Granulocytic-macrophagic and macrophagic colony stimulating factors elicit colonies of mast cells in mouse bone marrow agar culture. An electron microscope study

The aim of this paper is to describe a new methodology for the separation of human high-density lipoproteins (HDL) into apolipoprotein (apo) E-poor and apo E-rich subfractions by fast protein liquid chromatography (FPLC) using a heparin affinity column. Recoveries for apolipoproteins AI, AII, CI, CII, CIII, and E were 68.9, 74.7, 71.9, 73.5, 40.0, and 55.8%, respectively. We provide suggestive evidence that apo E-rich HDL is produced from apo E-poor HDL by the displacement of apo AI by apo E. Apo E-poor HDL was the predominant fraction. The molar ratio of apo E to apo AI in apo E-poor HDL was 0.02 and 0.01 for the subjects studied while in apo E-rich HDL it was 1.86 and 1.25. The molar ratios of the C apolipoproteins to apo AI are markedly different between the subfractions.     

In vivo metabolism of alpha-tocopherol in lipoproteins and liver: studies on rabbits in response to acute cholesterol loading

OBJECTIVE: To investigate the transport of alpha-tocopherol in lipoproteins of rabbits under normal diet and under acute loading of cholesterol. DESIGN: Two New Zealand White rabbits were fed 14C-alpha-tocopherol acetate in a single oral dose and the recovery of radiolabel in lipoproteins and plasma was monitored. Low density lipoprotein (LDL) from these animals was obtained and labeled with [3H] cholesteryl ester. Three other rabbits were injected with this double-labeled LDL in the native form; while three other animals received this LDL in the acetylated form. RESULTS: Plasma clearance, liver uptake and levels of radiolabel in high density lipoprotein (HDL) of animals injected with 14C[3H]acetyl LDL were significantly higher than those in animals injected with 14C[3H]native LDL. Larger particles of HDL, rich in apolipoprotein E (apoE) carried significantly higher levels of both labels in rabbits injected with acetylated LDL. CONCLUSION: These results provide evidence for in vivo mechanisms of "reverse alpha-tocopherol transport", analogous to "reverse cholesterol transport".     

Basis for rapid efflux of biosynthetic desmosterol from cells

Previous work shows that the efflux of biosynthetic desmosterol from cells is three times more efficient than that of cholesterol. To explain this difference, we labeled CHO-K1 cells with [3H]acetate precursor and measured sterols in the whole cells, plasma membranes and caveolae, and those released to high density lipoprotein (HDL3). The [3H]desmosterol-to-[3H]cholesterol ratio was similar in the plasma membrane and whole cells but was greater in HDL3, suggesting that the more efficient efflux of desmosterol is due to more rapid desorption from the plasma membrane. The ratio in caveolae was similar to that in whole cells, arguing against selective delivery of desmosterol to caveolae as an explanation for the more rapid efflux of this sterol. Additionally, to demonstrate that the enhanced release of desmosterol was not due to enhanced intracellular cycling, we made vesicles from CHO-cell plasma membranes labeled with [3H]desmosterol or [14C]cholesterol, and the rapid release of desmosterol was demonstrated in this system. To characterize sterol efflux from a simple lipid bilayer system, we measured the transfer of cholesterol and desmosterol between large unilamellar vesicles (LUV), and found that desmosterol transferred two to three times more rapidly than cholesterol. A similar differential was seen when HDL3 or low density lipoprotein (LDL) served as the acceptor. These results show that the greater efflux efficiency of biosynthetic desmosterol can be attributed to more efficient desorption from the plasma membrane, and that this difference is a property of the sterols' association with the lipid bilayer. In vivo, the rapid efflux of biosynthetic sterol intermediates, followed by efficient delivery to the liver, may constitute an important mechanism for preventing various types of pathology associated with these materials.     

ACTH regulates LDL receptor and CLA-1 mRNA in the rat adrenal cortex

Rat adrenocortical cells utilize both low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol for steroid hormone production. In addition to exogenous lipoprotein-derived cholesterol, cells produce cholesterol de novo. Adrenocorticotropin (ACTH) increases both steroid hormone secretion and uptake of LDL and HDL. We studied the expression of LDL receptor mRNA and CLA-1 (a putative HDL receptor) mRNA in cultured rat adrenocortical cells. ACTH increased the amounts of LDL receptor mRNA during 2 to 48 h of stimulation, the highest levels being detected after 2-4 h. Similar results were obtained with cyclic AMP (cAMP) derivatives, 8-bromo cAMP (8-Br cAMP) or dibutyryl cAMP. ACTH increased CLA-1 mRNA during 2 to 24 h of stimulation, the highest levels being detected after 4 h. In conclusion, ACTH up regulates both LDL and HDL receptor mRNA in rat adrenocortical cells.     

Diet, plasma lipoproteins and experimental atherosclerosis in baboons (Papio sp.)

Diet-induced hyperlipidaemia in baboons is similar to that in humans. As in humans, the ratio between low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol is a major determinant of atherosclerosis. Baboons, like humans and other non-human primates, vary in their lipaemic responses to dietary lipids. By selective breeding based on variability in plasma and lipoprotein cholesterol response to diet, lines of baboons with high and low responses of various lipoproteins have been developed. Genetic analyses suggest that lipoprotein patterns in response to dietary cholesterol and fat are heritable. Metabolic and molecular studies of high and low LDL and HDL cholesterol responses to dietary lipids have suggested that different mechanisms regulate plasma LDL cholesterol on the chow and on the high cholesterol-high fat (HCHF) diet. On the chow diet, plasma LDL cholesterol levels are positively associated with cholesterol absorption and negatively associated with hepatic LDL receptor levels and, thus, cholesterol absorption and LDL receptors seem to regulate plasma LDL cholesterol levels. However, when the animals consume a human-like fat- and cholesterol-enriched diet, plasma LDL cholesterol levels are not associated with either cholesterol absorption or hepatic LDL receptor mRNA levels, but are negatively associated with plasma 27-hydroxycholesterol concentrations, hepatic sterol 27-hydroxylase activity, and mRNA levels. Hepatic sterol 27-hydroxylase activity and mRNA levels are induced by dietary cholesterol and fat in low responding baboons more than in high responding baboons. Thus, the ability to induce sterol 27-hydroxylase determines the LDL cholesterol response in baboons. High HDL response baboons often have high levels of HDL1 in their plasma. Our studies suggest that the N-terminal fragment of apo C-I with 38 amino acids and a molecular weight of approximately 4 kDa acts as a cholesteryl ester transfer inhibitor peptide in high HDL1 baboons. The inhibitor peptide associates with apo A-1 in HDL to produce a modified apo A-1 protein with a molecular weight of approximately 31 kDa. The inhibitor peptide is a gene product and the presence of this peptide produces an antiatherogenic high HDL1 phenotype.     

Net transport of cholesterol from cells of the human EA.hy 926 endothelial cell line to high density lipoproteins

EA.hy 926 cells, a human endothelial cell line, show characteristics of differentiated endothelial cells. The cells express saturable binding of apo E-free 125I-high density lipoprotein3 (HDL3). Bmax increased from 71 to 226 ng HDL3 bound/mg cell protein after cholesterol loading of the confluent endothelial cells with cationized low density lipoprotein (LDL). The affinity did not change after cholesterol enrichment (Kd was 37 micrograms HDL3 protein/ml for control cells and 31 micrograms/ml for loaded cells). Incubation of cholesterol-loaded EA.hy 926 cells with native HDL and LDL had different effects on cellular cholesterol levels. Incubation with HDL decreased both esterified and unesterified cellular cholesterol, but LDL did not change total cellular cholesterol. However, LDL tended to increase cellular cholesteryl esters, with a concomitant decrease of unesterified cellular cholesterol. Incubation of endothelial cells with both HDL and LDL also resulted in decreased total cellular cholesterol levels. These data show that cationized LDL-loaded human endothelial EA.hy 926 cells can be used to study the net transport of cellular cholesterol to HDL, the first step in reverse cholesterol transport.     

Effects of insulin deficiency on lipoproteins and their hepatic receptors in Rico rats

The present study was designed to examine the effect of streptozotocin (STZ)-induced diabetes on the plasma lipoprotein profile and hepatic expression of the LDL receptor and HDL binding protein (HB2) in hypercholesterolemic Rico rats. The plasma level of HDL1 (density range 1.040-1.063), which is particularly high in this rat strain, decreased (-25%) 28 d after STZ administration (50 mg/kg). In contrast, the treatment increased (+54%) the plasma concentration of HDL2 (density range 1.063-1.210). These variations in the lipoprotein concentrations were associated with inverse changes in the hepatic protein levels of the LDL receptor (+118%) and HB2 (-46%). These results suggest that the hepatic expression of HB2, a putative HDL receptor, can influence the plasma level of apo Al-rich HDL as has already been shown for the LDL receptor for apo B/E containing lipoproteins.     

Apolipoprotein E polymorphism influences lipid phenotypic expression, but not the low density lipoprotein subfraction distribution in familial combined hyperlipidemia

The impact of apo E polymorphism on interindividual variation in plasma lipid, lipoprotein concentrations, and LDL subfraction profiles was studied in 201 well-defined patients (88 men and 103 women) with familial combined hyperlipidemia (FCH). When corrected for the concomitant influences of age, gender and obesity, the allelic variation in the apo E gene was shown to explain a statistically significant portion of the variability in lipid and (apo)lipoprotein concentrations. Carriers of the apo epsilon 2 allele exhibited a substantially higher plasma triglyceride concentration and a lower low density lipoprotein (LDL) cholesterol level, while subjects with the apo epsilon 4 allele had significant higher total plasma cholesterol and LDL cholesterol levels. In line with this observation, our FCH population was characterized by an over-representation of the apo E4 allele as compared with a Dutch standard population (chi 2 = 55.2, P < 0.0001). The contribution of apo E polymorphism to trait variability was different between sexes for plasma triglyceride, VLDL cholesterol, VLDL triglycerides, and high density lipoprotein (HDL) cholesterol levels. Apo E polymorphism had no impact on chemical composition of VLDL; for LDL particles the apo epsilon 2 allele was associated with a lower cholesterol to protein (C/P) ratio, whereas the opposite was true for the apo epsilon 4 allele. Despite the demonstrated impact of apo E polymorphism on plasma lipids and LDL chemical composition, in all phenotypic groups a dense LDL subfraction profile predominated. Thus, apo E polymorphism contributes to the lipid phenotypic expression in FCH, whereas further evidence was obtained that a dense LDL subfraction profile is an integral feature of FCH.     

Lipoproteins, apolipoproteins, and low-density lipoprotein size among diabetics in the Framingham offspring study

Diabetes mellitus has been shown to be associated with lipid abnormalities. Prior studies have indicated that women with diabetes have a risk of coronary heart disease similar to that of men. We compared lipid parameters in diabetic and nondiabetic participants in cycle 3 of the Framingham Offspring Study. Values for plasma total cholesterol (TC), triglyceride, lipoprotein, cholesterol, apolipoprotein (apo) A1, B, apo and lipoprotein(a) [Lp(a)] and low-density lipoprotein (LDL) particle size were analyzed in 174 diabetic and 3,757 nondiabetic subjects. Data from a total of 2,025 men and 2,042 women participating in the third examination (1983 to 1987) of the Framingham Offspring Study were subjected to statistical analysis. Male and female diabetics showed lower high-density lipoprotein (HDL) cholesterol, higher triglycerides, higher very-low-density lipoprotein (VLDL) cholesterol, lower apo A1, and higher LDL particle scores, indicating smaller size, than nondiabetics. Female diabetics also showed significantly higher TC and apo B values than nondiabetics. The results remained statistically significant after controlling for obesity and menopausal status. The presence of small dense LDL particles (pattern B) was highly associated with diabetes and hypertriglyceridemia in both sexes, and the relative odds for pattern B remained significant in women but not in men after adjustment for age and hypertriglyceridemia. No differences in apo E isoform distribution were found for diabetics and nondiabetics. Diabetes was not associated with elevated LDL cholesterol levels. In conclusion, diabetics have lower HDL cholesterol and higher triglyceride levels and are more likely to have small dense LDL particles. Diabetes is not a secondary cause of elevated LDL cholesterol. Lipid screening of diabetics should include full quantification of lipids for proper assessment of potential atherosclerotic risk.     

Antiatherogenic effects of adjuvant antiestrogens: a randomized trial comparing the effects of tamoxifen and toremifene on plasma lipid levels in postmenopausal women with node-positive breast cancer

PURPOSE: To evaluate whether a novel antiestrogen, toremifene, has similar antiatherogenic effects as tamoxifen. PATIENTS AND METHODS: Forty-nine postmenopausal patients with node-positive breast cancer were randomized in a trial that compared the effects of tamoxifen and toremifene on serum lipoproteins. Tamoxifen was given at 20 mg and toremifene at 60 mg orally per day for 3 years. Serum concentrations of apolipoprotein (apo) A-I, A-II, and B, and lipoprotein(a) [Lp(a)], cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol were measured before and after 12 months of antiestrogen therapy. RESULTS: Both antiestrogens significantly reduced serum total and LDL cholesterol and apo B levels. However, the response of HDL cholesterol to treatments was clearly different between the groups. Toremifene increased the HDL level by 14%, whereas tamoxifen decreased it by 5% (P = .001). As a consequence, both cholesterol-to-HDL and LDL-to-HDL ratios decreased more in the toremifene than tamoxifen group (P = .008 and P = .03, respectively). Toremifene also increased the apo A-I level (P = .00007) and apo A-I-to-A-II ratio (P = .018). Both tamoxifen and toremifene decreased the Lp(a) concentration significantly (change, 34% v 41%). CONCLUSION: These results provide positive evidence that toremifene has antiatherogenic properties with potency to improve all lipoproteins that are associated with increased coronary heart disease (CHD) risk.     

Pregnancy in a patient with homozygous familial hypercholesterolemia undergoing low-density lipoprotein apheresis by dextran sulfate adsorption

Pregnancy and delivery in homozygous familial hypercholesterolemic (HFH) patients is extremely rare. We describe the case of a woman with HFH treated with low-density lipoprotein (LDL) apheresis by dextran sulfate adsorption who became pregnant and reached delivery uneventfully. LDL apheresis was performed biweekly, and lipoprotein analyses in pre-apheresis samples showed progressive increases in triglyceride, total cholesterol, and apolipoprotein (apo) B plasma concentrations. The fractional catabolic rate (FCR) for LDL cholesterol, as estimated by the first-order disappearance constants (k values) of the recovery of LDL cholesterol concentration to basal values after each apheresis session, increased more than threefold from week-24 to week-4 (labor is considered as time 0). After delivery basal values were recovered, but normalization was slower for LDL cholesterol than for the other lipidic parameters. High-density lipoprotein (HDL) showed a different pattern: HDL3 remained stable throughout gestation, whereas HDL2 cholesterol and apo A-I had a maximum at midgestation, then declined, and finally increased again at late gestation. With the exception of this latter increase of HDL2, all the other changes in lipoprotein concentrations during pregnancy and postpartum were similar to those found in healthy women. Thus, LDL apheresis does not interfere with physiologic adaptations of lipoprotein metabolism during pregnancy in HFH patients.     

Specific and scavenger low-density lipoprotein receptors involved in the disturbed lipid metabolism of patients with non-insulin-dependent diabetes mellitus are independent of obesity

Comparative studies were performed on monocyte-derived macrophages (MDMs), prepared by a 72-hour incubation of blood monocytes obtained from patients with non-insulin-dependent diabetes mellitus (NIDDM) and age-matched obese and non-obese controls. The MDMs, after a 72-hour culturing, expressed both specific and scavenger low-density lipoprotein (LDL) receptors on their surfaces. To study the binding capacity of both receptor types, [125I]LDL and [125I] acetylated LDL (acLDL) were applied to cells and the labeled ligands were then monitored to estimate the rate of intracellular degradations. The LDL-induced inhibition of endogenous cholesterol synthesis and the acLDL-triggered apolipoprotein (apo) E secretion were also studied, as the biological marker of receptor activation. The results indicate that the binding capacities of both specific and scavenger LDL receptors were not reduced in MDMs of diabetic patients. However, the intracellular degradation after LDL incorporation was decreased. The LDL-induced inhibition of cholesterol synthesis and the acLDL-transmitted apo E secretion were also found to be decreased in the MDMs of patients with NIDDM as compared with the obese and non-obese control groups. The NIDDM-induced impaired signal transduction of both specific and scavenger LDL receptors suggests an unclarified functional alteration of both receptor structures.     

Cholesteryl ester transfer protein gene polymorphism is a determinant of HDL cholesterol and of the lipoprotein response to a lipid-lowering diet in type 1 diabetes

The TaqIB cholesteryl ester transfer protein (CETP) gene polymorphism (B1B2) is a determinant of HDL cholesterol in nondiabetic populations. Remarkably, this gene effect appears to be modified by environmental factors. We evaluated the effect of this polymorphism on HDL cholesterol levels and on the lipoprotein response to a linoleic acid-enriched, low-cholesterol diet in patients with type 1 diabetes. In 44 consecutive type 1 diabetic patients (35 men), CETP polymorphism, apolipoprotein (apo) E genotype, serum lipoproteins, serum CETP activity (measured with an exogenous substrate assay, n = 30), clinical variables, and a diet history were documented. The 1-year response to diet was assessed in 14 type 1 diabetic patients, including 6 B1B1 and 6 B1B2 individuals. HDL cholesterol was higher in 10 B2B2 than in 14 B1B1 homozygotes (1.63 +/- 0.38 vs. 1.24 +/- 0.23 mmol/l, P < 0.01). HDL cholesterol, adjusted for triglycerides and smoking, was 0.19 mmol/l higher for each B2 allele present. CETP activity levels were not significantly different between CETP genotypes. Multiple regression analysis showed that VLDL + LDL cholesterol was associated with dietary polyunsaturated:saturated fatty acids ratio (P < 0.02) and total fat intake (P < 0.05) in the B1B1 homozygotes only and tended to be related to the presence of the apo E4 allele (P < 0.10). In response to diet, VLDL + LDL cholesterol fell (P < 0.05) and HDL cholesterol remained unchanged in 6 B1B1 homozygotes. In contrast, VLDL + LDL cholesterol was unaltered and HDL cholesterol decreased (P < 0.05) in 6 B1B2 heterozygotes (P < 0.05 for difference in change in VLDL + LDL/HDL cholesterol ratio). This difference in response was unrelated to the apo E genotype. Thus, the TaqIB CETP gene polymorphism is a strong determinant of HDL cholesterol in type 1 diabetes. This gene effect is unlikely to be explained by a major influence on the serum level of CETP activity, as an indirect measure of CETP mass. Our preliminary data suggest that this polymorphism may be a marker of the lipoprotein response to dietary intervention.     

HDL causes mesangial cell mitogenesis through a tyrosine kinase-dependent receptor mechanism

Hypercholesterolemia and mesangial cell proliferation have been proposed to play a role in the progression of glomerulosclerosis in diabetic nephropathy and other renal diseases. Although LDL is mitogenic for and cytotoxic to mesangial cells, the effect of HDL on these cells is unknown. HDL stimulates fibroblast mitogenesis and is the principal cholesterol-bearing lipoprotein in the rat, the experimental model for studying the effect of hyperlipidemia on renal disease. Insulin is mitogenic in several cell systems, and its levels are increased in serum in non-insulin-dependent diabetes mellitus. This study investigates whether HDL acts as a growth factor in mesangial cells and whether it functions in parallel with insulin. It was found that HDL at protein concentrations between 10 and 500 microg/ml, both alone and in the presence of 100 nM insulin, increased DNA synthesis in mesangial cells (129 to 165% of control for HDL alone; 140 to 235% for HDL plus insulin), whereas HDL at 1000 microg/ml and greater inhibited mesangial cell proliferation. Insulin alone at 100 nM stimulated [3H]thymidine incorporation in the same cell system (145% of control); the mitogenic effect of insulin was additive to that of HDL. Purified apo A-I had a similar effect, but at significantly lower concentrations. Specific binding of HDL to mesangial cells was demonstrated (B(max) [binding constant] of 5.19 +/- 0.70 x 10(-7) micromol of HDL bound/mg cell protein and K(b) of 2.83 +/- 0.22 nM). Tetranitromethane alters apo A-I, preventing binding to its cognate receptor. Tetranitromethane-modified HDL did not bind to mesangial cells and had no effect on [3H]thymidine incorporation. Addition of HDL to mesangial cells caused an immediate transient increase in free intracellular calcium in several representative mesangial cells, similar to the response seen with platelet-derived growth factor. The mitogenic effect of HDL was not altered after attenuation of cellular protein kinase C activity, but the stimulatory effect of HDL alone and in combination with insulin on DNA synthesis was completely eliminated after inhibition of cellular tyrosine kinases by 24-h pretreatment with 0.25 microM herbimycin A. Thus, HDL binds to a specific apo A-I-dependent receptor, promotes DNA synthesis, and initiates second-messenger events by a tyrosine kinase-dependent and protein kinase C-independent mechanism.     

Decreased cholesterol efflux from fibroblasts of a patient without Tangier disease, but with markedly reduced high density lipoprotein cholesterol levels

A 51-yr-old woman without clinical evidence of Tangier disease, but with an extremely low high density lipoprotein (HDL) cholesterol level, was studied. No defect in the major structural protein of HDL, apolipoprotein AI (apo AI), was detected. A preponderance of small HDL particles in the patient's plasma suggested defective uptake of cellular cholesterol. Efflux of [3H]cholesterol from patient fibroblasts to normal apo AI was decreased 50%. Cholesterol efflux to HDL was also decreased, but efflux to trypsin-modified HDL was not. The patient's cells partitioned more exogenously provided [3H]cholesterol into free cholesterol and synthesized greater amounts of phosphatidylcholine than did normal or Tangier fibroblasts. Her fibroblasts did not differ from normal fibroblasts in sterol synthesis rate, cellular cholesterol and cholesterol ester content, or incorporation of oleate into cholesterol ester. The data indicate the presence of a defect in apolipoprotein-dependent cellular cholesterol efflux that differs from that seen in Tangier disease. These findings are the first evidence that other low HDL cholesterol syndromes, besides Tangier disease, may also be associated with cholesterol efflux abnormalities. The identification of mutant genes responsible for apolipoprotein-mediated efflux abnormalities should provide valuable insights into cellular mechanisms involved in the reverse cholesterol transport pathway.     

Schizophrenia and delusional disorder in older age: community prevalence, incidence, comorbidity, and outcome

Obesity is associated with dyslipidaemia characterised by increased fasting triglyceride and decreased high-density lipoprotein (HDL) concentrations. Causes for obesity-associated dyslipidaemia include insulin resistance, excessive caloric intake, increased free fatty acid production and disturbances in the counter-regulatory hormones. We examined the relationships between lipid parameters and obesity before and after adjustment of insulin resistance in 902 Hong Kong Chinese men. After adjustment for age, smoking and insulin resistance, increasing body mass index (BMI) and waist-to-hip ratio (WHR) remained closely associated with increased concentrations of triglyceride and apolipoprotein B (apo B), increased ratios between low-density lipoprotein (LDL) and HDL (LDL/HDL), and that between apo B and LDL (apo B/LDL), increased fasting and 2-h plasma glucose and insulin, as well as decreased concentrations of HDL, HDL2 and apolipoprotein A-I (apo A-I). On stepwise multiple regression analysis using age, BMI, WHR, insulin resistance and fasting plasma glucose as independent variables, BMI and WHR were the major determinants for the variance of triglyceride, HDL and its subfractions, LDL/HDL, apo B and apo B/LDL. Age was the most important predictor for total cholesterol and LDL. Insulin resistance only explained less than 1% of the variance in triglyceride and apo B. This was compared to a variance between 10 and 16% in these parameters as explained by BMI and/or WHR. In conclusion, obesity is associated with dyslipidaemia in Chinese men, characterised by increased plasma triglyceride, apo B, LDL/HDL, apo B/LDL, and decreased HDL, HDL2 and apo A-I concentrations. Obesity independent of insulin resistance, in particular central adiposity as reflected by increased WHR, was the most important independent variable for many of these lipid abnormalities. Our results emphasised the multifactorial linkage between obesity and dyslipidaemia.     

Polymorphism of apolipoprotein E, lipoprotein(a), and other lipoproteins in children with type I diabetes

We assessed the effect of particular apolipoprotein (apo) E phenotypes, lipoprotein(a) [Lp(a)], and other lipoproteins on the development of dyslipoproteinemia in 450 patients with type I diabetes, ages 13-14 years. The control group consisted of 450 healthy school children of both sexes, ages 13-14 years. Both groups were found to be normolipidemic, but the concentration of Lp(a) was significantly (P < 0.05) higher in the diabetic children than in the control group. Apo E 3/2 and apo E 4/4 phenotypes were more frequent in the group of diabetics. Diabetics with the apo E 3/3 phenotype had higher concentrations of very-low-density lipoprotein (VLDL) and Lp(a), and lower concentrations of low-density lipoprotein (LDL) than the apo E 3/3 nondiabetics. For apo E 3/2 phenotypes, total cholesterol, LDL cholesterol, LDL, apo A-I, and Lp(a) concentrations were higher in the diabetic children than in the control group; for apo E 4/3 phenotypes, this was true for triglycerides and VLDL cholesterol. The distribution of Lp(a) lipoprotein concentrations between 0.01 and > 0.5 g/L indicated a more frequent occurrence of higher Lp(a) values in diabetic children than in the control group. Results of this study indicate that an increased concentration of Lp(a) lipoprotein and apo E 3/2 and apo E 4/3 phenotypes contribute to the expression of dyslipoproteinemia in type I diabetes in childhood.     

Efflux of intracellular versus plasma membrane cholesterol in HepG2 cells: different availability and regulation by apolipoprotein A-I

We have compared the efflux of cholesterol from different cellular pools of human hepatoma cells HepG2 using intact cells or isolated membrane fractions. To label different pools, cells were incubated with either unesterified [14C]cholesterol that had been incorporated into high density lipoproteins ([14C]FC-HDL), low density lipoproteins ([14C]FC-LDL), or phosphatidylcholine liposomes ([14C]FC-PC), or with [14C]acetate. Cell fractionation revealed that labeling of cells with [14C]FC-PC resulted in the incorporation of [14C]cholesterol almost exclusively into the plasma membrane (PM), while incubation with [14C]FC-HDL resulted in the majority of [14C]cholesterol incorporation into the PM, but with a smaller component associated with lysosomes. Labeling with [14C]FC-LDL or [14C]acetate led to an accumulation of [14C]cholesterol predominantly in lysosomes or the endoplasmic reticulum (ER), respectively. When the kinetics of [14C]cholesterol efflux was analyzed after pulse-labeling of different cellular pools, half-times of cholesterol efflux from lysosomes and ER were significantly longer than that from PM. In another set of experiments, when both labeling and efflux times varied, efflux of [14C]cholesterol from the PM to human serum after 1.5 h pulse and chase incubations was double that from lysosomes and 8-fold that from ER. Extension of the incubation times from 1.5 to 3 h diminished the difference in cholesterol efflux from different membranes. Further incubation to 6 h almost abolished the different responses. Cell-free preparations of membranes, obtained from cells labeled with [14C]cholesterol, showed no differences in cholesterol efflux. No differences in the distribution of [14C]cholesterol released into serum among lipoprotein subfractions was observed. Pretreatment of the serum with Fab fragments of polyclonal rabbit anti-human apolipoprotein A-I antibodies reduced its ability to promote efflux of cholesterol from the ER by 77%, but had no effect on cholesterol efflux from the PM. Fab fragments of non-immune IgG had no effect on the efflux of both ER and PM cholesterol. We conclude that the availability of cellular cholesterol for efflux from HepG2 cells is strongly influenced by its subcellular location, and is regulated by apolipoprotein A-I.     

Cellular cholesterol transport and efflux in fibroblasts are abnormal in subjects with familial HDL deficiency

Familial high density lipoprotein (HDL) deficiency (FHD) is a genetic lipoprotein disorder characterized by a severe decrease in the plasma HDL cholesterol (-C) level (less than the fifth percentile). Unlike Tangier disease, FHD is transmitted as an autosomal dominant trait. FHD subjects have none of the clinical manifestations of Tangier disease (lymphoid tissue infiltration with cholesteryl esters and/or neurological manifestations). Plasmas from FHD subjects contain pre-beta-migrating HDLs but are deficient in alpha-migrating HDLs. We hypothesized that a reduced HDL-C level in FHD is due to abnormal transport of cellular cholesterol to the plasma membrane, resulting in reduced cholesterol efflux onto nascent HDL particles, leading to lipid-depleted HDL particles that are rapidly catabolized. Cellular cholesterol metabolism was investigated in skin fibroblasts from FHD and control subjects. HDL3- and apolipoprotein (apo) A-I-mediated cellular cholesterol and phosphatidylcholine efflux was examined by labeling cells with [3H]cholesterol and [3H]choline, respectively, during growth and cholesterol loading during growth arrest. FHD cells displayed an approximately 25% reduction in HDL3-mediated cellular cholesterol efflux and an approximately 50% to 80% reduction in apoA-I-mediated cholesterol and phosphatidylcholine efflux compared with normal cells. Cellular cholesterol ester levels were decreased when cholesterol-labeled cells were incubated with HDL3 in normal cells, but cholesterol ester mobilization was significantly reduced in FHD cells. HDL3 binding to fibroblasts and the possible role of the HDL binding protein/vigilin in FHD were also investigated. No differences were observed in 125I-HDL3 binding to LDL-loaded cells between FHD and control cells. HDL binding protein/vigilin mRNA levels and its protein expression were constitutively expressed in FHD cells and could be modulated ( approximately 2-fold increase) by elevated cellular cholesterol in normal cells. In conclusion, FHD is characterized by reduced HDL3- and apoA-I-mediated cellular cholesterol efflux. It is not associated with abnormal cellular HDL3 binding or a defect in a putative HDL binding protein.     

Increases in dietary cholesterol and fat raise levels of apoprotein E-containing lipoproteins in the plasma of man

Previous studies from this laboratory have determined that diets containing the usual amounts of fat to which are added 750-1500 mg/day cholesterol elevate the plasma cholesterol concentration by variable amounts, depending upon the ratio of polyunsaturated to saturated fatty acids (P/S ratio) of the diet. Diets with P/S ratios of 0.25-0.4 are accompanied by elevations of low density lipoprotein (LDL) cholesterol, whereas diets with a P/S ratio of 2.5 produce no significant changes in cholesterol levels. On the low P/S ratio diets, the structure, composition, and interaction with cultured fibroblasts of LDL are not significantly changed. Plasma high density lipoprotein (HDL) cholesterol levels remain constant, but HDL2 increase relative to HDL3. In the present study, not only dietary cholesterol but also total dietary fat was altered. Six normal young men were fed a basal diet consisting of 18% protein, 51% carbohydrate, and 30% fat, containing 250 mg/day cholesterol. After 2 weeks, an experimental diet consisting of 18% protein, 42% carbohydrate, and 39% fat, containing 1760 mg/day cholesterol, was fed for 4 weeks. The P/S ratios of both diets were about 0.4. Plasma samples were taken twice during each dietary period from 12- to 14-h-fasted subjects and analyzed for their contents of lipoprotein lipids. Plasma levels of LDL and HDL cholesterol increased by 30 and 13 mg/dl, respectively; total and very low density lipoprotein (VLDL) triglyceride concentrations were unaltered. The plasma concentrations of apoproteins (apo) B, E. and A-I, but not A-II, were elevated. Plasma samples also were studied by zonal ultracentrifugation, gel permeation column chromatography, and Pevikon electrophoresis. Although on zonal ultracentrifugation the total concentrations of LDL were increased, the flotation properties and chemical compositions of LDL were not changed. By contrast, HDL2 and HDL3L concentrations increased, and HDL2 became enriched with cholesteryl esters. On gel permeation chromatography, with the subjects on the basal diet, plasma cholesterol eluted in two peaks, corresponding to LDL and HDL. The sizes of the peaks increased on the experimental diet. ApoE eluted in two peaks: one at the leading edge of LDL (corresponding to VLDL or IDL) and the other in the area between LDL and HDL, corresponding to HDLC. On the experimental diet, the apoE peak between LDL and HDL increased. On Pevikon electrophoresis apoE migrated between the LDL and HDL bands. This apoE peak was increased on the experimental diet. These findings suggest that increasing the concentrations of both dietary cholesterol and total fat can increase the levels of plasma LDL, HDL2, and HDLC in fasting normal subjects. Thus, the concentrations of some putatively atherogenic as well as antiatherogenic lipoproteins increased in plasma, and the apparent paradox between the epidemiological and metabolic behaviors of some lipoproteins remains. Clearly, more work is needed to resolve the roles of various lipoproteins in plasma in atherosclerosis.