Differential effects of isolated lipoproteins from normal and hypercholesterolemic rhesus monkeys on cholesterol esterification and accumulation in arterial smooth muscle cells in culture.

Whole serum obtained from hypercholesterolemic rhesus monkeys was found to stimulate cholesterol esterification and cholesteryl ester accumulation in rhesus monkey arterial smooth muscle cells in culture to a significantly greater extent than normocholesterolemic serum. This was true even when the cholesterol concentration of the culture medium was equalized. Isolation and characterzation of the low density lipoproteins (LDL) from rhesus monkeys indicated that the LDL from hypercholesterolemic animals was 33% larger than LDL from normocholesterolemic animals due principally to an increase in the amount of cholesteryl ester per molecule. As a result, LDL from hypercholesterolemic animals transported over 50% more cholesterol per molecule than did normal LDL. The LDL of altered composition from hypercholesterolemic animals, when added to smooth muscle cells in culture, was nearly twice as effective in stimulating cholesterol esterification and cholesteryl ester accumulation than was LDL of normal composition. Results suggest that at least part of the exaggerated ability of whole hypercholesterolemic serum to stimulate the esterification and accumulation of cholesterol in cells in culture is due to the presence of LDL of altered composition.

Metabolism by cells in culture of low-density lipoproteins of abnormal composition from non-human primates with diet-induced hypercholesterolemia.

Diet-induced hypercholesterolemia in non-human primates results in the production of a low-density lipoprotein (LDL) of abnormal size and composition. This LDL from hypercholesterolemic monkeys has been shown to be more atherogenic than the same amount of LDL from normocholesterolemic animals. Previous studies have demonstrated that hypercholesterolemic LDL is approximately twice as effective as normal LDL in stimulating cholesterol accumulation and esterification in arterial smooth muscle cells in culture. The purpose of the present study was determine whether this effect was secondary to differences in metabolism of the normal and hypercholesterolemic LDL. for this, the metabolism of 125I-labeled normal and hypercholesterolemic LDL from rhesus and cynomolgus monkeys was compared in several lines of skin fibroblasts and smooth muscle cells. Both normal and hypercholesterolemic LDL bound with high affinity to the same cell surface receptor. However, the affinity for binding of hypercholesterolemic LDL was about twice that of normal LDL (apparent dissociation constant for binding, Kd, was 2.63 micrograms protein/ml and 4.35 micrograms protein/ml, respectively). Conversely, only about 50% as many particles of hypercholesterolemic were able to bind to the receptor, compared with normal LDL. Those cells with the greatest capacity to metabolize LD generally accumulated the most cholesterol with either hypercholesterolemic or normal LDL. In all cell lines, nearly twice as much cholesterol accumulated in cells incubated with hypercholesterolemic LDL compared with normal LDL, and this differential could not be explained by differences in metabolism of the two lipoproteins, suggesting that some cholesterol entered the cells independent of the uptake of the intact LDL molecule. LDL receptors appear necessary for this to occur, since no difference in cholesterol accumulation was observed in cells genetically deficient in LDL receptors.

Quantitative analysis of the calcium-sensing receptor messenger RNA in parathyroid adenomas.

BACKGROUND: In primary hyperparathyroidism, hypercalcemia fails to suppress adequately secretion of parathyroid hormone by the parathyroid gland, which may result from failure of the cell-surface calcium receptor (CaR) to sense calcium correctly. Quantification of mRNA concentrations should provide important information on the role of expression of Call in primary hyperparathyroidism. METHODS: We have developed a quantitative reverse transcriptase-polymerase chain reaction assay with a competitive template (CaR-M). Amplified cDNAs for CaR and CaR-M are quantified, and the concentration of CaR mRNA is determined from the ratio of CaR-M/CaR versus known CaR-M concentrations. RESULTS: In parathyroid adenomas (n = 12) the CaR mRNA was 19.2 +/- 2.4 (mean +/- SE) fg/ng total RNA (range, 7.4 to 32.8 fg/ng). Extracellular ionized calcium levels ranged from 1.38 to 1.74 mmol/L (normal 1.19 to 1.31 mmol/L) and parathyroid hormone from 69 to 345 pg/ml (normal, 14 to 65 pg/ml). In spite of the wide variability in CaR expression in the various adenomas, there was no correlation between mRNA and either extracellular ionized calcium (r2 = 0.013) parathyroid hormone levels (r2 = 0.001). Normal human parathyroid glands gave values of 8.0 and 16.6 fg/ng, whereas normal bovine parathyroid glands had a mean of 20 +/- 0.6 fg/ng (n = 4). CONCLUSIONS: There is no apparent relationship between CaR mRNA levels in adenomas and preoperative Ca and PTH levels. Our findings suggest that defective Ca sensing in adenomas may involve post-translational modification or signal transduction distal to the receptor. Our highly sensitive assay for CaR mRNA should prove useful in examining further the role of CaR in Ca sensing in parathyroid tissue.

Enhanced cholesterol delivery to cells in culture by low density lipoproteins from hypercholesterolemic monkeys.

Previous studies have shown that the large molecular weight low density lipoprotein (LDL) of abnormal composition isolated from hypercholesterolemic nonhuman primates stimulates greater cholesterol accumulation in cells in culture than does the same amount of normal LDL. The purpose of the present study was to determine if a correlation existed over a range of LDL molecular weights with cholesterol accumulation in cells in culture, if the differential in cholesterol accumulation was the result of increased delivery of cholesterol to the cells and to evaluate the extent to which this differential was dependent on a functional LDL receptor pathway. Monkey and human skin fibroblasts were incubated for 24 hours with LDL isolated from individual normal or hypercholesterolemic cynomolgus monkeys having LDL molecular weights ranging from 2.58-6.39 x 10(6), and the cellular free and esterified cholesterol contents were determined. There was no correlation of LDL molecular weight with accumulation of cellular free or esterified cholesterol with LDL from normal animals having molecular weight of 2.58 to 3.08 X 10(6) or from hypercholesterolemic animals with LDL molecular weights greater than 4.5 x 10(6). There was a positive and significant correlation of LDL molecular weight with the accumulation of cellular free and esterified cholesterol with LDL molecular weights of 3.0 x 4.5 x 10(6). These differences were present when the LDL were added at equivalent protein or cholesterol concentrations and cannot be entirely explained by the increased amounts of cholesterol in LDL particles of larger molecular weight. The enhanced cellular cholesterol accumulation with hypercholesterolemic LDL seems to be the result of increased delivery of LDL cholesterol to the cells as shown by the increased rate of suppression of cellular sterol synthesis and LDL receptor activity, the increased stimulation of cholesterol esterification, and the increased accumulation of cellular 3H-cholesterol from LDL labeled with 3H-cholesteryl oleate. This difference in cellular cholesterol accumulation requires that the LDL must be both bound and internalized by a functional LDL receptor pathway, since cells that lack LDL receptors or are unable to internalize their LDL receptors do not show increased accumulation of cholesterol when incubated with hypercholesterolemic LDL.

Cholesterol efflux from cells enriched with cholesteryl esters by incubation with hypercholesterolemic monkey low density lipoprotein.

The mass efflux of free and esterified cholesterol was studied in skin fibroblasts loaded with cholesterol by incubation with low density lipoproteins (LDL) isolated from normal or hypercholesterolemic cynomolgus monkeys. Cells incubated with hypercholesterolemic LDL accumulated 2-3 times more cholesteryl ester than did cells incubated with the same amount of normal LDL. Cholesteryl oleate was the principal cholesteryl ester species to accumulate in cells incubated with both normal and hypercholesterolemic LDL. Efflux of this accumulated cholesterol was absolutely dependent on the presence of a cholesterol acceptor in the culture medium. Lipoprotein-deficient serum (LPDS) was the most potent promoter of cholesterol efflux tested, with maximum efflux occurring at LPDS concentrations greater than 1.5 mg protein/ml. Upon addition of efflux medium containing LPDS, there was a reduction in both the free and esterified cholesterol concentration of the cells. Greater than 90% of the cholesteryl esters that were lost from the cells appeared in the culture medium as free cholesterol, indicating that hydrolysis of cholesteryl esters preceded efflux. Efflux was not inhibited by chloroquine, however, suggesting a mechanism independent of lysosomes. Loss of cellular free cholesterol was maximum by 6 hr and changed very little thereafter up to 72 hr. Cholesteryl ester loss from cells decreased in a log linear fashion for efflux periods of 6-72 hr, with an average half-life for cholesteryl ester efflux of 30 hr, but with a range of 20-50 hr, depending upon the specific cell line. The rate of efflux of cellular cholesteryl esters was similar for cells loaded with normal or hypercholesterolemic LDL. In cells loaded with cholesteryl esters, cholesterol synthesis was suppressed and cholesterol esterification and fatty acid synthesis were enhanced. During efflux, cholesterol synthesis remained maximally suppressed while cholesterol esterification decreased for the first 24 hr of efflux, then plateaued at a level approximately 5-fold higher than control levels, while fatty acid synthesis was slightly stimulated. There was little difference in the rate of efflux of individual cholesteryl ester species. There was, however, the suggestion that reesterification of cholesterol principally to palmitic acid occurred during efflux. Since the rate of cellular cholesteryl ester efflux was similar regardless of whether the cells had been loaded with cholesterol by incubation with normal LDL or hypercholesterolemic LDL, the greater accumulation of cholesterol in cells incubated with hypercholesterolemic LDL cannot be explained by differences in rates of efflux.-St. Clair, R. W., and M. A. Leight. Cholesterol efflux from cells enriched with cholesteryl esters by incubation with hypercholesterolemic monkey low density lipoprotein.

Dietary fish oil modification of cynomolgus monkey low density lipoproteins results in decreased binding and cholesteryl ester accumulation by cultured fibroblasts.

Dietary fish oil (FO) has been reported to increase low density lipoprotein (LDL) receptor function resulting in lower plasma LDL concentrations in the rat (Ventura et al. J. Clin. Invest. 84: 528-537, 1989). The purpose of this study was to determine whether dietary FO, as compared to lard, affected the receptor-mediated uptake of LDL by cultured skin fibroblasts. Plasma LDL was isolated by combined ultracentrifugation and column chromatography from cynomolgus monkeys fed diets enriched in FO or lard and the effect of these two dietary fats on the binding of LDL and esterified cholesterol (EC) accumulation by cultured fibroblasts was determined. There was no difference in total plasma or LDL cholesterol concentrations between diet groups. The monkeys fed FO had significantly smaller LDL which, on average, contained less protein, phospholipid (PL), and free and esterified cholesterol compared to the LDL from monkeys fed the lard diet. FO LDL were less effective than lard LDL in competing for binding, internalization, and degradation of a standard 125I-labeled LDL by fibroblasts (11.0 +/- 2.4 vs. 3.0 +/- 0.8 micrograms LDL protein/ml for 50% displacement of binding, respectively; P = 0.013). FO versus lard LDL also resulted in less accumulation of cellular EC after a 24-h incubation with fibroblasts (7.7 +/- 0.2 vs. 13.0 +/- 0.4 micrograms EC/mg protein, respectively; P = 0.0001). In general, cellular EC accumulation was proportional to LDL particle size and LDL apoE/B molar ratio; however, LDL from the lard group resulted in greater EC accumulation even when LDL particle size and apoE content were nearly equivalent between diet groups. When LDL were isolated from the same animals by sequential ultracentrifugation, the lard LDL apoE was reduced 22% compared to column isolated LDL and this resulted in a 32% decrease in cellular EC accumulation. However, for FO LDL, apoE content was reduced 34% by sequential ultracentrifugation but this only resulted in a 10% decrease in EC accumulation. These results suggested that lard LDL contained more receptor-active apoE than FO LDL. We conclude that isocaloric substitution of fish oil for lard in the diet of cynomolgus monkeys results in LDL particles that bind less avidly to LDL receptors and in less EC accumulation in fibroblasts. The decreased binding of LDL from the FO group appears related to their decreased size and CE content as well as the decreased content of receptor-active apoE relative to the lard group.

Metabolism of low density lipoproteins by pigeon skin fibroblasts and aortic smooth muscle cells. Comparison of cells from atherosclerosis-susceptible and atherosclerosis-resistant pigeons.

Aortic smooth muscle cells from atherosclerosis-susceptible White Carneau (WC) pigeons lack a functional low density lipoprotein (LDL) receptor pathway. The purpose of the present study was to determine if atherosclerosis-resistant Show Racer pigeons (SR) shared this lack of an LDL receptor pathway and if LDL from normal and hypercholesterolemic pigeons were metabolized similarly. The amount of LDL bound, internalized, and degraded by skin fibroblasts, embryo fibroblasts, and aortic smooth muscle cells from WC and SR pigeons were similar and averaged from 2% to 25% of that seen with monkey smooth muscle cells incubated with the same LDL. LDL uptake by pigeon cells was due largely to nonspecific processes, while specific uptake predominated in monkey cells. A similar lack of specific uptake was obtained with LDL from normal and hypercholesterolemic pigeons. Sterol synthesis and HMG-CoA reductase activity were 10- to 35-fold higher in pigeon cells than in monkey cells incubated in serum-containing medium. LDL had little effect on cholesterol esterification and cholesteryl ester accumulation in pigeon cells. These results indicate that despite major changes in the size and composition of LDL from hypercholesterolemic pigeons, this LDL, like normal pigeon and monkey LDL, was not metabolized by specific uptake processes by pigeon cells. Cells from both WC and SR pigeons lack a functional LDL receptor pathway.

Dietary fish oil-induced decrease in low density lipoprotein binding to fibroblasts is mediated by apolipoprotein E.

In a previous study we demonstrated that isocaloric substitution of fish oil (FO) for lard in the diet of cynomolgus monkeys resulted in low density lipoproteins (LDL) that were poorer competitors for binding of a standard 125I-labeled LDL and led to less cholesteryl ester accumulation in skin fibroblasts (Linga, V., et al. 1993. J. Lipid Res. 34: 769-778). The decreased binding and cholesteryl ester accumulation by FO LDL appeared related to the LDL apolipoprotein E (apoE) content. We hypothesized that FO LDL had reduced binding to skin fibroblasts due to a decrease in receptor active apoE. To test this hypothesis and determine the relative contribution of apoE versus apolipoprotein B (apoB) in binding of LDL to skin fibroblasts, LDL from cynomolgus monkeys fed lard or FO-containing diets were isolated, characterized, radioiodinated, and tested for binding in the absence or presence of a 10-fold molar excess of monoclonal antibody to the receptor binding domain of apoE (1D7) or apoB-100 (MB47). FO LDL were smaller, contained less apoE (E/B molar ratio = 0.48 +/- 0.03 vs. 1.85 +/- 0.22; P < 0.001), and had a weaker binding affinity (Kd = 11.3 +/- 1.6 vs. 3.8 +/- 0.80 microgram/ml; P < 0.01) compared to the lard counterparts. Furthermore, the apoE/B molar ratio of LDL appeared inversely related to the Kd for binding to skin fibroblasts. Incubation of LDL with skin fibroblasts in the presence of a 10-fold molar excess of monoclonal antibody directed at the receptor binding domain of apoB-100 (MB47) eliminated 96 +/- 3% of binding of FO LDL, but eliminated only 43 +/- 18% of binding for lard LDL. Incubation with a 10-fold molar excess of monoclonal antibody to the receptor-binding domain of apoE (1D7) eliminated only 23 +/- 6% of FO LDL binding to fibroblasts relative to a no-antibody control, but for lard LDL 44 +/- 11% of binding to fibroblasts was eliminated. Both antibodies together blocked all binding of LDL from both diet groups. In a fluid phase precipitation assay > 75% of the LDL particles from both diet groups was precipitated with saturating amounts of MB47, indicating that the proportion of LDL particles expressing this epitope was the same for both diet groups. The same assay using 1D7 showed approximately 4-fold greater precipitation of LDL in the lard versus FO group.(ABSTRACT TRUNCATED AT 400 WORDS)