Effects of different doses of atorvastatin on human apolipoprotein B-100, B-48, and A-I metabolism

Nine hypercholesterolemic and hypertriglyceridemic subjects were enrolled in a randomized, placebo-controlled, double-blind, crossover study to test the effect of atorvastatin 20 mg/day and 80 mg/day on the kinetics of apolipoprotein B-100 (apoB-100) in triglyceride-rich lipoprotein (TRL), intermediate density lipoprotein (IDL), and LDL, of apoB-48 in TRL, and of apoA-I in HDL. Compared with placebo, atorvastatin 20 mg/day was associated with significant reductions in TRL, IDL, and LDL apoB-100 pool size as a result of significant increases in fractional catabolic rate (FCR) without changes in production rate (PR). Compared with the 20 mg/day dose, atorvastatin 80 mg/day caused a further significant reduction in the LDL apoB-100 pool size as a result of a further increase in FCR. ApoB-48 pool size was reduced significantly by both atorvastatin doses, and this reduction was associated with nonsignificant increases in FCR. The lathosterol-campesterol ratio was decreased by atorvastatin treatment, and changes in this ratio were inversely correlated with changes in TRL apoB-100 and apoB-48 PR. No significant effect on apoA-I kinetics was observed at either dose of atorvastatin. Our data indicate that atorvastatin reduces apoB-100- and apoB-48-containing lipoproteins by increasing their catabolism and has a dose-dependent effect on LDL apoB-100 kinetics. Atorvastatin-mediated changes in cholesterol homeostasis may contribute to apoB PR regulation.     

Cholesterol turnover and metabolism in two patients with abetalipoproteinemia

Total body turnover of cholesterol was studied in two patients with abetalipoproteinemia, a 32-year-old man and a 31-year-old woman. The patients received [14C]cholesterol intravenously, and the resulting specific activity-time curves (for 40 and 30 weeks, respectively) were fitted with a three-pool model. Parameters were compared with those from studies of cholesterol turnover in 82 normal and hyperlipidemic subjects. A three-pool model gave the best fit for the abetalipoproteinemic patients, as well as for the 82 previously studied subjects, suggesting general applicability of this model. Cholesterol production rates in the two abetalipoproteinemic subjects (0.82 and 0.89 g/day) were close to values predicted for persons of their body weight. Thus, total body turnover rate of cholesterol was quite normal in abetalipoproteinemia, confirming previous reports. Very low values (9.2 and 8.4 g) were found for M1, the size of the rapidly exchanging compartment pool 1, in the two abetalipoproteinemic subjects. These values were well below the values predicted (from the comparison study population) for normal persons of this size with low plasma cholesterol levels. For one patient, total body exchangeable cholesterol was very low, although not significantly below the predicted values for a person of his size. In the second patient, the observed estimate for total body exchangeable cholesterol was well within the range of values predicted for persons of her size with low to extremely low cholesterol levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of intestinal alkaline phosphatase in serum apolipoprotein B-48 level and its association with ABO and secretor blood group types

Serum levels of intestinal alkaline phosphatase (IAP), a protein implicated in transcellular transport of chylomicrons, vary among ABO blood groups. In rat enterocytes, IAP is associated with chylomicron secretion, but the rat expresses only blood group A. It is not known whether chylomicron secretion may be affected in humans who express multiple blood group types. Serum samples from 40 healthy subjects were obtained after overnight fast and 3h after a high-fat meal, and assayed for IAP and apolipoprotein B-48 (apoB-48), both proteins exclusive to intestine, although only apoB-48 is found in chylomicrons. The two proteins were greater in subjects without blood antigen A (B and O) than in those with this antigen (A and AB); 2.4- and 4.7-fold for IAP and 1.5- and 2.0-fold for apoB-48 before and after the meal, respectively. Moreover, IAP and apoB-48 levels were strongly correlated in the subjects with the secretor phenotype (r > 0.81). These results indicate that IAP is strongly involved in chylomicron formation and fatty acid metabolism might change among ABO blood type. In addition, ABO blood type classification in apoB-48 measurement would improve the diagnostic value in the evaluation of metabolic syndrome.     

Evidence of increased secretion of apolipoprotein B-48-containing lipoproteins in subjects with type 2 diabetes

Patients with type 2 diabetes have high levels of triglyceride-rich lipoproteins (TRLs), including apolipoprotein B-48 (apoB-48)-containing TRLs of intestinal origin, but the mechanism leading to overaccumulation of these lipoproteins remains to be fully elucidated. Therefore, the objective of this study was to examine the in vivo kinetics of TRL apoB-48 and VLDL, intermediate density lipoprotein (IDL), and LDL apoB-100 in type 2 diabetic subjects (n = 11) and nondiabetic controls (n = 13) using a primed-constant infusion of l-[5,5,5-D(3)]leucine for 12 h in the fed state. Diabetic subjects had significantly higher fasting glycemia, higher fasting insulinemia, higher plasma triglyceride, and lower HDL-cholesterol levels than controls. Compared with controls, diabetic subjects had increased TRL apoB-48, VLDL apoB-100, and IDL apoB-100 pool sizes as a result of increased production rates (PRs) and reduced fractional catabolic rates of these lipoprotein subfractions. Furthermore, multiple linear regression analyses revealed that the diabetic/control status was an independent predictor of TRL apoB-48 PR and represented nearly 35% of its variance. These results suggest that the overaccumulation of TRLs seen in patients with type 2 diabetes is attributable to increased PRs of both intestinally derived apoB-48-containing lipoproteins and TRL apoB-100 of hepatic origin and to decreased catabolism of these subfractions.     

Apolipoprotein B-100: immunolocalization and synthesis in human intestinal mucosa

Despite the evidence that the human small intestine produces two separate species of apoB mRNA encoding for B-100 and B-48, there is a paucity of data concerning the expression of the latter form in this organ. Using a high resolution immunogold approach, with specific polyclonal antibodies and a panel of monoclonal antibodies (2D8, 3A10, 4G3), both forms of apoB (B-48 and B-100) were revealed over enterocytes of pediatric intestinal samples. Intense labeling was observed over microvilli, apical smooth membrane vesicles, multivesicular bodies, the basolateral membrane, as well as the trans Golgi region. Only low labeling was found over the rough endoplasmic reticulum (rER). Similar patterns of apoB distribution characterized both duodenal and jejunal regions. The presence of labeling over the Golgi apparatus and rER suggests a synthetic activity of both forms of apoB by the epithelial cells. To test this hypothesis, human intestine was incubated with [3H]leucine, homogenized, and subjected to immunoprecipitation for apoB. Immunoprecipitates contained radioactivity mainly in apoB-48 with relatively small amounts in apoB-100 when examined by NaDodSO4-polyacrylamide gel electrophoresis. These findings were further supported by the biochemical determination of apoB-100 and apoB-48 in chylomicron particles isolated from thoracic duct lymph of a human donor. Taken together, our data suggest that the human intestine is able to synthesize and to express the apoB-100.     

Familial defective apolipoprotein B-100: a lesson from homozygous and heterozygous patients

Familial defective apolipoprotein B-100 (FDB) is a genetic disorder caused by a substitution of glutamine for arginine at residue 3500 of the apolipoprotein B-100 molecule. We have identified 23 heterozygotes and one homozygote for FDB (frequency 1:20) in a group of 510 patients with hypercholesterolemia. Mean age of the patients (18 females and 6 males) was 46 years. The diagnosis of FDB was based on point mutation PCR analysis of exon 26 of the apo B gene. Plasma lipids in heterozygous patients were: total cholesterol 8.76+/-1.2 mmol/l, triglycerides 1.42+/-0.5 mmol/l, HDL-cholesterol 1.43+/-0.3 mmol/l, LDL-cholesterol 6.69+/-1.2 mmol/l, apoB 1.69+/-0.4 g/l, Lp(a) 0.26+/-0.2 g/l. The most frequent apoE genotype was 3/3 (19 patients), apoE 3/4 genotype was found in 3 patients and one person had apoE 2/3. Xanthelasma palpebrarum was present in 4 patients and tendon xanthomas in 3 patients including the homozygote. Premature manifestation of coronary heart disease was revealed in 3 patients. Sixteen patients were treated with statins, a combination of statin and resin was used in 2 patients (including the homozygote), whereas six patients were treated with the diet only. We conclude that although the plasma lipid levels of total and LDL cholesterol in FDB patients are lower than in patients with familial hypercholesterolemia, the patients with FDB suffer from premature atherosclerosis. The therapeutic approach to FDB individuals and patients with familial hypercholesterolemia is very similar.     

Apolipoprotein C-II deficiency: detection of immunoreactive apolipoprotein C-II in the intestinal mucosa of two patients

Recent data suggest that mutant immunoreactive forms of apolipoprotein C-II (apoC-II) can be detected in the plasma of patients with the apoC-II deficiency syndrome. We studied the possible presence of apoC-II mutants in the plasma of two patients with apoC-II deficiency by immunological means. The patients were hypertriglyceridemic, and apoC-II was undetectable in plasma as determined by radial immunodiffusion, electroimmunoassay, and immunonephelometry. Furthermore, apoC-II was undetectable either by electrophoresis or by immunoblotting in the plasma of the probands, while apoC-II was present in the plasma of their parents, although at less than half-normal concentration. Immunochemical localization of apoC-II, however, showed that the apoprotein could be detected within the enterocytes obtained from the intestinal mucosa of the patients. From these data we conclude that the patients synthesize apoC-II, at least in the intestine.     

Familial defective apolipoprotein B-100: a mutation of apolipoprotein B that causes hypercholesterolemia

Familial defective apolipoprotein B-100 is a genetic disorder of apolipoprotein B-100 that causes moderate to severe hypercholesterolemia. A single amino acid mutation in apolipoprotein B diminishes the ability of low density lipoproteins to bind to the low density lipoprotein receptor. Low density lipoproteins accumulate in the plasma because their efficient receptor-mediated catabolism is disrupted. This mutation has been identified in the United States, Canada, and Europe and is estimated to occur at a frequency of approximately 1/500 in these populations. Thus, it appears that this newly described disorder may be a significant genetic cause of hypercholesterolemia in Western societies.     

The relationship of serum lipoprotein lipase mass with fasting serum apolipoprotein B-48 and remnant-like particle triglycerides in type 2 diabetic patients.

BACKGROUND: There have been no previous reports showing specifically the relation between lipoprotein lipase (LPL) and apolipoprotein (apo) B-48 or remnant metabolism. In this study, we have clarified the relationships of LPL mass in pre-heparin with serum apo B-48 measured by enzyme-linked immunosorbent assay, triglycerides (TG), and remnant-like particle triglycerides (RLP-TG). MATERIAL AND METHODS: Seventy-nine type 2 diabetic subjects [age, 55+/-13; body mass index (BMI), 25+/-5.0 kg/m2; fasting plasma glucose (FPG), 7.39+/-2.22 mmol/l, HbA1c, 6.5+/-1.3%, total cholesterol (TC), 5.36+/-1.09 mmol/l, TG, 2.32+/-2.53 mmol/l; HDL-C, 1.22+/-0.44 mmol/l; serum LPL mass, 45+/-22 ng/ml; apo B-48, 6.6+/-6.3 microg/ml] were recruited in this study. Fasting serum apo B-48 were measured by ELISA using anti-human apo B-48 monoclonal antibodies (MoAb) and LPL mass by ELISA using anti-bovine milk LPL MoAb. RLP-TG levels were measured using monoclonal antibodies to apo B-100 and apo A-1. RESULTS: There was no relationship of LPL mass to age, BMI, FPG, and HbA1c. Serum LPL mass was correlated inversely with TG (r=-0.529 p<0.0001) and positively with HDL-C (r=0.576, p<0.0001). Also, LPL mass showed inverse correlations with apo B-48 (r=-0.383 p<0.0001) and RLP-TG (r=-0.422 p<0.0001, n=51). Multiple regression analysis with TG, apo B-48, or RLP-TG as dependent variables, and age, gender, BMI, plasma glucose, and LPL mass as independent variables showed that LPL mass was associated independently with TG, apo B-48, or RLP-TG. CONCLUSION: The decrease in LPL protein mass could cause an increase in serum apo B-48 and RLP-TG levels, which is related to the retardation of remnant metabolism.     

Enrichment of apolipoprotein B-48 in the LDL density class following in vivo inhibition of hepatic lipase

In order to explore the in vivo function of hepatic lipase, rats were injected with goat anti-rat hepatic lipase serum which produced a complete and specific inhibition of heparin-releasable hepatic lipase. In the fasting rats, protein, phospholipid and free cholesterol expressed as either mass or percent weight increased significantly in low-density lipoprotein (LDL) and high-density lipoprotein 2 (HDL-2) fractions. These three constituents were not affected in the VLDL and HDL-3 lipoproteins. In the fat-loaded (1 ml corn oil) rat, 6 h post inhibition of hepatic lipase triacylglycerol, phospholipid and free cholesterol concentrations in the d less than 1.006 fraction were 2.5-fold higher in the inhibited animals than in the control rats. The composition of the d less than 1.006 fraction was also affected. Expressed as percent mass, protein was lower (5.2 +/- 1.2 vs. 10.3 +/- 1.5, P less than 0.001) as was cholesteryl ester (1.7 +/- 0.7 vs. 2.6 +/- 0.4, P less than 0.01); triacylglycerol was elevated (77.2 +/- 4.0 vs. 72.6 +/- 2.4, P less than 0.025), as was free cholesterol (3.0 +/- 0.6 vs. 2.4 +/- 0.2, P less than 0.025). Overall, inhibition lowered the ratio of surface-to-core constituents suggesting a larger mean particle diameter. SDS-polyacrylamide gel electrophoresis showed the intermediate- and low-density lipoprotein from treated rats to be significantly enriched in apolipoprotein B-48. In the LDL fraction, apolipoprotein B-48 accounted for 62 +/- 14% of the total apolipoprotein B in the inhibited rats, vs. 12 +/- 2% in the control rats. The above results support the previously described in vivo function of hepatic lipase as a phospholipase. In addition, the results demonstrate a role of hepatic lipase in the catabolism of chylomicrons. Since removal of apolipoprotein B-48-containing lipoproteins is dependent upon apolipoprotein E, their appearance in the LDL fraction implies a masking of apolipoprotein E-binding determinants.     

Behavior of fetal intestinal organ culture explanted onto a collagen substratum

A model of organ culture of 18 day old fetal rat intestine (Quaroni, 1985) was modified and characterized in the present work with the purpose of developing an in vitro model for the study of intestinal epithelial cell behaviour. Fragments of this intestine were kept in suspension culture for 7 days and then explanted onto collagen (type I) matrix. Within a day, the fragments became anchored to the substratum and a circular monolayer grew out to about 1 cm diameter. In the fragments, an outer layer of absorptive epithelial cells came to enclose a stroma, which was polarized into a loose (mesenchymal) and a dense portion. The dense portion contained a mixture of smooth muscle cells and primitive stem-type epithelial cells ('p-cells'). After explantation, at the contact point with the matrix, the epithelium broke up and the mesenchyme grew into the matrix and anchored the fragment. The epithelial edges now became continuous with the developing monolayer. Radioautography with tritiated thymidine indicated a constant cell renewal in epithelium and monolayer apparently from foci of p-cells, a reserve population of which was seen to be sequestered among the smooth muscle cells. Activated stem cells could differentiate into three mature epithelial phenotypes, each differentiation pathway apparently being determined by the type of underlying stroma. Immunohistochemistry using gold- and fluorescein-labeled monoclonal antibodies indicated that adult differentiation-specific markers (e.g. brush border enzymes) were present in the fragment epithelium but not in the monolayer cells. On the other hand, the monolayer cells could be induced to express some of these markers by contact with mesenchymal cells or by co-culturing with fibroblastic cell lines. Matrigel substratum mixed with collagen (type I) supported the appearance in monolayer of strands positive for amino-peptidase and lactase. The model thus appears to be suitable for the in vitro study of epithelial renewal and differentiation, and it has already provided some results in this respect.     

Measurement of human apolipoprotein B-48 and B-100 kinetics in triglyceride-rich lipoproteins using [5,5,5-2H3]leucine.

A primed-constant infusion of deuterated leucine was used in humans to determine the maximal level of enrichment at plateau of apolipoprotein (apo)B-48 and apoB-100 which are synthesized in the intestine and liver, respectively, and to compare the kinetics of these two proteins under identical conditions. Eight normal subjects (four post-menopausal females and four males) over the age of 40 were studied in the constantly fed state over a 20-h period by providing small hourly feedings of identical composition. [5,5,5-2H3]Leucine (10 mumol/kg body weight followed by 10 mumol/kg body weight per hour) was infused over 15 h intravenously. The enrichment of deuterated leucine in apoB-48 and apoB-100 triglyceride-rich lipoproteins isolated by ultracentrifugation (d less than 1.006 g/ml) was determined during the entire infusion period. The plateau level of enrichment in triglyceride-rich lipoprotein apoB-48 was 3.96 +/- 1.41 tracer/tracee ratio (%) which was 39.7% of the plasma leucine enrichment level. The plateau level of enrichment in triglyceride-rich lipoprotein apoB-100 was 7.23 +/- 1.17 tracer/tracee ratio (%) which was 72.5% of the plasma leucine enrichment level. Mean fractional secretion rates of triglyceride-rich lipoprotein apoB-48 and apoB-100 were 4.39 +/- 2.00 and 5.39 +/- 1.98 pools per day, respectively, with estimated residence times of 5.47 and 4.45 hours, respectively. The data indicate that in the fed state there is about a twofold difference in the plateau enrichment of an intestinally derived protein, as compared to one of hepatic origin, most likely attributable to differences in the enrichment of the intracellular leucine in the two organs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a critical lysine residue in apolipoprotein B-100 that mediates noncovalent interaction with apolipoprotein(a)

We have previously shown that lipoprotein(a) (Lp(a)) assembly involves an initial noncovalent interaction between sequences within apolipoprotein(a) (apo(a)) kringle IV types 5-8 and the amino terminus of apolipoprotein B-100 (sequences between amino acids 680 and 781 in apoB-100), followed by formation of a disulfide bond. In the present study, citraconylation of lysine residues in apoB-100 abolished the ability of the modified low density lipoprotein to associate with apo(a), thereby demonstrating a direct role for lysine residues in apoB in the first step of Lp(a) assembly. To identify specific lysine residues in the amino terminus of apoB that are required for the noncovalent interaction, we initially used an affinity chromatography method in which recombinant forms of apo(a) (r-apo(a)) were immobilized on Sepharose beads. Assessment of the ability of carboxyl-terminal truncations of apoB-18 to bind to r-apo(a)-Sepharose revealed that a 25-amino acid sequence in apoB (amino acids 680-704) bound specifically to apo(a) in a lysine-dependent manner; citraconylation of the lysine residues in the apoB derivative encoding this sequence abolished the binding interaction. Using fluorescence spectrometry, we found that a synthetic peptide corresponding to this sequence bound directly to apo(a); the peptide also reduced covalent Lp(a) formation. Lysine residues present in this sequence (Lys(680) and Lys(690)) were mutated to alanine in the context of apoB-18. We found that the apoB-18 species containing the Lys(680) mutation was incapable of binding to r-apo(a)-Sepharose columns, whereas the apoB-18 species containing the Lys(690) mutation exhibited slightly reduced binding to these columns. Taken together, our data indicate that Lys(680) is critical for the noncovalent interaction of apo(a) and apoB-100 that precedes covalent Lp(a) formation.     

The assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins in McA-RH7777 cells

We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [(3)H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 +/- 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum.     

Thrombin cleavage of apolipoprotein Bh of rabbit LDL: structural comparisons with human apolipoprotein B-100.

Rabbit plasma low density lipoprotein (LDL) contains one major apolipoprotein of apparent molecular weight of 320 kDa, designated apolipoprotein (apo) Bh, while another component termed apoB1 of apparent molecular weight of 220 kDa is found in chylomicrons. The fragments generated by thrombin digestion of the protein moieties of rabbit and human LDL were separated by polyacrylamide gradient gel electrophoresis and compared. As in the human species, the enzyme produced limited cleavage patterns of rabbit LDL apoB. Within the first 2 h, two fragments (Tr1 and Tr2, with apparent molecular weights 280,000 and 44,000, respectively) appeared. Longer incubations led to the production of two additional peptides, Tr3 and Tr4 (apparent molecular weights 180,000 and 96,000, respectively). Ten monoclonal antibodies, developed against rabbit LDL and designated P01 to P10, were found to react with rabbit apoB. Some also cross-reacted with human apoB. Epitope mapping, performed with these antibodies, showed that Tr3 and Tr4 were derived from the further degradation of Tr1. The rabbit is one of the most frequently used animals in atherosclerosis research. Its LDL receptor has been characterized and there exists a strain of homozygous LDL receptor-deficient rabbits referred to as WHHL rabbits. Despite this, little has been done to characterize the structure of rabbit apoB; only a short region has been sequenced and shown to be the carboxyl-terminal region, the rabbit apoB1. The molecular weight of human apoB (550,000) is much larger than rabbit apoBh. In both species, a primary and secondary thrombin cleavage occur, but the size of the fragments produced is very different between the two species. Identification of the thrombolytic fragments of the rabbit apoB have afforded the opportunity to compare the structures of both apoB species.     

Degradation of newly synthesized apolipoprotein B-100 in a pre-Golgi compartment

The synthesis and secretion of apolipoprotein (apo) B-100 have been studied in a human hepatoblastoma cell line, the Hep G2 cells. Pulse-chase analysis showed that apoB-100 was not quantitatively recovered in the culture medium. To reveal the intracellular degradation of apoB-100 prior to secretion, cells were incubated with 1 microgram/ml Brefeldin A (BFA) which impeded protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus and the fate of apoB-100 retained in the cells was traced at 37 degrees C. A significant amount of intracellular apoB-100 (40-60%/h) was degraded during the chase period, whereas apoA-1 remained intact. ApoB-100 degradation was temperature dependent, no degradation was observed below 20 degrees C. This degradation process was not inhibited by chloroquine, leupeptin, pepstatin, and chymostatin, suggesting that lysosomal proteases were not involved and that apoB-100 was degraded in a pre-Golgi compartment which is either part of, or closely related to, the ER. Preincubation of cells with low density lipoproteins (LDL) induced a 22-32% increase in the degradation of apoB-100. This result raised the possibility that secretion of apoB-100 might be regulated through the intracellular degradation of apoB-100. These results suggest the existence of the degradation pathway for apoB-100 in a pre-Golgi compartment and an unique regulatory mechanism for apoB-100 secretion.