Gustav Schonfeld

Effects of dietary cholesterol and fatty acids on plasma lipoproteins.

The effects of dietary cholesterol and fatty acids on low density and high density lipoproteins (LDL and HDL) were studied in 20 young men. After 2-3 wk of evaluations on ad lib. diets, basal diets, which consisted of 15% protein, 45% carbohydrates, 40% fat, and 300 mg/day of cholesterol, were given for 4-5 wk (Basal). The ratio of dietary polyunsaturated to saturated fatty acids (P/S) for different groups of subjects were 0.25, 0.4, 0.8, or 2.5. 750 and 1,500 mg/d of cholesterol were added to the basal diets as 3 and 6 eggs, respectively. Total cholesterol and LDL cholesterol were lower in all subjects on the basal diets than on the ad lib. diets. Addition of 750 mg cholesterol to the diet with P/S = 0.25-0.4 raised LDL cholesterol by 16 +/- 14 mg/dl to 115% of basal diet values (n = 11, P less than 0.01); 1,500 mg increased LDL cholesterol by 25 +/- 19 mg/dl to 125% (n = 9, P less than 0.01). On the diet with P/S = 0.8, 750 mg produced insignificant increases in LDL cholesterol, but 1,500 mg produced increases of 17 +/- 22 mg/dl to 115% of basal (n = 6, P less than 0.02). On the P/S = 2.5 diet, neither 750 nor 1,500 mg produced significant changes. Thus, both the cholesterol contents and P/S ratios of diets were important in determining LDL levels. The lipid and apoprotein compositions, flotation rates, molecular weights, and binding by cellular receptors of LDL were virtually unchanged by the addition of cholesterol to the diets high in saturated fat. These diets, therefore, caused an increase in the number of LDL particles of virtually unchanged physical and biological properties. On the diet with low P/S ratio, HDL2 rose, whereas this effect was absent on diets with high P/S ratios. The response of LDL to dietary manipulations is consonant with epidemiologic data relating diets high in cholesterol and saturated fat to atherogenesis. The response of HDL2, however, is opposite to that of its putative role as a negative risk factor. Further work is needed to clarify this interesting paradox.

Estrogen Up-regulates Apolipoprotein E (ApoE) Gene Expression by Increasing ApoE mRNA in the Translating Pool via the Estrogen Receptor α-Mediated Pathway

The antiatherogenic property of estrogens is mediated via at least two mechanisms: first by affecting plasma lipoprotein profiles, and second by affecting the components of the vessel wall. Raising plasma apolipoprotein E (apoE) in mice protects them against diet-induced atherosclerosis (Shimano, H., Yamada, N., Katsuki, M., Gotoda, T., Harada, K., Murase, T., Fukuzawa, C., Takaku, F., and Yazaka, Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1750–1754). It is possible that estrogen may be antiatherogenic at least in part by increasing plasma apoE levels. Therefore, we studied the regulation of apoE by estrogen. A survey of 15 inbred strains of mice showed that some mouse strains responded to injections or subcutaneously implanted pellets of estradiol by raising their apoB and apoE levels and some did not. We performed detailed studies in two “responder” strains, C57L and C57BL, and two “non-responder” strains, C3H and BALBc. Responders increased their plasma apoE levels 2.5-fold. Non-responders’ levels were altered ±10%. In the responders the distribution of apoE among the plasma lipoproteins shifted from high density lipoprotein toward the apoB-containing lipoprotein fractions. In nonresponders the shift was toward high density lipoprotein. Hepatic apoE mRNA levels and relative rates of apoE mRNA transcription were unchanged in all strains, suggesting that apoE regulation occurred at posttranscriptional loci. Therefore, we measured apoE synthesis in fresh liver slices and on isolated hepatic polysomes. Two-fold increases were noted but only in responders accompanied by selective 1.5-fold increases in polysomal apoE mRNA levels. Similar increases in apoE synthesis were also observed in castrated C57BL mice given either physiological or pharmacological replacement doses of estradiol, but not testosterone, suggesting that the effect of estradiol was specific on the distribution of apoE mRNA in the translationally active polysomal pool. Next, we examined whether the effects of estrogen on apoE translation were mediated by estrogen receptors (ER). ER-α knock-out mice and their wild-type littermates were administered estradiol. As expected, apoE levels and hepatic apoE synthesis increased more than 2-fold in the wild-type littermates, but only 20% increases in the plasma apoE and hepatic synthesis were observed in the ER knock-out mice. Hepatic apoE mRNA levels did not change in either the wild-type or the ER knock-out mice. Thus, estradiol up-regulates apoE gene expression by increasing levels of apoE mRNA in the polysomal translating pool. Furthermore, the increased polysomal recruitment of apoE mRNA is largely mediated by estrogen receptors.

Assay of Total Plasma Apolipoprotein B Concentration in Human Subjects

We have developed a double antibody radioimmunoassay (RIA) for human apolipoprotein B (ApoB). The assay measures not only the ApoB content of beta-lipoproteins (low density lipoproteins [LDL]) but also that contained in the other lipoproteins in plasma. Purified lymph and plasma chylomicrons and plasma very low density lipoproteins (VLDL) produced displacement curves in the assay system which paralleled those produced by pure LDL. Thus, the ApoB found in chylomicrons, VLDL, and LDL were immunologically identical. ApoB accounted for about 25 and 35%, respectively, of the total protein of chylomicrons and VLDL by RIA. VLDL and LDL preparations from normal and hyperlipoproteinemic subjects also produced parallel displacement curves, suggesting that the ApoB of normal and hyperlipoproteinemic subjects were immunologically identical. High density lipoproteins and abetalipoproteinemic plasma displaced no counts, nor did the sera of several animal species produce any useful displacement curves in this system. The fasting total plasma ApoB concentration of normal subjects was 83+/-16 mg/dl (mean+/-SD). ApoB levels were high in Type II (162+/-16), and less so in Type IV (112+/-24) and Type V (105+/-17).When plasma ApoB concentration in Type IV patients was graphed against plasma glycerides, two subpopulations, which may represent different genetic or biochemical subgroups, were apparent.ApoB concentration in individuals on constant diet and drug regimen was stable over weeks to months. Greater than 90% of ApoB of normal and Type II subjects was in the d > 1.006 plasma fraction. By contrast, only 50-80% of ApoB was in the d > 1.006 fraction in Types IV and V. Thus, hypertriglyceridemia was associated primarily with a redistribution of ApoB to the lighter density fractions; by contrast, in hypercholesterolemia absolute ApoB concentration was markedly increased.

Beta-carotene inhibits atherosclerosis in hypercholesterolemic rabbits.

Oxidatively damaged LDL may be of central importance in atherogenesis. Epidemiological evidence suggests that high dietary intakes of beta-carotene and vitamin E decreases the risk for atherosclerotic vascular disease, raising the possibility that lipid-soluble antioxidants slow vascular disease by protecting LDL from oxidation. To test this hypothesis, we fed male New Zealand White rabbits a high-cholesterol diet or the same diet supplemented with either 1% probucol, 0.01% vitamin E, 0.01% all-trans beta-carotene, or 0.01% 9-cis beta-carotene; then we assessed both the susceptibility of LDL to oxidation ex vivo and the extent of aortic atherosclerosis. As in earlier studies, probucol protected LDL from oxidation and inhibited lesion formation. In contrast, vitamin E modestly inhibited LDL oxidation but did not prevent atherosclerosis. While beta-carotene had no effect on LDL oxidation ex vivo, the all-trans isomer inhibited lesion formation to the same degree as probucol. Moreover, all-trans beta-carotene was undetectable in LDL isolated from rabbits fed the compound, although tissue levels of retinyl palmitate were increased. The effect of all-trans beta-carotene on atherogenesis can thus be separated from the resistance of LDL to oxidation, indicating that other mechanisms may account for the ability of this compound to prevent vascular disease. Our results suggest that metabolites derived from all-trans beta-carotene inhibit atherosclerosis in hypercholesterolemic rabbits, possibly via stereospecific interactions with retinoic acid receptors in the artery wall.

Exercise training decreases plasma cholesteryl ester transfer protein.

To assess the effect of exercise on the plasma concentration of cholesterol ester transfer protein (CETP) and its possible influence in mediating the exercise-associated redistribution of cholesterol among plasma lipoproteins, we measured plasma CETP in 57 healthy normolipidemic men and women before and after 9 to 12 months of exercise training. The training protocol resulted in significant changes in VO2max (mean +/- SD, +5.3 +/- 3.5 mL.kg-1 x min-1), body weight (-2.5 +/- 3.5 kg), plasma triglycerides (-25.7 +/- 36.3 mg/dL), high-density lipoprotein cholesterol (HDL-C) (+2.6 +/- 6.2 mg/dL), and ratios of total cholesterol to HDL-C (-0.30 +/- 0.52) and low-density lipoprotein cholesterol (LDL-C) to HDL-C (-0.18 +/- 0.45) (all P < or = .05) but no change in lipoprotein(a). CETP concentration (in milligrams per liter) fell significantly in response to training in both men (n = 28, 2.47 +/- 0.66 to 2.12 +/- 0.43; % delta = 14.2%; P < .005) and women (n = 29, 2.72 +/- 1.01 to 2.36 +/- 0.76; % delta = 13.2%; P < .047). The CETP change was observed both in subjects who lost weight (n = 28, delta mean weight = -5.0 kg; delta CETP = -0.42 +/- 0.79; % delta = 15.4%; P < .009) and in those who were weight stable (n = 29, delta mean weight = -0.12 kg; delta CETP = -0.29 +/- 0.78; % delta = 10.4%; P < .055).(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein C-II and C-III levels in hyperlipoproteinemia

Abstract Apoprotein C-II (ApoC-II) functions as a modulator of the hydrolysis of lipoprotein triglycerides by increasing the activity of lipoprotein lipase. Apoprotein C-III (ApoC-III) appears to have an inhibitory effect on ApoC-II-stimulated hydrolysis. Because of the physiologic importance of these apoproteins, we have developed double antibody radioimmunoassays that are capable of measuring the levels of ApoC-II and ApoC-III in human whole plasma and in isolated lipoproteins. ApoC-II and ApoC-III were isolated from very low density lipoproteins by column chromatography. Fractions that were homogeneous on polyacrylamide disc gel electrophoresis and isoelectric focusing and that had the appropriate amino acid compositions were used as antigens, assay standards, and labels. The assays contained 125 I-ApoC-II or 125 I-ApoC-III (Na 125 I + lactoperoxidase), rabbit antihuman ApoC-II (or ApoC-III), samples of plasma, lipoproteins, or apoprotein standards, goat antirabbit IgG, and 0.05 M barbital pH 8.6, 0.01% Triton X-100. The assays had the specificities, accuracies, precisions, and immunologic indentities between standards and samples required of all such assays. All of the ApoC-II in plasma in isolated lipoproteins was detected by the ApoC-II assay. ApoC-III 1 and ApoC-III 2 were equally reactive, and all of the ApoC-III 0 , ApoC-III 1 , and ApoC-III 2 in plasma and in lipoproteins was detected by the ApoC-III assay. ApoC-II and ApoC-III levels were measured in 171 fasting plasmas taken from normal subjects and patients with type II–V hyperlipidemia. “Normal” values for white subjects were approximately 4 and 16 mg/dl for ApoC-II and ApoC-III, respectively. Levels were several fold elevated in the hyperlipidemias. In normal subjects ∼40% of ApoC-II and ApoC-III were found in the d d = 1.006–1.063, ∼45% in the d = 1.063–1.21. These distributions were altered in hyperlipidemia. However, in all cases the d >1.21 fraction contained

Oleic acid stimulation of apolipoprotein B secretion from HepG2 and Caco-2 cells occurs post-transcriptionally

HepG2 and Caco-2 cells were studied to compare the regulation of liver and intestinal apolipoprotein (apo) biosynthesis and secretion by fatty acids. Incubation with fatty acid consistently stimulated apoB production by both HepG2 and Caco-2 cells. Media concentrations of apoB, determined by radioimmunoassay, were approx. 3-fold greater for cells incubated for 24 h in serum-free medium containing oleic acid bound to albumin than for cells incubated with albumin alone. Oleic acid also resulted in a 2-3-fold accumulation of cellular triacylglycerol for HepG2 cells and Caco-2 cells. Cellular apoB and media and cellular apoA-I concentrations were not affected by oleic acid. Immunoblotting with a monoclonal antibody against human apoB confirmed a greater mass of apoB in media from HepG2 and Caco-2 cells incubated with oleic acid. Radiolabeled apoB-100 was also increased in media from HepG2 and Caco-2 cells incubated with [35S]methionine for 24 h in the presence of oleic acid, suggesting enhanced apoB synthesis. However, apoB mRNA concentrations were unchanged in response to oleic acid. Gel filtration of media by fast protein liquid chromatography (FPLC) revealed a redistribution of apoB from LDL-sized particles to VLDL or chylomicrons in media from Caco-2 cells incubated with oleic acid, whereas apoB remained in LDL for HepG2 cells. ApoA-I in media from HepG2 and Caco-2 cells eluted as free or lipid-poor apoA-I, and the apoA-I distribution was unaltered by incubation with oleic acid. These data demonstrate that HepG2 and Caco-2 cells maintained in supplemented serum-free medium respond to oleic acid by a similar post-transcriptional increase in apoB synthesis, but different packaging of apoB into triacylglycerol-rich lipoproteins.

Probucol attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits.

1 Probucol was administered to rabbits fed a cholesterol‐enriched (2% wt/wt) diet to determine potential anti‐atherogenic effects in a preparation in which the disease process is due to elevated plasma concentrations of cholesterol ester‐rich very low density lipoproteins (CER‐VLDL). 2 Probucol was supplemented to the diet at 1% wt/wt which resulted in plasma concentrations rising steadily to 53 ± 8 μg ml−1 after 14 days, with no significant changes during continued administration. Dietary consumption and body weight gains were comparable in the drug‐treated and control groups during the observation period. 3 Probucol treatment did not significantly affect plasma concentrations of total cholesterol, unesterified cholesterol, triglycerides or phospholipids. 4 The concentration of CER‐VLDL in plasma and its physicochemical characteristics were not significantly changed during administration of probucol. CER‐VLDL from both control and probucol‐treated animals was a potent stimulant of the augmentation of the intracellular incorporation of [3H]‐oleate into cholesteryl‐[3H]‐oleate in cultured macrophages. 5 Despite the lack of effect of probucol on concentrations of plasma lipids and the cell interaction characteristics of CER‐VLDL, administration of the drug markedly decreased the extent of intimal aortic surface area covered by grossly discernible atherosclerotic lesions from 55.6 ± 11.8% to 11.6 ± 1.9% in thoracic sections, and from 49.1 ± 10.2% to 7.2 ± 0.4% in abdominal sections. Furthermore, probucol treatment significantly reduced the deposition of total cholesterol in vascular tissue. 6 Probucol reduced the extent of aortic atherosclerosis produced by diet‐induced hypercholesterolemia in rabbits. This reduction occurred in the absence of any significant change in the characteristics of plasma lipoproteins that were determined. These results indicate that either there is a role of oxidation in the disease process of this animal model of atherosclerosis or that probucol is acting via a presently undefined mechanism.

Alterations in levels and interrelations of plasma apolipoproteins induced by diet

Diets high in carbohydrates increase the rates of hepatic secretion of VLDL in man. VLDL particles are secreted in greater numbers; they also tend to contain more triglycerides and to be larger in size. Comparable changes in intestinal secretion of chylomicrons and VLDL accompany fat absorption. We studied the metabolism of the ApoC apoproteins under these circumstances because in vitro studies suggest that these apoproteins may play important roles in the catabolism of VLDL. ApoA-I and ApoB were also examined because of their importance in HDL and VLDL structure, respectively. The fasting plasmas of 16 normolipemic young adults were examined before and after they had been fed high carbohydrate diets for 4-5 days. ApoA-I and ApoB were measured by radioimmunoassay. The relative proportions of ApoC-II and ApoC-III in VLDL were determined by disc gel electrophoresis. Lipids in plasma and in individual lipoproteins were determined chemically. Lipoproteins were isolated by ultracentrifugal and precipitation methods. VLDL-TG and VLDL-chol and VLDL-protein rose by factors of 2.4, 1.67, and 1.88, respectively. (Final value divided by initial value.) Since TG rose more than the others, VLDL became enriched by TG. LDL-chol fell by a factor of 0.78 while LDL-TG and LDL-ApoB remained constant, i.e., LDL became relatively enriched in TG and ApoB. HDL-TG rose by 1.42 and HDL-chol fell by 0.74, i.e., HDL too became TG enriched. Plasma Apo-A-I fell by 0.84. Thus, there were alterations in both the levels and compositions of all lipoproteins. The proportion of ApoC-II relative to ApoC-III in VLDL increased in each subject on the carbohydrate diet (mean rise was 1.55-fold). The increases in ApoC were detectable after as little as 48 hr of diet, and the same results were obtained in two people who were each tested twice on two separate occasions several months apart. Thus, the changes in ApoC were reproducible. Proportions of ApoC-II were greater in SF greater than 400 than in SF 20-400 subfractions of VLDL on ad lib diets. Proportions of ApoC-II rose in both density subfractions on carbohydrate diets. The changes in both subfractions suggest that rises of ApoC-II in total VLDL (SF greater than 20) fraction were not due solely to the accumulation of SF greater than 400 particles of unaltered ApoC composition in postdiet plasma. The changes following carbohydrate could have been due to secretion of altered lipoproteins or due to diet induced alterations in apoprotein catabolism. Three subjects were given 150 gm of corn oil to drink. Lipoproteins were analyzed, as above, at 0 and 3-12 hr after the drink. In spite of several-fold rises in the SF greater than 20 TG and proteins, the relative proportions of ApoC-II and ApoC-III remained constant. Thus, an acute dietary fat load and 2-4 days of carbohydrate diets provoked different responses. The fluctuations in the levels and relative proportions of ApoC support the notion that the ApoC group may have importance in vivo in the metabolism of VLDL.

Plasma apoprotein and lipoprotein lipid levels in vegetarians

Abstract Low-fat, low-cholesterol, high-P:S-ratio diets greatly affect plasma lipid levels. There is no information as to whether such diets affect only lipoprotein levels or also levels of apoproteins and lipoprotein compositions. It is important to have information on the latter to understand diet-induced changes in the metabolism of lipoproteins. Since vegetarians regularly eat an extremely low-cholesterol, low-fat, and-high-P:S ratio diet, they represent an ideal group to study. Fifty-eight vegetarians who eat no animal products and live on a farm commune were examined. Venous bloods were drawn after 12–14 hr fasts and analyzed for lipoprotein-lipids by Lipid Research Clinic procedures and for apoA-I and apoB by radioimmunoassay. Their normal dietary intake was evaluated with 24-hr food diaries. They averaged 2200 kcal/day with 17% protein, 32% fat, and 51% carbohydrate. Negligible amounts of cholesterol (