G. Hollander

Metabolism of HDL-cholesteryl ester in the rat, studied with a nonhydrolyzable analog, cholesteryl linoleyl ether

Intralipid was sonicated with [3H]cholesteryl linoleyl ether (a nonhydrolyzable analog of cholesteryl linoleate) and incubated with rat HDL and d greater than 1.21 fraction of rabbit serum at a ratio of 0.012 mg triacylglycerol to 1 mg HDL protein. 25% of [3H]cholesteryl linoleyl ether was transferred to HDL. The labeled HDL was injected into donor rats and was screened for 4 h. [125I]HDL was subjected to the same protocol as the 3H-labeled HDL, including screening. The screened, labeled sera were injected into acceptor rats and the disappearance of radioactivity from the circulation was compared. The t1/2 in the circulation of [125I]HDL was about 10.5 h, while that of [3H]cholesteryl linoleyl ether-HDL was about 8 h. The liver and carcass were the major sites of uptake of [3H]cholesteryl linoleyl ether-HDL and accounted for 29-41% (liver) and 30% (carcass) of the injected label. Maximal recovery of [3H]cholesteryl linoleyl ether in the liver was seen 48 h after injection, and thereafter there was a progressive decline of radioactivity, which reached 7.8% after 28 days. The maximal recovery of [125I]HDL in the liver was about 9%. Pretreatment of the acceptor rats with estradiol for 5 days resulted in a 20% increase in the hepatic uptake of [3H]cholesteryl linoleyl ether-HDL and a 5-fold increase in adrenal uptake. The present findings indicate that in the rat the liver is the major site of uptake of HDL cholesteryl ester and that part of the HDL cholesteryl ester may be cleared from the circulation separately from the protein moiety. On the basis of our previous findings (Stein, Y., Kleinman Y, Halperin, G., and Stein, O. (1983) Biochim. Biophys. Acta 750, 300-305) the loss of the [3H]cholesteryl linoleyl ether from the liver after 14-28 days was interpreted to indicate that the labeled [3H]cholesteryl linoleyl ether had been taken up by hepatocytes.

Cholesteryl ester transfer activity in hamster plasma: increase by fat and cholesterol rich diets

We investigated the presence of cholesteryl ester transfer activity (CETA) in plasma of hamsters kept on various dietary regimens. In hamsters kept on a regular diet, CETA activity was about 5 units/4 mg protein of d greater than 1.21 g/ml fraction of plasma, as compared to about 35 units present in human d greater than 1.21 g/ml fraction. Addition of 15% margarine or butter alone or together with 2% cholesterol resulted in a 2-3-fold increase in plasma CETA. The increase in plasma CETA was correlated with plasma cholesterol levels (r = 0.78; P less than 0.001) and plasma triacylglycerol levels (r = 0.56, P less than 0.001). Hamsters consuming the cholesterol + butter-supplemented diets had the highest plasma CETA, cholesterol and triacylglycerol levels, while CETA in plasma of rats and mice remained nondetectable even after 4 weeks on the diet. The causal relation between hypercholesterolemia, hypertriglyceridemia and evaluation in CETA in hamsters remains to be elucidated.

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.

Relative resistance of the hamster to aortic atherosclerosis in spite of prolonged vitamin E deficiency and dietary hypercholesterolemia. Putative effect of increased HDL

UNLABELLED Male golden hamsters were rendered hypercholesterolemic by feeding diets enriched with cholesterol and fat. In the first series of experiments, 5% butter and 1% cholesterol were added to a chow diet and plasma cholesterol levels were maintained at 350-390 mg/dl over the entire experimental period. Groups of hamsters and their age controls consuming the chow diet, were killed after 7, 15 and 20 months when the aorta was examined for atherosclerosis by determination of cholesterol mass. In the controls, aortic total cholesterol (TC) increased with age by 28% and esterified cholesterol increased to 11% of TC. In the hypercholesterolemic animals aortic TC was only 28% higher than in the controls and cholesteryl ester was also 11.5% of TC. In the second series, one group of hamsters were fed a semi-purified diet deficient in vitamin E, containing 1% cholesterol and 10% lard; a second group received the same diet, but supplemented with vitamin E. Controls consumed local chow. After 7 months on the vitamin E deficient diet plasma alpha-tocopherol was 0.05 mg/l, in those supplemented with vitamin E it was 20 mg/l, while in the controls it was 3.3 mg/l. Plasma thiobarbituric acid reactive substances (TBARS) were higher in the vitamin E deficient group and there was a greater propensity of lipoproteins (d < 1.063 g/ml) to peroxidation in vitro than in the vitamin E supplemented group. Plasma cholesterol was 366 mg/dl in the vitamin E deficient, 336 mg/dl in the vitamin E supplemented group, and 64 mg/dl in controls. Aortic cholesterol was 79.1 in vitamin E supplemented and 84.4 micrograms/10 mg dry weight in vitamin E deficient hamsters. In both series of experiments, HDL amounted to 36-41% of plasma TC in the hypercholesterolemic animals and 59-62% in the controls. IN CONCLUSION the hamster appears to be quite resistant to atherosclerosis in face of sustained hypercholesterolemia, even in the presence of increased peroxidative stress caused by vitamin E deficiency. This relative resistance could be related to commensurate increase in plasma HDL which was observed in both series of experiments. Since vitamin E deficiency did not enhance aortic cholesteryl ester deposition, the protective effect of HDL seems to be related to its role in reverse cholesterol transport, rather than in prevention of peroxidation.

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.

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.

Divergent fate of unsaturated and saturated ceramides and sphingomyelins in rat liver and cells in culture

The metabolism of sphingomyelins and ceramides with defined labeled fatty acids was compared after injection in vivo or incubation with cultured cells. The liver was the major site of uptake of sphingomyelins and ceramides with 18:2 or 16:0 fatty acids, but with both sphingolipids a higher recovery of radioactivity was found with 16:0 species. The distribution of radioactivity among liver lipids showed that 1.5 h after injection of 18:2 sphingomyelin, only 21% of the label was found as sphingomyelin, and this value was 37% in the case of 16:0 sphingomyelin. There was a very marked difference in the metabolism of 18:2 and 16:0 ceramides. After injection of 18:2 ceramide only 14% of the radioactivity was recovered as sphingomyelin, and this value was more than 50% with 16:0 ceramide. [14C]18:2 ceramide was converted also to glucoceramide and hydrolyzed more extensively than 16:0 ceramide. These observations were extended to sphingomyelins and ceramides with other fatty acids, using Hep-G2 cells in culture. Significantly more radioactivity was recovered as labeled sphingomyelin after incubation with 16:0, 18:0, 20:0 and 24:0 sphingomyelins than with 18:1 and 18:2 sphingomyelins, while more labeled phosphatidylcholine and phosphatidylethanolamine were found with the unsaturated sphingomyelins. In analogy to the findings in vivo, in the Hep-G2 cells more 16:0, 18:0 and 24:0 ceramides were converted to sphingomyelin than 18:1 or 18:2 ceramides. These differences were also seen with cultured macrophages, in which a more marked reutilization for sphingomyelin formation was found with the saturated ceramide series. The sphingomyelin liposomes were tested also for their capacity to mobilize cholesterol, and a rise in plasma unesterified cholesterol occurred after injection of 18:2 sphingomyelin. Marked enhancement of cholesterol efflux from cholesterol ester-loaded macrophages was also seen with 18:1 and 18:2, 20:0 sphingomyelin in the presence of delipidated high-density lipoprotein. The present results demonstrate that the metabolic fate of sphingolipids is related to their fatty acid composition. While ceramides with saturated fatty acids are predominantly reutilized for sphingomyelin formation, those with unsaturated fatty acids undergo probably more rapid hydrolysis with liberation of fatty acids and channeling into glycerolipids.

Cholesterol removal by peritoneal lavage with phospholipid-HDL apoprotein mixtures in hypercholesterolemic hamsters

Syrian hamsters were rendered hypercholesterolemic by supplementation of their diet with 1% cholesterol and 15% butter. The hamsters were injected intraperitoneally (i.p.) with about 20 mg of phospholipid liposomes containing trace amounts of [3H]cholesteryl linoleyl ether ([ 3H]CLE) alone or combined with 10 mg delipidated high-density lipoprotein (apoHDL). After 2 h the peritoneal cavity was washed repeatedly with up to 15 ml phosphate-buffered saline. 60%-70% of [3H]CLE were retained after i.p. injection without apoHDL, 30-50% in the presence of apoHDL. The amount of free cholesterol recovered in the peritoneal lavage was significantly higher when apoHDL was combined with 18:2 sphingomyelin or dilinoleyl phosphatidylcholine liposomes, when compared to either liposomes or apoHDL alone. It is suggested that supplementation of dialysate with HDL apolipoproteins and phospholipids in patients undergoing continuous peritoneal dialysis could be of use in a cholesterol depletion regimen.

Metabolism of 3-[3H]sphingosine sphingomyelin labeled with [14C]palmitic or [14C]linoleic acid by Hep G2 cells and rat liver in vivo

The metabolism of sphingomyelin labeled with 3-[3H]sphingosine and [14C]16:0 or [14C]18:2 fatty acid was studied in cultured Hep G2 cells or macrophages and after injection into rats. In pulse-chase experiments, the loss of 3H and 14C-label was more rapid when the cells had been pulsed with 18:2 than with 16:0 sphingomyelin. At the end of 24 h chase, the labeled ceramide contained more [14C]18:2 fatty acid than [14C]16:0. In addition, the 3H-label derived from 3-[3H]sphingomyelin was recovered also as free sphingosine. After injection in vivo, more [3H]sphingosine-labeled sphingomyelin was present in the liver 3 and 24 h after injection of 16:0 than after injection of 18:2 sphingomyelin. The ratio of [3H]ceramide derived from 16:0 sphingomyelin to that derived from 18:2 sphingomyelin as percent of injected dose was 1.84 3 h after injection and 1.31 after 24 h. The ratio of 3H/14C in liver ceramide was 6.4 3 h after injection of 18:2 sphingomyelin and 3.4 after 16:0 sphingomyelin. The present results show that 3-[3H]sphingomyelin is metabolized quite extensively and that the fate of the sphingosine moiety is related to the type of fatty acid present in the phospholipid. These findings indicate that there is little or no reutilization of 18:2 ceramide for sphingomyelin formation and suggest that sphingosine derived from 18:2 sphingomyelin is channeled primarily for catabolism.

Removal of cholesteryl ester from hepatic reticuloendothelial cells in vivo is not enhanced by plasma cholesteryl ester transfer protein

The putative role of cholesteryl ester transfer protein (CETP) in the removal of cholesteryl ester from hepatic reticuloendothelial cells in vivo was studied in hamsters. The parameter tested was retention of [3H]cholesteryl linoleyl ether ([3H]CLE), a nonhydrolysable analog of cholesteryl ester, in the liver after injection of [3H]CLE labeled acetylated LDL, which is targetted to nonparenchymatous littoral cells. In hamsters fed laboratory chow, plasma cholesteryl ester transfer activity (CETA) was 10.6 +/- 0.9 units and the retention of [3H]CLE in the liver 28 days after injection was 86% of the 4 h value. It was about 55% in rats fed the same diet, in which CETA was not detectable. When the diet was supplemented with 2% cholesterol and 15% margarine, CETA activity in hamsters increased 2-fold, yet no change in retention of [3H]CLE in liver was seen after 28 days. In rats, the retention of [3H]CLE in the liver was also not changed by the dietary fat supplementation. These results do not support the role of CETP in vivo in removal of cholesteryl ester from intact reticuloendothelial cells.