Cholesteryl Ester Synthesis In Macrophages Research Articles

Inhibiting low-density lipoprotein glycation ameliorates increased cholesteryl ester synthesis in macrophages and hypercholesterolemia and aortic lipid peroxidation in streptozotocin diabetic rats

Increased nonenzymatic glycation of apolipoprotein (apo) B–containing lipoproteins impairs uptake and metabolism by the high-affinity low-density lipoprotein receptor and is one of the postsecretory modifications contributory to accelerated atherosclerosis in diabetes. The present study evaluated in vitro and in vivo effects of 2,2-chlorophenylaminophenylacetate to probe the influence of glycated lipoprotein on cholesterol homeostasis. This compound prevented the increased formation of glycated products in low-density lipoprotein incubated with 200 mmol/L glucose and the increased cholesteryl ester synthesis in THP-1 macrophages induced by apo B–containing lipoproteins preincubated with high glucose concentration. The elevated circulating concentrations of glycated lipoprotein and cholesterol and higher vascular levels of lipid peroxidation products observed in streptozotocin diabetic rats compared with nondiabetic controls were significantly reduced in diabetic animals treated for 6 months with test compound. These results are the first to demonstrate that inhibiting nonenzymatic glycation of apo B–containing lipoproteins ameliorates abnormalities contributory to hypercholesterolemia and atherogenic risk in diabetes.

Discovery and Combinatorial Synthesis of Fungal Metabolites Beauveriolides, Novel Antiatherosclerotic Agents

For discovery of a new type of antiatherosclerotic agents, a cell-based assay of lipid droplet accumulation using primary mouse peritoneal macrophages was conducted as a model of macrophage-derived foam cell accumulation, which occurs in the early stage of atherosclerogenesis. During the screening of microbial metabolites for inhibitors of lipid droplet accumulation, 13-membered cyclodepsipeptides, known beauveriolide I and new beauveriolide III, were isolated from the culture broth of fungal Beauveria sp. FO-6979, a soil isolate, by solvent extraction, ODS column chromatography, silica gel column chromatography, and preparative HPLC. The structure including the absolute stereochemistry of beauveriolide III was elucidated as cyclo-[(3 S,4 S)-3-hydroxy-4-methyloctanoyl- l-phenylalanyl- l-alanyl- d-alloisoleucyl] by spectral analyses, amino acid analyses, and synthetic methods. Furthermore, the absolute stereochemistry was confirmed by the total synthesis of beauveriolides. Study on the mechanism of action revealed that beauveriolides inhibited macrophage acyl-CoA:cholesterol acyltransferase (ACAT) activity to block the synthesis of cholesteryl ester (CE), leading to a reduction of lipid droplets in macrophages. There are two ACAT isozymes in mammals, ACAT1 and ACAT2. ACAT1 is ubiquitously expressed in most tissues and cells including macrophages, while ACAT2 is expressed predominantly in the liver (hepatocytes) and the intestine (enterocytes). Interestingly, beauveriolides inhibited both ACAT1 and ACAT2 to a similar extent in an enzyme assay that utilized microsomes but inhibited ACAT1 selectively in intact cell-based assays. Beauveriolides proved orally active in both low-density lipoprotein receptor and apolipoprotein E knockout mice, reducing the atheroma lesion of heart and aorta without any side effects such as diarrhea or cytotoxicity to adrenal tissues as observed for many synthetic ACAT inhibitors. To obtain more potent inhibitors, a focused library of beauveriolide analogues was prepared by combinatorial chemistry in which solid-phase assembly of linear depsipeptides was carried out using a 2-chlorotrityl linker, followed by solution-phase cyclization, yielding 104 beauveriolide analogues. Among them, diphenyl derivatives were found to show 10 times more potent inhibition of CE synthesis in macrophages than beauveriolide III. Furthermore, most analogues showed selective ACAT1 inhibition or inhibition of both ACAT1 and ACAT2, but interestingly certain analogues gave selective ACAT2 inhibition. These data indicated that subtle structural differences of the inhibitors could discriminate the active sites of the ACAT1 and ACAT2 isozymes. Efforts of further analogue synthesis would make it possible to obtain highly selective ACAT1/ACAT2 inhibitors.

Inhibition of Lipid Droplet Accumulation in Macrophages by Triterpenoids Produced from Torametes orientaris

Four triterpenoids 1-4 isolated from the fruit body of Torametes orientaris were found to inhibit lipid droplet accumulation in macrophages. From the biochemical analysis, compounds 2 and 3 inhibited selectively cholesteryl ester synthesis in macrophages, while compounds 1 and 4 showed inhibition of both cholesteryl ester and triacylglycerol syntheses.

New Beauveriolides Produced by Amino Acid‐Supplemented Fermentation of Beauveria sp. FO‐6979

AbstractFor Abstract see ChemInform Abstract in Full Text.

Bew Beauveriolides Produced by Amino Acid-supplemented Fermentation of Beauveria sp. FO-6979.

Five new beauveriolides were isolated from the acetone extracts of Beauveria sp. FO-6979 mycelia fermented in amino acid-supplemented media. The structures were elucidated by spectroscopic studies including NMR experiments and chemical degradation. All the beauveriolides are cyclodepsipeptides consisting of one 3-hydroxy-4-methyl fatty acid, two L-amino acids and one D-amino acid in common. Beauveriolide VII with the structure of cyclo-[3-hydroxy-4-methyloctanoyl-L-phenylalanyl-L-alanyl-D-valyl] inhibited lipid droplet formation and cholesteryl ester synthesis in macrophages, but the other beauveriolides showed only slight or almost no effect on lipid droplet formation.

Role of estrogen in the regulation of cholesteryl ester synthesis in macrophages: the interaction between native and modified low density lipoprotein and human monocyte-derived macrophages

Objectives: To study the effects of estrogen on the changes in cholesterol esterification induced by native and modified low density lipoprotein (LDL) in macrophages. Design and methods: Human monocyte-derived macrophages (HMDM) were used, and the influence of the presence of 17β estradiol in the short term, and during the maturation of the cells, on the esterification of cholesterol from native (nLDL), acetylated (acLDL) and oxidized (oxLDL) LDL was determined. Results: In the short-term (6 h), 17β estradiol (1.5 × 10 −6M) did not affect the esterification of cholesterol from acLDL or oxLDL, but with native LDL (nLDL) a 1.5-fold increase was observed. In contrast, long-term exposure of HMDM during maturation to 17β-estradiol (1.5 × 10 −9M - 1.5 × 10 −5M) decreased cholesterol esterification in the presence of oxLDL and acLDL, but not nLDL. Conclusions: These results suggest that both the time of exposure and the concentration of estrogen used influence its effects on the interaction between HMDM and LDL, and thus on macrophage foam cell formation.

Copper-Modified Low Density Lipoprotein from Diabetic Patients Enhances Cholesteryl Ester Synthesis by Mouse Peritoneal Macrophages.

In this study, the capability of superoxide production by LDL either without stimulation or under the stimulation of Fe-ADP and the manner of modification of low density lipoprotein (LDL) by copper (Cu++) in diabetic patients were examined and compared with those of healthy subjects. The amount of superoxide generated by diabetic LDL was similar to that of healthy subjects both at the basal level and following stimulation by Fe-ADP. LDL from healthy subjects and diabetic patients was modified in the presence of 10μM CuSO4. When the level of thiobarbituric acid-reactive substance was determined in the Cu++-modified LDL, no difference was found. On the other hand, when the incorporation of [14C]oleate into cholesteryl ester (CE) by mouse peritoneal macrophages was stimulated by the Cu++-modified LDL, we found that the modified diabetic LDL significantly enhanced the CE synthesis compared with the modified healthy LDL (p<0.01). To clarify the mechanism of the enhanced CE synthesis, the rate of degradation of [125I]Cu++-modified diabetic LDL was measured. We found that this rate was similar to that of healthy subjects. These data suggest that the degree of oxidation does not differ among patients although diabetic LDL is modified by Cu++ in a manner such that it preferentially promotes the activity of CE synthesis in macrophages by some unknown intracellular metabolic mechanism.

Interaction of very-low-density lipoprotein isolated from type I (insulin-dependent) diabetic subjects with human monocyte-derived macrophages

Very-low-density lipoproteins (VLDL) (density < 1.006 g/mL) were isolated from type I (insulin-dependent) diabetic patients in good to fair glycemic control and from age-, sex-, and race-matched, nondiabetic, control subjects. VLDL were incubated with human, monocyte-derived macrophages obtained from nondiabetic donors, and the rates of cellular cholesteryl ester synthesis and cholesterol accumulation were determined. VLDL isolated from diabetic patients stimulated significantly more cholesteryl ester synthesis than did VLDL isolated from control subjects (4.04 ± 1.01 v 1.99 ± 0.39 nmol 14C-cholesteryl oleate synthetized/mg cell protein/20 h; mean ± SEM, P < .05). The stimulation of cholesteryl ester synthesis in macrophages incubated with VLDL isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation ( P < .05). The increases in cholesteryl ester synthesis and accumulation in macrophages were mediated by a significant increase in the receptor mediated, high affinity degradation (2.55 ± 0.23 v 2.12 ± 0.20 μg degraded/mg cell protein/20 h) and accumulation (283 ± 35 v 242 ± 33 ng/mg cell protein/20 h) of 125I-VLDL isolated from diabetic patients compared with VLDL from control subjects. To determine if changes in VLDL apoprotein composition were responsible for the observed changes in cellular rates of cholesteryl ester synthesis and accumulation, we also examined the apoprotein composition of the VLDL from both groups. There were no significant differences between the apoproteins B, E, and C content of VLDL from both groups. We also determined the chemical composition of VLDL isolated from both groups of subjects. The free cholesterol content of VLDL isolated from diabetic patients increased significantly and, thus, also contributed to the increased rates of cellular cholesteryl ester synthesis and accumulation. In conclusion, we have shown that VLDL isolated from Type I diabetic patients interact abnormally with human macrophages, and this altered interaction may contribute to the increased prevalence of atherosclerosis in diabetes.

Glycosylation of low-density lipoprotein enhances cholesteryl ester synthesis in human monocyte-derived macrophages.

Glucose can react with the lysine residues of low-density lipoproteins (LDLs) and convert the lipoprotein to a form with a receptor-mediated uptake by cultured cells that is impaired. However, in contrast to other modified lipoproteins taken up by both murine and human macrophages via the scavenger-receptor pathway that may induce the formation of foam cells, glycosylated LDL is not recognized by murine macrophages, and thus far, it has not been shown to lead to marked intracellular accumulation of cholesterol in human macrophages. This study illustrates that glycosylated LDL incubated with human monocyte-derived macrophages, at a concentration of 100 micrograms LDL/ml medium, stimulates significantly more cholesteryl ester (CE) synthesis than does control LDL (10.65 +/- 1.5 vs. 4.8 +/- 0.13 nmol.mg-1 cell protein.20 h-1; P less than .05). At LDL concentrations similar to those of plasma, the rate of CE synthesis in macrophages incubated with glycosylated LDL is more markedly enhanced than that observed in cells incubated with control LDL (3-fold increase). The marked stimulation of CE synthesis in human macrophages exposed to glycosylated LDL is paralleled by a significant increase in CE accumulation in these cells (P less than .001). The increase in CE synthesis and accumulation seem to be mediated by an increase in the degradation of glycosylated LDL by human macrophages. Glycosylated LDL enters the macrophages and is degraded by the classic LDL-receptor pathway in slightly smaller amounts than control LDL, but its degradation by pathways other than the classic LDL receptor or scavenger receptor is markedly enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of cholesteryl ester synthesis in human monocyte-derived macrophages by low-density lipoproteins from type 1 (insulin-dependent) diabetic patients: the influence of non-enzymatic glycosylation of low-density lipoproteins.

Diabetes mellitus is an independent risk factor in the development of atherosclerosis. In this study we aimed to demonstrate whether there is an abnormal interaction between low-density lipoproteins from diabetic patients and human macrophages. We measured cholesteryl ester synthesis and cholesteryl ester accumulation in human monocyte-derived macrophages (obtained from non-diabetic donors) incubated with low density lipoproteins from Type 1 (insulin-dependent) diabetic patients in good or fair glycaemic control. Low density lipoproteins from the diabetic patients stimulated more cholesteryl ester synthesis than low density lipoproteins from non-diabetic control subjects (7.19 +/- 1.19 vs 6.11 +/- 0.94 nmol/mg cell protein/20 h, mean +/- SEM, p less than 0.05). The stimulation of cholesteryl ester synthesis by low density lipoproteins isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (p less than 0.02). There were no significant differences in the lipid composition of low density lipoproteins between the diabetic and control groups. Non-enzymatic glycosylation of low density lipoproteins was higher in the diabetic group (p less than 0.01) and correlated significantly with cholesteryl ester synthesis (r = 0.58). Similarly, low-density lipoproteins obtained from non-diabetic subjects and glycosylated in vitro stimulated more cholesteryl ester synthesis in macrophages than control low density lipoproteins. The increase in cholesteryl ester synthesis and accumulation by cells exposed to low density lipoproteins from diabetic patients seems to be mediated by an increased uptake of these lipoproteins by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein E is the determinant that mediates the receptor uptake of beta-very low density lipoproteins by mouse macrophages.

Beta very low density lipoproteins (beta-VLDL) from cholesterol-fed animals from patients with Type III hyperlipoproteinemia are internalized by a receptor-mediated process in mouse macrophages. Once internalized, the cholesteryl esters of beta-VLDL are hydrolyzed in lysosomes, and the released cholesterol is re-esterified, resulting in a massive accumulation of cholesteryl esters. In the present study, competitive binding experiments demonstrated that canine apo E HDLc (lipoproteins that contain almost exclusively apolipoprotein E) inhibited the receptor-mediated uptake of 125I-beta-VLDL. The incorporation of human apo E into beta-VLDL was also shown to modulate binding. Reductively methylated beta-VLDL (methyl beta-VLDL) were not taken up by macrophages and did not stimulate cholesteryl ester synthesis. When unmodified human apo E-3 was incorporated into the lipoprotein in place of the canine methyl apo E, these hybrid beta-VLDL (methyl beta-VLDL [E-3]) were internalized and degraded and were as effective as native beta-VLDL in stimulating cholesteryl ester synthesis in macrophages. In the reverse experiment, the incorporation of methyl apo E-3 into native canine beta-VLDL (beta-VLDL [methyl E-3]) reduced the binding activity of the beta-VLDL and abolished their ability to stimulate cellular cholesteryl ester synthesis. Canine beta-VLDL into which apo E-2(Arg158----Cys) had been incorporated had less ability to stimulate cholesteryl ester synthesis (20%) than did native beta-VLDL, but they were more active than beta-VLDL [methyl E-2] or beta-VLDL [methyl E-3], which had virtually no activity. These results demonstrate that apo E is the determinant mediating the receptor binding and uptake of beta-VLDL by mouse macrophages.

Stimulation of cholesteryl ester synthesis in macrophages by extracts of atherosclerotic human aortas and complexes of albumin/cholesteryl esters.

Cholesteryl ester-rich particles extracted from human atherosclerotic plaques were shown to increase the rate of incorporation of [14C]oleate into cholesteryl [14C]oleate and to cause massive accumulation of cholesteryl esters in monolayers of mouse peritoneal macrophages. This stimulation showed saturation kinetics and susceptibility to competition by polyanions (polyinosinic acid, fucoidin, dextran sulfate), suggesting that cell surface binding was required. Cellular uptake and lysosomal hydrolysis of the cholesteryl esters were also required, as indicated by the finding that stimulation of cholesteryl ester formation was prevented by the lysosomal inhibitor, chloroquine. The cholesterol esterification-stimulating activity of the aortic extracts was excluded on a 2% agarose column and floated in the density range of 1.006 to 1.063 g/ml. Cholesterol-rich extracts from human adrenal glands and liver did not stimulate cholesteryl ester formation in macrophages. The aortic extracts did not stimulate cholesteryl ester synthesis in human fibroblasts. Complexes of 125I-labeled albumin and cholesteryl linoleate formed in vitro were taken up and degraded in macrophages, but not in fibroblasts, by a process resembling the uptake of the aortic extracts. The current data suggest that macrophages express mechanisms for internalizing certain types of cholesteryl ester-rich lipid/protein complexes, including those present in atherosclerotic plaques.

Cholesteryl ester synthesis in macrophages: stimulation by beta-very low density lipoproteins from cholesterol-fed animals of several species

Animals fed cholesterol accumulate several types of cholesterol-rich lipoproteins in their plasma and ultimately develop cholesteryl ester deposition in tissue macrophages. Previous studies in the cholesterol-fed dog have shown that one class of cholesterol-rich lipoproteins. beta-migrating very low density lipoproteins (beta-VLDL, density < 1.006 g/ml), possesses a unique ability to produce cellular cholesteryl ester accumulation when incubated with mouse peritoneal macrophages in vitro. This accumulation results from the receptor-mediated uptake of beta-VLDL with subsequent lysosomal hydrolysis of the lipoprotein and re-esterification of the liberated cholesterol. In the current studies, we demonstrate that beta-VLDL obtained from cholesterol-fed animals of several other species, including monkeys, rabbits, and rats, also causes cholesteryl ester accumulation in monolayers of mouse peritoneal macrophages, as monitored by an increase in the rate at which the cells incorporate exogenous [14C]oleate into cholesteryl [14C]oleate. Like canine beta-VLDL, the beta-VLDL from these three other species were effective at low concentrations and exhibited saturation kinetics, suggesting that they, too, entered macrophages by receptor-mediated endocytosis. Very low density lipoprotein (VLDL) from normal animals and low density lipoprotein (LDL) from normal and cholesterol-fed monkeys, rats, and rabbits did not stimulate cholesteryl ester synthesis in mouse peritoneal macrophages. In addition to their effects on mouse macrophages, the beta-VLDL from cholesterol-fed dogs and rabbits stimulated cholesteryl ester synthesis in cultured human monocytes. The current findings suggest that beta-VLDL from cholesterol-fed animals has the general property of stimulating cholesteryl ester synthesis and accumulation in macrophages.