Absence of familial defective apolipoprotein B-100 in Finnish patients with elevated serum cholesterol

Familial defective apolipoprotein B-100 is a genetic disorder which is associated with elevated plasma LDL levels. It appears to result from a G----A mutation at nucleotide 10,708 in exon 26 of the apolipoprotein B-100 gene leading to a substitution of glutamine for arginine at amino acid residue 3500. We explored the possible role of this point mutation as a cause of elevated plasma cholesterol among the Finns, a genetically isolated population in which both hypercholesterolemia and coronary heart disease are common: 552 hyperlipidemic patients from Western and Southern Finland were screened either by assaying patient sera with monoclonal antibody MB47 or by amplifying the region of the apo B gene containing the nucleotide 10,708 followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes. Not a single individual with this particular mutation could be found. We conclude that familial defective apo B-100 is not a common cause of elevated plasma cholesterol in this population.     

Human apolipoprotein B-100: cloning, analysis of liver mRNA, and assignment of the gene to chromosome 2.

Human apolipoprotein B-100 (apo B-100) is the major apolipoprotein of low density lipoproteins and the principal ligand for interaction with the low density lipoprotein receptor. The human apo B-100 gene has been inserted into a lambda gt-11 expression vector, and the apo B-100 cDNA clones have been identified by screening with a monospecific apo B-100 antiserum, by screening with synthetic oligonucleotides based on the amino acid sequence of peptides isolated from apo B-100, and by immunoblot analysis of the expressed protein with a monoclonal antibody to apo B-100. The complete nucleotide and derived-amino acid sequence of a 1.7-kilobase cDNA clone of apo B-100 was determined. The 560-amino acid residues of apo B-100 contain no unique linear or repeating sequences of amino acids. The computer-predicted conformation of the apo B-100 protein contains segments of helical structure; however, a large portion of the protein is organized into beta-structure. The beta-structure may be important in lipid-apo B-100 interactions in low density lipoprotein and may contribute to the insolubility of delipidated apo B-100 in aqueous buffers. RNA blot hybridization analysis of liver mRNA utilizing a Nco I/HindIII apo B-100 cDNA probe revealed that the apo B-100 mRNA is 15-18 kilobases long, which is of sufficient size to code for a 250-387 kDa apolipoprotein, the proposed molecular size of delipidated plasma apo B-100. The gene for human apo B-100 has been localized to chromosome 2 by filter hybridization of human-mouse somatic cell hybrids utilizing a 400-base-pair Nco I/HindIII apo B-100 cDNA probe. This location is in contrast to the low density lipoprotein receptor that has been localized to chromosome 19. The cloning of human apo B-100 has provided new insights into the structure and physicochemical properties of apo B-100 and will facilitate studies on the factors modulating apo B-100 biosynthesis and the expression of the apo B-100 gene in patients with dyslipoproteinemias.     

Apolipoprotein B subspecies in chylomicrons isolated from a patient with chyluria

The diagnosis and the clinical course of a 17-year-old white male with chyluria are reported. Cloudy, milky urine appeared spontaneously, in the absence of edema or any signs or symptoms of parasitic infection. Pedal lymphangiography demonstrated the presence of a lymphatic renal fistula, and digital subtraction angiography showed aneurysmal dilatation of the aorta at the level of the renal arteries. This case provided an opportunity to ascertain which of the forms of apolipoprotein B were present in lymph chylomicrons. Apolipoprotein B is needed for chylomicron secretion. It exists in several forms--B-100, B-74, B-48, and B-26. After a meal consisting of fat, chylomicrons in which apolipoprotein B-48 was virtually the only apolipoprotein B present appeared in the urine, while apolipoprotein B-100 was the only apolipoprotein B present in the plasma very low-density lipoproteins. Chyluria disappeared two weeks after institution of a low-fat diet. This case illustrates an interesting, rare cause of chyluria. Because of the presence of chyluria, it was also demonstrated that chylomicrons in which apolipoprotein B-48 is virtually the only apolipoprotein B present are a physiologically normal product of the intestine.     

Treatment of rheumatoid arthritis with monoclonal CD4 antibody M-T151. Clinical results and immunopharmacologic effects in an open study, including repeated administration

Recent experimental and clinical data point to the T helper lymphocyte subset as playing a central role in the pathogenesis of rheumatoid arthritis (RA). Thus, a therapeutic strategy aimed specifically at the CD4 T cell subset is warranted. We treated patients with active RA for 7 days with a daily dose of 20 mg of CD4 monoclonal antibody M-T151, administered intravenously over 30 minutes. There were no negative side effects. According to changes in the combined parameters of Ritchie articular index, pain assessment, grip strength, and morning stiffness, 6 patients had a good response. Clinical improvement was greatest approximately 2 weeks after termination of the therapy and lasted from 4 weeks to 6 months. Of the serologic parameters of inflammation, only the C-reactive protein level improved in the patients with a favorable response. Close immunologic monitoring revealed a transient, selective depletion of CD4+ T cells after each infusion. During the entire treatment period, residual circulating CD4+ cells were found to be coated with CD4 antibody, whereas free antibody was detected in the serum only for approximately 8 hours after each infusion. Immediately after infusion, soluble CD4 antigen appeared in the serum. In addition to the cell-bound CD4 antibody, complement components could be detected on the surface of the remaining CD4+ cells. The proliferative response of peripheral blood mononuclear cells to purified protein derivative was significantly diminished 4 weeks after cessation of antibody treatment. Six patients showed a weak antibody response to mouse immunoglobulin. In 4 of the responders who received a second course of therapy (2 of them as outpatients), a therapeutic effect was noted that was similar to that after the first course. Only 1 patient, who had low titers of serum IgE anti-mouse Ig antibodies, showed a mild anaphylactic reaction at the end of the second course of therapy. Treatment of RA with the monoclonal CD4 antibody M-T151 seems to be a promising alternative, although the optimal dose and the regimen of administration are still to be defined.     

Cys4057 of apolipoprotein(a) is essential for lipoprotein(a) assembly.

Lipoprotein(a) contains one copy each of apolipoprotein B-100 and apolipoprotein(a). It has been hypothesized that a disulfide bond might exist between Cys4057 of apolipoprotein(a) and Cys3734 in apolipoprotein B-100. To investigate the role of Cys4057 for lipoprotein(a) assembly, wild-type and in vitro mutagenized apolipoprotein(a) cDNA plasmids were expressed in the human hepatocarcinoma line HepG2. The mutant plasmids encoded apolipoprotein(a) species with Cys4057 exchanged to either serine or glycine. Untransfected HepG2 cells, although able to secrete apolipoprotein B-100-containing lipoproteins, do not synthesize detectable amounts of apolipoprotein(a). After transfection of wild-type plasmid, almost all apolipoprotein(a) in the culture supernatant was present in lipoprotein(a)-like particles as demonstrated by immunoblotting, density-gradient centrifugation, and ELISA. The same analysis performed with supernatants of cells transfected with plasmids mutated in codon 4057 revealed free apolipoprotein(a) glycoprotein without detectable amounts of lipoprotein-associated apolipoprotein(a). Our results strongly suggest the existence of a disulfide bridge between Cys4057 of apolipoprotein(a) and apolipoprotein B-100 within recombinant lipoprotein(a) particles. Furthermore, they indicate that disulfide bridge formation is essential for assembly of the lipoprotein(a)-like complex produced by HepG2 cells and suggest a similar role of Cys4057 during lipoprotein(a) assembly in vivo.     

Relationships between the responses of triglyceride-rich lipoproteins in blood plasma containing apolipoproteins B-48 and B-100 to a fat-containing meal in normolipidemic humans.

The concentration of triglyceride-rich lipoproteins containing apolipoprotein (apo) B-48 (chylomicrons) and apo B-100 (very low density lipoproteins) was measured in blood plasma of healthy young men after an ordinary meal containing one-third of daily energy and fat. Plasma obtained in the postabsorptive state and at intervals up to 12 hr after the meal was subjected to immunoaffinity chromatography against a monoclonal antibody to apo B-100 that does not bind apo B-48 and a minor fraction of apo B-100 rich in apo E. Measurements of the concentrations of components of the total and unbound triglyceride-rich lipoproteins separated from plasma by ultracentrifugation showed that about 80% of the increase in lipoprotein particle number was in very low density lipoproteins containing apo B-100 and only 20% was in chylomicrons containing apo B-48 that carry dietary fat from the intestine. The maximal increments and the average concentrations of apo B-48 and B-100 during the 12 hr were highly correlated (r2 = 0.80), suggesting that preferential clearance of chylomicron triglycerides by lipoprotein lipase leads to accumulation of hepatogenous very low density lipoproteins during the alimentary period. The composition of the bulk of very low density lipoproteins that were bound to the monoclonal antibody changed little and these particles contained about 90% of the cholesterol and most of the apo E that accumulated in triglyceride-rich lipoproteins. The predominant accumulation of very low density lipoprotein rather than chylomicron particles after ingestion of ordinary meals is relevant to the potential atherogenicity of postprandial lipoproteins.     

Comparative effects of rosuvastatin and gemfibrozil on glucose, insulin, and lipid metabolism in insulin-resistant, nondiabetic patients with combined dyslipidemia

To evaluate the pharmacologic intervention most likely to decrease cardiovascular disease risk in insulin-resistant patients with combined dyslipidemia, 39 patients with this abnormality were assessed before and after 3 months of treatment with gemfibrozil (1,200 mg/day) or rosuvastatin (40 mg/day) with regard to: (1) steady-state plasma glucose concentration at the end of a 180-minute infusion of octreotide, insulin, and glucose; (2) fasting lipid, lipoprotein, and apolipoprotein concentrations; and (3) daylong glucose, insulin, triglyceride, and remnant lipoprotein cholesterol concentrations in response to breakfast and lunch. The 2 groups were similar at baseline in age, gender, body mass index and in measurements of carbohydrate and lipoprotein metabolism. Neither gemfibrozil nor rosuvastatin enhanced insulin sensitivity or lowered daylong glucose and insulin concentrations in insulin-resistant patients with combined dyslipidemia, but both drugs significantly decreased fasting triglyceride concentrations. However, only rosuvastatin treatment significantly (p <0.05 to <0.001) reduced fasting low-density lipoprotein cholesterol, apolipoprotein B-100, apolipoprotein C-III, apolipoprotein C-III:B particles, the apolipoprotein B-100:apolipoprotein A-I ratio, and increased apolipoprotein A-I (p <0.05). The degree of improvement in fasting and postprandial remnant lipoprotein cholesterol concentrations was significantly greater (p <0.05) in rosuvastatin-treated patients, and this difference in the relative effectiveness of the drugs was also true of the decrease in non-high-density lipoprotein cholesterol concentrations.     

Cyclic AMP-mediated cytoskeletal effects in adrenal cells are modified by serum, insulin, insulin-like growth factor-I, and an antibody against urokinase plasminogen activator

In adrenocortical cells in culture, increased intracellular cyclic AMP resulting from exposure to agents such as ACTH and cholera toxin causes a change in cell morphology termed 'retraction' or 'rounding'. The breakdown of actin-containing stress fibers in rounding suggested a role for microfilaments in steroidogenesis. Previously, we showed that cultured bovine adrenal cells under standard conditions (medium with 10% fetal bovine serum) do not round in response to intracellular cyclic AMP. Here, we show that these cells do round in defined, serum-free medium. Rounding was maximal within 1 h of addition of 1 nM cholera toxin and after 10 h most cells remained rounded. Cycloheximide at 100 micrograms/ml did not inhibit the response to cholera toxin. The rounding response was abolished when 10% fetal bovine serum, horse serum, or ether-extracted fetal bovine serum was included in the medium. The inhibitory effect of serum was not mimicked by growth factors with the exception that insulin and insulin-like growth factor-I (IGF-I), while not preventing rounding, accelerated the return of cells to a flattened morphology. A monoclonal antibody against urokinase plasminogen activator completely prevented rounding whereas a monoclonal antibody against tissue plasminogen activator had only a slight effect. Fluorescence visualization of F-actin with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin showed that rounding in cultured bovine adrenocortical cells resembles that defined earlier for human and rat adrenocortical cells and includes depolymerization of actin microfilaments. These cytoskeletal changes in adrenal cells are unlikely to play a role in steroidogenesis; however, they may be involved in tissue remodeling occurring as part of the indirect mitogenic effects of ACTH.     

Division and differentiation of natural antibody-producing cells in mouse spleen

B-1a cells reside in both the peritoneal cavity and the spleen. LPS stimulates splenic B-1a to differentiate to plasma cells producing natural IgM specific for microbial and self antigens. However, there are conflicting views as to whether the B-1a cells divide before this differentiation occurs, and hence how the resident B-1a population is maintained in the spleen. Studies here resolve this dispute in favor of both sides: we show that (some or all) B-1a cells resident in the spleen respond to LPS by differentiating to plasma cells immediately, without dividing; however, we also show that additional B-1a cells immigrate into the spleen after LPS stimulation and divide at least once before differentiating. Importantly, the studies we presently describe reveal the complex cell migration and differentiation events that collectively underlie the rapid production of natural antibodies in response to in vivo LPS stimulation. Thus, the studies present a different view of the roles that B-1a cells play in the early phases of the innate immune response.     

NK-104, a potent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, decreases apolipoprotein B-100 secretion from Hep G2 cells.

Intracellular cholesterol biosynthesis may play a key role in supplying cholesterol (as cholesteryl ester) for the neutral core of very low density lipoprotein (VLDL), thus modulating the secretion of apolipoprotein B-100 (apo B-100) from hepatocytes. The effect of compound NK-104 was studied, a new competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA-reductase), on apo B-100 synthesis and secretion from the human hepatoma cell line Hep G2. Cells were preincubated with NK-104 (0.01-5 microM) in the presence or absence of oleate (0.8 mM). Apo B-100 in the medium was determined by an enzyme-linked immunosorbent assay (ELISA). Incubation of Hep G2 with NK-104 resulted in a marked inhibition of cholesterogenesis (up to 95%), determined as incorporation of [14C]acetate into sterols, and decreased in a dose-dependent manner apo B-100 secretion, both in basal conditions (from 110 to 82 ng/mg cell protein, P < 0.01) and after incubation with oleate (from 227 to 165 ng/mg cell protein, P < 0.01). Density gradient for distribution of apo B-100 secreted, showed that this decrease was essentially due to a reduction of apo B-100 associated with lipoproteins in the density range of low density lipoproteins (LDL). Pulse chase experiment demonstrated that NK-104 did not affect the synthetic rate of apo B-100 but increased intracellular degradation of newly synthesized protein. The compound had only marginal effect on the mass of intracellular triglyceride but significantly decreased intracellular mass of free cholesterol and cholesteryl ester (P < 0.01). It is speculated that the ability of compound NK-104 to decrease apo B-100 secretion from Hep G2 cells is due to a decreased intracellular cholesterol availability.     

Chromosomal localization of the human apolipoprotein B gene and detection of homologous RNA in monkey intestine.

A cDNA clone of the human apolipoprotein B-100 was used as a hybridization probe to detect homologous sequences in both flow-sorted and in situ metaphase chromosomes. The results indicate that the gene encoding this protein is on the distal end of the short arm of chromosome 2 (2p23-2p24). RNA isolated from monkey small intestine contained sequences (6.5 and 18 kilobases) homologous to the cDNA of apolipoprotein B-100. These results are consistent with the hypothesis that one gene codes for both the intestinal (B-48) and the hepatic (B-100) forms.     

Effects of a thyromimetic on apolipoprotein B-100 in rats

We have studied the effects of a cardiac sparing thyromimetic, CGS 23425, on postprandial levels of triglycerides, abundance of apolipoprotein B (apo B) protein and hepatic apo B mRNA expression in rats. When compared with control rats, triglyceride clearance was significantly accelerated by treatment with CGS 23425. A full return to baseline values was achieved within 8 h after ingesting a large quantity of fat, as compared to >24 h in control animals. The abundance of apo B-100 protein in CGS 23425-treated hyperlipidemic rats decreased in a dose-dependent manner, but levels of apo B-48 were not significantly affected. Like L-tri-iodothyronine (L-T(3)), treatment with 30 microg/kg CGS 23425 for 6 or 9 days decreased the levels of apo B-100 protein by 80% and 40% respectively. This change was paralleled by a 27% reduction in hepatic apo B-100 mRNA. To investigate a potential mechanism of CGS 23425 action, we measured in vitro apo B mRNA editing activity in hepatocellular extract from control or CGS 23425-treated rats. Treatment with CGS 23425 increased activity of the hepatic apo B-100 editosome, apobec-1. In human hepatoma cells which lack apobec-1 activity, apo B-100 mRNA levels remained the same in cells treated with or without the agent. In summary, these observations show that CGS 23425 decreases the levels of apo B-100 in rats. This action of CGS 23425 involves apo B-100 mRNA editing activity.     

Metabolism of lipoproteins containing apolipoprotein B-100 in blood plasma of rabbits: heterogeneity related to the presence of apolipoprotein E

Apolipoprotein B-100 is a constant component of very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL) in mammalian blood plasma. We have found that each of these classes of lipoproteins includes particles that contain apolipoprotein E (B,E particles) as well as particles that lack this protein (B particles). These two species can be separated by immunosorption on columns of anti-apolipoprotein E bound to Sepharose. We have injected radioiodinated VLDL, IDL, and LDL intravenously into recipient rabbits and have determined the concentration of radioiodine in apolipoprotein B-100 in B,E and B particles in whole-blood plasma obtained at intervals for 24 hr. We have developed a multicompartmental model that is consistent with this new information and with current concepts of lipoprotein metabolism. The model indicates that all apolipoprotein B-100 enters the blood as VLDL, of which about 90% is in B,E particles. Most VLDL B,E particles are removed rapidly from the blood, and only a small fraction is converted to IDL and eventually to LDL (overall conversion is approximately 2%). By contrast, a much smaller fraction of VLDL B particles is removed directly, and approximately 27% is converted to LDL. In addition, some B,E particles are converted to B particles as VLDL are converted to LDL, so that most LDL particles lack apolipoprotein E. Fractional rates of irreversible removal of B,E and B particles in IDL and LDL are similar. Our results indicate that the presence of apolipoprotein E is a major determinant of the metabolic fate of VLDL particles and support the hypothesis that polyvalent binding of particles containing several molecules of apolipoprotein E promotes receptor-dependent endocytosis of hepatogenous lipoproteins and limits their conversion to lipoproteins of higher density.     

Alterations in apolipoprotein B-48 in the postprandial state in NIDDM

Summary The intestine is a major site of cholesterol synthesis and produces apolipoprotein B-48, which is critical for intestinal cholesterol absorption and secretion. The purpose of this study was to examine postprandial changes in apolipoprotein B-48 in diabetes. Six non-insulin-dependent diabetic patients and six non-diabetic control subjects were given a high-fat meal (1300 kcal) and blood samples were taken pre- and postprandially, from which the triglyceride-rich lipoprotein fraction was isolated by ultracentrifugation (density<1.006 g/ml). Apolipoprotein B-48 was separated on 4–15% gradient gels and quantified as a percentage of the fasting concentration by densitometric scanning. Total protein, triglyceride and cholesterol in the triglyceride-rich lipoprotein fraction, blood glucose, and serum insulin were also measured. Diabetic patients exhibited a postprandial triglyceride-rich apolipoprotein B-48 profile significantly different from that of control subjects (p<0.05). The triglyceride and total protein concentration in the triglyceride-rich lipoprotein fraction mirrored the post-prandial profile and apolipoprotein B-48 in both groups. Significantly different patterns for triglyceride (p<0.02) and total protein (p<0.05) following the fat-rich meal were observed in the two groups. Fasting and postprandial triglyceride-rich lipoprotein cholesterol and total apolipoprotein B were significantly higher in diabetic patients than in control subjects (p<0.05). Since apolipoprotein B-48 is the structural protein of intestinally-derived lipoprotein particles, these studies suggest an abnormality in intestinal lipoprotein metabolism in diabetes.Abbreviations TRL Triglyceride-rich lipoprotein - Apo-B48 apolipoprotein B-48 - apo-B100 apolipoprotein B-100 - apo E apolipoprotein E - VLDL very low density lipoprotein - NIDDM non-insulin-dependent diabetes     

Increased levels of lipoprotein (a) in Crohn's disease: a relation to thrombosis?

OBJECTIVE: Lipoprotein (a) is recognized as a risk factor for arterial and venous thrombosis, a property that might be related to its structural similarity to plasminogen. Since patients with inflammatory bowel disease frequently suffer from thromboembolic events, we studied the role of lipoprotein (a) in conjunction with lipids and apolipoproteins in Greek patients with ulcerative colitis and Crohn's disease. METHODS: Lipoprotein (a), total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, apolipoprotein A-1 and apolipoprotein B-100 were determined in sera from 129 consecutive fasting Greek patients with inflammatory bowel disease (66 with ulcerative colitis and 63 with Crohn's disease) and from 66 matched healthy controls. RESULTS: In Crohn's disease patients, the mean serum lipoprotein (a) level was significantly higher than in control patients (41.2 mg/dl vs 22.9 mg/dl; P = 0.005). Mean apolipoprotein A-1 and apolipoprotein B-100 levels were significantly lower in Crohn's disease patients than in the controls. In ulcerative colitis patients the mean levels of lipoprotein (a) and apolipoprotein A-1 were not significantly different to the controls, but the levels of apolipoprotein B-100 were significantly lower. Raised levels of lipoprotein (a) of > 30 mg/dl were found in 29 Crohn's disease patients (46%), 15 ulcerative colitis patients (23%) and 11 control patients (17%). Patients with active Crohn's disease had significantly higher mean lipoprotein (a) and lower apolipoprotein A-1 than patients with non-active disease. CONCLUSIONS: Our results suggest that Crohn's disease patients have different lipoprotein (a) and apolipoprotein patterns compared to ulcerative colitis patients and healthy controls. These changes in Crohn's disease patients may possibly expose them to a higher risk of thrombosis.     

The expression of membrane-bound CD14 renders mouse B-1 cells susceptible to LPS

The surface expression of CD14 on mouse B-1 cells and its role on their response to lipopolysaccharide (LPS) were studied by using the murine TH2.52 B-1 cell line and peritoneal B-1 cells. TH2.52 cells with the B-1 phenotype were found to express membrane-bound CD14. Furthermore, CD14 was expressed on physiological peritoneal CD5+ B-1 cells. The stimulation of CD14-expressing TH2.52 cells with a low concentration of LPS resulted in the activation of nuclear factor (NF)-B and a mitogen-activated protein kinase (MAPK). The LPS-induced NF-B and MAPK activation was markedly inhibited by anti-CD14 antibody. These results suggest that B-1 cells may respond to LPS via membrane-bound CD14.     

Heterogeneity of apolipoprotein B: isolation of a new species from human chylomicrons.

Low density lipoproteins and the triglyceride-rich lipoproteins of human serum each contain proteins of high molecular weight termed apolipoprotein B, which have previously been thought to be identical. We have isolated four species of apolipoprotein B with unique molecular weights and amino acid compositions. We have assigned numerical designations to these species in a centile system based upon their relative apparent Mr in NaDodSO4. One which we term B-100, with an apparent Mr of 549,000 +/- 7650 (SD) determined by NaDodSO4 gel electrophoresis, predominates in low density and very low density lipoproteins and is also present in chylomicrons from thoracic duct lymph or from plasma. Substantial amounts of two large proteins designated B-74 (apparent Mr 407,000 +/- 5790) and B-26 (apparent Mr 144,500 +/- 8970), which appear to be complementary fragments or constituents of the B-100 protein, are found in the low density lipoproteins of many individuals. A distinct protein, B-48, with an apparent Mr of 264,000 +/- 8150 is a major and constant constituent of chylomicrons from thoracic duct lymph or from plasma.