 micrograms of cholesterol. The lipoproteins tested were native or modified LDL labeled with trace amounts of [3H]free cholesterol ([3H]FC). The amount of label or of cholesterol mass recovered at various time intervals after injection was normalized to that found after 10 min (designated time 0). In mice, the highest recovery of the [3H]cholesterol 24 h after injection was found with cationized LDL, and ranged between 78% and 84%, whereas retention of native LDL did not exceed 24%. Based on results of 9 experiments with cationized LDL, the loss of [3H]FC was mono-exponential between 1 and 14 days and the t1/2 was about 4 days. The disappearance curve of cholesterol mass showed an initial slow and a later more rapid component, the latter with a t1/2 of 4 days. The initial lag is most probably due to the presence of cholesteryl ester, which needs to be hydrolyzed prior to egress. This assumption was verified by injection of cat-LDL labeled with [3H]cholesteryl oleate and finding a similar lag as well as evidence of [3H]cholesteryl ester hydrolysis. Histological examination of the injected muscle 1-4 days after injection of cat LDL showed infiltration with mononuclear cells in an area limited to the site of injection. The presently described model system, which mimics to some extent events occurring during atherogenesis, permits quantitative evaluation of egress of deposited cholesterol and may allow to study the role of HDL in such a process.

Scavenger receptor activity and expression of apolipoprotein E mRNA in monocyte-derived macrophages of young and old healthy men.

The aim of this study was to compare some aspects of lipid metabolism in monocyte-derived macrophages isolated from young males, aged 18-24 years, and old males, aged 74-90 years, who were found healthy in accordance with the Senieur protocol. The parameters tested were metabolism of 125I-acetylated low-density lipoproteins (LDL) and oxidized LDL, incorporation of [3H]cholesterol into cholesteryl ester and expression of apolipoprotein E (apo E) mRNA. Cell association and degradation of 125I-acetylated LDL by macrophages of old and young subjects, respectively, was 15,978 +/- 2492 and 9300 +/- 1416 ng/mg cell protein per 24 h. Incorporation of [3H]cholesterol into cellular [3H]cholesteryl ester in the presence of acetylated LDL in cells isolated from old subjects was twice that in cells from young subjects. The macrophages from both age groups metabolized less 125I-oxidized LDL than 125I-acetylated LDL. Cell association and degradation of 125I-oxidized LDL in cells from old and young subjects, respectively, was 6779 +/- 1398 and 3219 +/- 643 ng/mg cell protein per 24 h. Expression of apo E mRNA was determined by reverse transcriptase polymerase chain reaction. In the basal state, it was 5.8 +/- 0.4 and 2.4 +/- 0.2 photo-stimulated luminescence (PSL) units in cells from the old and young subjects, respectively, and increased after exposure to acetylated LDL. In conclusion, these findings suggest that a combination of higher scavenger receptor activity and increased expression of apo E mRNA in macrophages could contribute to (a) enhanced metabolism of modified LDL and (b) more efficient removal of cholesterol from arteries, thus leading to healthy old age.

Effects of interactions of apolipoprotein A-II with apolipoproteins A-I or A-IV on [3H]cholesterol efflux and uptake in cell culture.

Conflicting evidence has accumulated with years regarding the putative negative effect of apolipoprotein A-II on apo A-I mediated cholesterol efflux. In this study, this question was reexamined and in addition to the interaction of apo A-II with apo A-I, its possible effect on apo E and apo A-IV was investigated as well. Free cholesterol (FC) donors were the main components of atheroma, namely, mouse peritoneal macrophages (MP), bovine aortic smooth muscle (SMC) and fibroblasts labeled with [3H]FC. Acceptors of FC were dioleoylphosphatidylcholine (DOPC) liposomes containing apo A-I, rh-apo A-IV or rh-apo E alone or together with apo A-II. When [3H]FC labeled MP were incubated for 2 or 4 h with equimolar concentrations of apo A-I, A-II, A-IV or E, the lowest [3H]cholesterol efflux occurred with apo A-II. Exposure of [3H]FC MP to liposomes containing apo A-I/A-II at 1:2 M/M (keeping the total protein concentration at 50 micrograms/ml), resulted in a lower [3H]FC efflux as compared to apo A-I alone. However, when apo A-I or apo A-IV protein concentration was kept constant and supplemented with apo A-II, a lower [3H]FC efflux was found only at 1:3 M/M of apo A-I/A-II. Apo A-II added to apo E had no effect on FC efflux. With aortic SMC and fibroblasts, no inhibitory effect of addition of apo A-II to apo A-I or apo A-IV on cholesterol efflux was seen at apo A-I/A-II of 1:1 or 1:2 M/M. The uptake of macrophage derived [3H]FC by SMC or HepG2 cells was studied using the serum-free efflux media, containing PC liposomes + apolipoproteins, from 3H-labeled macrophages. The cellular uptake of [3H]FC was higher when apo A-II had been added to apo A-I or apo A-IV than when the apolipoproteins were added alone. In conclusion, apo A-II was found to be less effective in cholesterol efflux and to interfere with the action of A-I only when the cholesterol donors were macrophages and when the relative amount of apo A-I to apo A-II was low. This was not the case when SMC or fibroblasts served as cholesterol donors. In the presence of apo A-II, enhanced [3H]cholesterol delivery to cells was seen which could contribute to the proatherogenic activity of apo A-II.

Effect of transforming growth factor-beta on lipoprotein lipase in rat mesenchymal heart cell cultures.

The effect of recombinant transforming growth factor-beta 2 (rTGF-beta 2) on lipoprotein lipase (LPL) synthesis was studied in mesenchymal rat heart cell cultures. Addition of rTGF-beta 2 to culture medium containing 20% serum resulted in a time-dependent decrease in LPL activity. With 10 ng/ml a 30% fall occurred after 12 h and only 20% of enzyme activity remained after 24 h with 5 or 10 ng/ml. The minimal effective dose of rTGF-beta 2 was 0.1 ng/ml and a 20% decrease occurred after exposure for 24 h. Antibodies specific to TGF-beta 2 blocked this effect. The decrease in enzymic activity was accompanied by a decrease in enzyme mass and LPL mRNA. Addition of rTGF-beta 2 was effective only during the first week in culture, when enzyme activity was increasing but not after 12 days when the cultures were overconfluent, and the enzyme activity was high.

Murine macrophages secrete factors that enhance uptake of non-lipoprotein [3H]cholesteryl ester by aortic smooth muscle cells.

We have recently demonstrated that macrophage conditioned medium (MP medium) and beta VLDL enhance cholesterol esterification in cultured aortic smooth muscle cells by LDL receptor mediated and other pathways (Stein, O. et al. (1993) Arteroscl. Thromb. 13, 1350-1358). In view of the presence of extracellular non-lipoprotein cholesteryl ester (in the form of lipid droplets) in the atheroma, the effect of MP medium on the cellular uptake of liposomal cholesteryl linoleyl ether (CLE) or cholesteryl ester (CE) was studied. After 4 h incubation in MP medium, the uptake of liposomal [3H]CLE was up to 10-fold higher than in the presence of control medium of the same composition but not conditioned with macrophages (DV medium). Similar results were seen also with HSF derived from LDL receptor negative donors. The MP medium-stimulated uptake of liposomal [3H]CE resulted also in hydrolysis of 70-90% of the labeled compound, indicating that the [3H]CE was intracellular. While the MP medium effect on liposomal [3H]CLE uptake was evident after 4 h, its effect on [3H]cholesterol esterification by SMC in the presence of beta VLDL could be demonstrated only after 24 h. Addition of apoE to MP medium resulted in a small (30-40%) increase in the uptake of liposomal [3H]CLE; however, it was augmented more than 4-fold when apoE was added to DV medium. The MP medium effect on the uptake of liposomal [3H]CLE was interfered with by heparin, anti-LPL antibody or heparinase, while these treatments did not affect [3H]cholesterol esterification in the presence of beta VLDL. These results suggest that the interaction between SMC and two potential sources of lipids in atheroma, i.e., lipoproteins and non-lipoprotein lipid droplets, could be governed by different components of the MP medium. In the case of the lipid droplets, as modeled here in the form of liposomes, macrophage-derived lipoprotein lipase could play a major role in cholesteryl ester transfer into SMC.

Macrophage-conditioned medium and beta-VLDLs enhance cholesterol esterification in SMCs and HSFs by LDL receptor-mediated and other pathways.

Thioglycolate-elicited mouse peritoneal macrophages were incubated for 24 hours in serum-free Dulbecco-Vogt medium containing 0.5% fatty acid-poor bovine serum albumin. This conditioned medium, designated MP medium, was used for experiments with bovine aortic smooth muscle cells (SMCs) or human skin fibroblasts (HSFs). Dulbecco-Vogt medium of the same albumin content but without macrophages served as a control medium. In SMCs labeled from plating the [3H]cholesterol and incubated with hypercholesterolemic rabbit beta-very-low-density lipoprotein (beta-VLDL) in Dulbecco-Vogt medium for 24 hours, there was an increase in cellular [3H]cholesteryl ester (CE) content compared with cells incubated without lipoprotein. When MP medium was used for the incubation of SMCs with beta-VLDL, cellular [3H]cholesteryl ester content increased threefold compared with cells incubated with Dulbecco-Vogt medium. A smaller increase in cholesterol esterification in the presence of MP medium was also encountered with low-density lipoprotein (LDL). The MP medium-induced increase in [3H]cholesterol esterification was not evident up to 6 hours of incubation. Similar results were also obtained with HSFs. The increase in [3H]cholesterol esterification with MP medium in the presence of beta-VLDL was also elicited in cells obtained from LDL receptor-negative donors with familial hypercholesterolemia (FH-HSF), even though in these cells significantly less [3H]cholesteryl ester was formed in the presence of beta-VLDL. MP medium contains numerous agents that could be responsible for the increase in cellular [3H]cholesteryl ester induced by lipoproteins. The first considered was lipoprotein lipase, but lack of inhibition of the MP medium effect by antiserum to lipoprotein lipase did not support this possibility.(ABSTRACT TRUNCATED AT 250 WORDS)

Can lipoprotein lipase be the culprit in cholesteryl ester accretion in smooth muscle cells in atheroma?

Bovine aortic smooth muscle cells and human skin fibroblasts were incubated with beta-very low density lipoprotein (beta VLDL) isolated from cholesterol-fed rabbits and labeled with [3H]cholesteryl oleate. Addition of lipoprotein lipase resulted in a 3.2-4.8-fold increase in cell associated radioactivity of which 45-61% was in free cholesterol, i.e., derived after intracellular hydrolysis. After exposure of smooth muscle cells to beta VLDL for up to 9 days and 60 min sodium heparin wash at 4 degrees C to remove extracellular surface bound lipoprotein, cellular cholesterol increase was 2 micrograms in controls and in the presence of lipoprotein lipase (LPL) it was tenfold higher. Addition of [3H]cholesteryl ester labeled beta VLDL during the last 48 h of incubation showed that 30-40% of total cellular label was in free cholesterol. This value represents the minimal cellular uptake of the added lipoprotein cholesteryl ester. Addition of recombinant apolipoprotein (apo) E to smooth muscle cells incubated with beta VLDL and [3H]oleate induced no further increase in [3H]cholesteryl oleate. We propose that following LPL-mediated binding of beta VLDL to heparan sulphate, this complex either undergoes endocytosis, or translocation of cholesteryl ester into the smooth muscle cells (SMC) occurs without endocytosis of the entire particle. The present results indicate that in the aortic wall macrophage-derived lipoprotein lipase could play a role in cholesteryl ester accretion in smooth muscle cells during atherogenesis.

Persistence of increased cholesteryl ester in human skin fibroblasts is caused by residual exogenous sphingomyelinase and is reversed by phospholipid liposomes.

Human skin fibroblasts (HSF) were exposed to sphingomyelinase 50 or 5 mU/ml for 60 min, washed with 5 mM EDTA or 20% serum and with phosphate-buffered saline, and postincubated for 24 h in the presence of [14C]16:0 sphingomyelin (SP) liposomes. The recovery of up to 48% of label in the medium in ceramide provided evidence of persistence of sphingomyelinase activity. The rate of hydrolysis of [14C]16:0 SP remained the same irrespective of whether the liposomes were added immediately after the wash, or 3 or 6 h thereafter. In HSF labeled with [3H]cholesterol exposure to 50 mU/ml of sphingomyelinase for 60 min resulted in an increase in labeled cholesteryl ester (CE) at 6 and 24 h of postincubation. Addition of sphingomyelin liposomes reduced markedly the fraction of cellular labeled cholesteryl ester recovered after 24 h, while phosphatidylcholine liposomes were not effective. When the enzyme concentration had been reduced 5-20 fold the effect of sphingomyelin liposomes on cellular 3H-CE was evident already after 6 h of postincubation and some effect was seen also with phosphatidylcholine liposomes. Increase in the concentrations of SP liposomes to 150 micrograms/ml restored labeled cholesteryl ester to control values at 24 h. A significant reduction occurred also with 18:1 phosphatidylcholine liposomes but labeled cholesteryl ester remained 50-100% higher when compared with 18:1 or 18:2 SP. No correlation was seen between the rates of cholesteryl ester decrease and free cholesterol efflux into the medium. The inability to remove residual sphingomyelinase by regular washing procedures exaggerates and prolongs the recovery period of sphingomyelin during postincubation and delays the return of the cholesteryl ester pool to control levels. This can be counteracted by addition of phospholipid liposomes that can compete for the enzyme with the plasma membrane sphingomyelin and also substitute the hydrolyzed molecule in the plasma membrane to impede cholesterol flow to cell interior.

Regulation of lipoprotein lipase by dibutyryl cAMP, cholera toxin, Hepes and heparin in F1 heart-cell cultures.

Regulation of lipoprotein lipase was studied in mesenchymal rat heart-cell cultures. Treatment of the cultures with dibutyryl cyclic AMP or with cholera toxin resulted in an increase in LPL activity and a comparable increase in LPL mRNA. When the cells were exposed to 100 mM Hepes for 24 h, total enzyme activity rose 2-fold and LPL mRNA increased 2.4-fold. After 72 h, there was a 3-fold increase in LPL mRNA and a 4-fold rise in cellular LPL activity, while medium activity increased 20-fold. Exposure of the cultures to heparin for 24 h resulted in a 3.2-fold increase in total activity and a 36-fold increase in medium activity. This increase was not accompanied by any rise in LPL mRNA. Addition of actinomycin D to control dishes for 24 h resulted in a 33% reduction in LPL mRNA and a 43% reduction in enzyme activity. These values were 71% and 56%, respectively, in Hepes-treated cells, indicating that no stabilization of LPL mRNA occurred under these conditions. It can be concluded that in mesenchymal rat heart-cells in culture cAMP and cholera toxin upregulate lipoprotein lipase at the level of transcription. The increase in LPL activity after 24 h exposure to Hepes could be compatible with transcriptional regulation, while exposure to heparin is not accompanied by a change in LPL mRNA.

Modulation of sphingomyelinase-induced cholesterol esterification in fibroblasts, CaCo2 cells, macrophages and smooth muscle cells.

The present study has focused on three questions concerning the effect of sphingomyelinase on release of free cholesterol from the plasma membrane and its intracellular translocation: (i) Can one change the direction of the flow of cholesterol? (ii) Can one modulate the flow? (iii) May such a mechanism be relevant in atherogenesis? (i) The results obtained show that even in the presence of potent nonlipoprotein cholesterol acceptors in the medium, the intracellular flow of cholesterol is not reduced as measured by cholesterol esterification. Moreover, in sphingomyelinase-treated cells, cholesterol efflux in presence of nonlipoprotein acceptors was not enhanced even when intracellular esterification was inhibited. (ii) Modulation of the sphingomyelinase induced cholesterol flow can be obtained by 100 microM verapamil which reduces it. In human skin fibroblast, interference with the delivery of free cholesterol to its site of esterification was found in the presence of brefeldin A. (iii) Aortic smooth muscle cells in culture are sensitive to low concentrations of sphingomyelinase and the increase in esterified cholesterol is evident also after exposure to the enzyme for 24 h. The present results suggest that in the plasma membrane, free cholesterol bound to sphingomyelin may be in a compartment which renders it more available for transport to the cell interior than for efflux. In view of the sensitivity of aortic smooth muscle cells to sphingomyelinase, this mechanism for enhanced esterification of cholesterol could be relevant to the transformation of arterial smooth muscle cells into foam cells in the process of atherogenesis.

Dissimilar effects of Brefeldin A on cholesteryl ester and triacylglycerol metabolism in CaCo2 and HepG2 cells as compared to peritoneal macrophages.

The effect of Brefeldin A (BFA) on lipid metabolism was studied in two cell lines and in primary cultures of peritoneal macrophages. In both CaCo2 and HepG2 cells, which are models for human liver and intestine, addition of BFA resulted in a 2-10-fold increase in recovery of labeled cholesteryl ester when the cells had been prelabeled with free cholesterol or with [3H]oleic acid. This effect was linear for up to 6 h and could be elicited with doses of BFA as low as 0.03 micrograms/ml. The increase in cholesteryl ester induced by BFA was completely abolished by the ACAT inhibitor (Sandoz 58-035) and partly by forskolin. Intracellular hydrolysis of labeled cholesteryl ester was studied in the presence of the ACAT inhibitor and while in the controls 30-40% was hydrolyzed in 6 h, the values were 7-16% in the BFA treated cells. The slower rate of hydrolysis in the BFA treated cells could not account for the entire increase of cholesteryl ester and there was also no decrease in cholesteryl ester secretion. Even though activation of acyl-CoA:cholesterol acyltransferase by BFA was not demonstrated in cell homogenates, we hypothesize that in the intact cell the BFA induced increase in cholesteryl ester might have been related to the pronounced increase in modified endoplasmic reticulum which results from the dispersion of the Golgi apparatus. In the macrophages, BFA at doses of 0.25-1 micrograms/ml resulted in a 90% reduction in the incorporation of [3H]oleic acid into triacyglycerol. Incorporation of [3H]oleic acid into triacyglycerol in CaCo2 cells was not affected by BFA. In view of the ever-increasing use of BFA in cell biology, it seems important to emphasize that BFA may affect different pathways of lipid metabolism in various cells.