Characteristics and functional relevance of apolipoprotein-A1 and cholesterol binding in mammary gland tissues and epithelial cells

Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of (125)I-apoA-I and (3)H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular (3)H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell(®) plates. The amounts of isolated EPM and the maximal binding capacity of (125)I-apoA-I to EPM differed depending on the MG's physiological state, while the kinetics of (3)H-cholesterol and (125)I-apoA-I binding were similar. (3)H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of (125)I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of (125)I-apoA-I ranged between 40-74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and (125)I-apoA-I binding. The ABCA1 inhibitor Probucol displaced (125)I-apoA-I binding to EPM and reduced (3)H-cholesterol efflux in MeBo. Time-dependent (3)H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell(®) plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of (3)H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the importance of the apoA-I/ABCA1 pathway in MG cholesterol transport and suggest its role in influencing milk composition and directing cholesterol back into the bloodstream.

Alpha tocopherol supplementation elevates plasma apolipoprotein A1 isoforms in normal healthy subjects

Plasma α-tocopherol (AT) concentrations are inversely related to cardiovascular (CV) risk; however, intervention studies with AT have failed to show any consistent benefit against CV disease (CVD). Proteomics offers the opportunity to examine novel effects of AT supplementation on protein expression and therefore improve our understanding of the physiological roles of AT. Thus, to investigate the effects of AT supplementation on the plasma proteome of healthy subjects we have undertaken a double-blind, randomised, parallel design supplementation study in which healthy subjects (n = 32; 11 male and 21 female) consumed AT supplements (134 or 268 mg/day) or placebo capsules for up to 28 days. Plasma samples were obtained before supplementation and after 14 and 28 days of supplementation for analysis of changes in the plasma proteome using 2-DE and MALDI-MS. Using semiquantitative proteomics, we observed that proapolipoprotein A1 (identified by MS and Western blotting) was altered at least two-fold. Using quantitative ELISA techniques, we confirmed a significant increase in plasma apolipoprotein A1 concentration following supplementation with AT which was both time and dose dependent (p < 0.01 after 28 days supplementation with 268 mg AT/day). These data demonstrate the time and dose sensitivity of the plasma proteome to AT supplementation. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

Paradoxical decrease in HDL-cholesterol and apolipoprotein A1 with simvastatin and atorvastatin in a patient with type 2 diabetes

Statins are agents widely used to lower LDL-cholesterol (LDL-C) in primary and secondary prevention of coronary heart disease. The five statins available in the UK (simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin) differ in many of their pharmacologic properties. In addition to lowering LDL-C, statins also increase HDL-cholesterol (HDL-C) moderately. There have been rare reports of significant HDL-C decreases in patients commenced on fibrates and when thiazolidinediones are added to fibrates. This is known as a 'paradoxical HDL-C decrease' as both groups of agents usually increase HDL-C. This phenomenon has never been clearly documented following statin therapy. We now describe a patient with type 2 diabetes who showed this paradoxical fall in HDL-C (baseline HDL-C: 1.8 mmol/L; on simvastatin 40 mg HDL-C 0.6 mmol/L; on atorvastatin 20 mg HDL-C 0.9 mmol/L) with a similar decrease in apolipoprotein A1. No similar decrease was observed with pravastatin and rosuvastatin therapy. This phenomenon appeared to be associated with statin treatment and not a statin/fibrate combination. Our patient clearly demonstrated a paradoxical HDL-C fall with simvastatin and atorvastatin, but not pravastatin or rosuvastatin. Simvastatin and atorvastatin share many pharmacokinetic properties such as lipophilicity while pravastatin and rosuvastatin are relatively hydrophilic and are not metabolized by cytochrome P450 3A4. However, these characteristics do not explain the dramatic reductions in HDL-C observed.

Long-term orange juice consumption is associated with low LDL-cholesterol and apolipoprotein B in normal and moderately hypercholesterolemic subjects

Abstract. Background: This study investigated the hypothesis that long-term orange juice consumption (≥ 12 months) was associated with low risk factors for cardiovascular disease in adult men and women with normal and moderately high cholesterol blood levels. Methods. The sample consisted of 103 men (18-66 y) and 26 women (18-65 y); all were employees of an orange juice factory with daily access to free orange juice. The results showed that 41% of the individuals consumed 2 cups (480 mL) of orange juice per day for at least twelve months, while 59% of the volunteers are non-consumers of orange juice. Results: Orange juice consumers with normal serum lipid levels had significantly lower total cholesterol (-11%, p <0.001), LDL-cholesterol (-18%, p < 0.001), apolipoprotein B (apo B) (-12%, p < 0.01) and LDL/HDL ratio (-12%, p < 0.04) in comparison to non-consumers, as did the consumers with moderate hypercholesterolemia: lower total cholesterol (-5%, p <0.02), LDL-cholesterol (-12%, p <0.03), apolipoprotein B (-12%, p <0.01) and LDL/HDL ratio (-16%, p <0.05) in comparison the non-consumers counterparts. Serum levels of homocysteine, HDL- cholesterol and apolipoprotein A-1, body composition and the dietary intake of food energy and macronutrients did not differ among orange juice consumers and non-consumers, but vitamin C and folate intake was higher in orange juice consumers. Conclusion: Long-term orange juice consumers had lower levels of total cholesterol, LDL-cholesterol, apo B and LDL/HDL ratio and an improvement of folate and vitamin C in their diet. © 2013 Aptekmann and Cesar; licensee BioMed Central Ltd.

Avaliação do polimorfismo genético das apolipoproteínas A1 e A5 em pacientes HIV positivos com síndrome lipodistrófica no estado do Pará

Introdução: A dislipidemia é uma das alterações metabólicas do uso da terapia antirretroviral (TARV) em pacientes HIV positivos com Síndrome Lipodistrófica. Objetivo: Avaliar os polimorfismos genéticos das apolipoproteínas A1 e A5 em pacientes HIV positivos com lipodistrofia, em uso de TARV e sua associação com a dislipidemia. Métodos: Estudo do tipo transversal analítico, que utilizou um protocolo de pesquisa, que estudou as condições sóciodemográfica, clínicas, fatores de risco (atividade física, tabagismo, etilismo, frequência alimentar), exames bioquímicos para dislipidemia e avaliação dos polimorfismos das apolipoproteínas A1 e A5. Resultados: Dos 105 pacientes HIV positivos estudados, 63,8% eram homens, com idade média de 44,5 (± 9,4) anos; 70,5% relataram ser solteiros e possuir renda familiar de até três salários mínimos (77,1%). Os fatores de risco observados foram: tabagismo (21%), etilismo (43,8%), sedentarismo (69,5%), Diabetes mellitus (16,2%), excesso de peso (22,9%) e risco cardiovascular (39,1%). A forma de lipodistrofia mais prevalente foi à síndrome mista (51,4%). O consumo alimentar verificou a frequência de frutas (60,8%), legumes e verduras (36,3%), leite e derivados (75%), diariamente. Doces e guloseimas (31,4%), embutidos (11,7%) e lanches gordurosos (26,4%) mais de duas vezes na semana. E o hábito do consumo de carnes com gordura aparente (56,9%). Em relação à classificação da dislipidemia observou que a maioria dos pacientes possuía hipertrigliceridemia isolada (30,5%) e hiperlipidemia mista (32,4%). Observou-se que a hipertrigliceridemia isolada está associada com o gene da apolipoproteína A5 (rs3135506, rs619054 e rs662799), não sendo influenciada pelas formas clínicas de lipodistrofia. Não foi encontrada a presença do polimorfismo da apolipoproteína A1 (Lys107-0must2) nos pacientes em estudo. Conclusão: Os principais fatores para hipertrigliceridemia foram o sexo masculino e os polimorfismos do gene da apolipoproteína A5 (rs3135506, rs619054 e rs662799).

Type 2 diabetes is associated with reduced ATP-binding cassette transporter A1 gene expression, protein and function

Objective Increasing plasma glucose levels are associated with increasing risk of vascular disease. We tested the hypothesis that there is a glycaemia-mediated impairment of reverse cholesterol transport (RCT). We studied the influence of plasma glucose on expression and function of a key mediator in RCT, the ATP binding cassette transporter-A1 (ABCA1) and expression of its regulators, liver X receptor-α (LXRα) and peroxisome proliferator-activated receptor–γ (PPARγ). Methods and Results Leukocyte ABCA1, LXRα and PPARγ expression was measured by polymerase chain reaction in 63 men with varying degrees of glucose homeostasis. ABCA1 protein concentrations were measured in leukocytes. In a sub-group of 25 men, ABCA1 function was quantified as apolipoprotein-A1-mediated cholesterol efflux from 2–3 week cultured skin fibroblasts. Leukocyte ABCA1 expression correlated negatively with circulating HbA1c and glucose (rho = −0.41, p<0.001; rho = −0.34, p = 0.006 respectively) and was reduced in Type 2 diabetes (T2DM) (p = 0.03). Leukocyte ABCA1 protein was lower in T2DM (p = 0.03) and positively associated with plasma HDL cholesterol (HDL-C) (rho = 0.34, p = 0.02). Apolipoprotein-A1-mediated cholesterol efflux correlated negatively with fasting glucose (rho = −0.50, p = 0.01) and positively with HDL-C (rho = 0.41, p = 0.02). It was reduced in T2DM compared with controls (p = 0.04). These relationships were independent of LXRα and PPARγ expression. Conclusions ABCA1 expression and protein concentrations in leukocytes, as well as function in cultured skin fibroblasts, are reduced in T2DM. ABCA1 protein concentration and function are associated with HDL-C levels. These findings indicate a glycaemia- related, persistent disruption of a key component of RCT.

High-density lipoprotein and the risk of recurrent venous thromboembolism

BACKGROUND - High-density lipoprotein (HDL) protects against arterial atherothrombosis, but it is unknown whether it protects against recurrent venous thromboembolism. METHODS AND RESULTS - We studied 772 patients after a first spontaneous venous thromboembolism (average follow-up 48 months) and recorded the end point of symptomatic recurrent venous thromboembolism, which developed in 100 of the 772 patients. The relationship between plasma lipoprotein parameters and recurrence was evaluated. Plasma apolipoproteins AI and B were measured by immunoassays for all subjects. Compared with those without recurrence, patients with recurrence had lower mean (±SD) levels of apolipoprotein AI (1.12±0.22 versus 1.23±0.27 mg/mL, P<0.001) but similar apolipoprotein B levels. The relative risk of recurrence was 0.87 (95% CI, 0.80 to 0.94) for each increase of 0.1 mg/mL in plasma apolipoprotein AI. Compared with patients with apolipoprotein AI levels in the lowest tertile (<1.07 mg/mL), the relative risk of recurrence was 0.46 (95% CI, 0.27 to 0.77) for the highest-tertile patients (apolipoprotein AI >1.30 mg/mL) and 0.78 (95% CI, 0.50 to 1.22) for midtertile patients (apolipoprotein AI of 1.07 to 1.30 mg/mL). Using nuclear magnetic resonance, we determined the levels of 10 major lipoprotein subclasses and HDL cholesterol for 71 patients with recurrence and 142 matched patients without recurrence. We found a strong trend for association between recurrence and low levels of HDL particles and HDL cholesterol. CONCLUSIONS - Patients with high levels of apolipoprotein AI and HDL have a decreased risk of recurrent venous thromboembolism. © 2007 American Heart Association, Inc.

High-density lipoprotein deficiency and dyslipoproteinemia associated with venous thrombosis in men

Background-Although dyslipoproteinemia is associated with arterial atherothrombosis, little is known about plasma lipoproteins in venous thrombosis patients. Methods and Results-We determined plasma lipoprotein subclass concentrations using nuclear magnetic resonance spectroscopy and antigenic levels of apolipoproteins AI and B in blood samples from 49 male venous thrombosis patients and matched controls aged <55 years. Venous thrombosis patients had significantly lower levels of HDL particles, large HDL particles, HDL cholesterol, and apolipoprotein AI and significantly higher levels of LDL particles and small LDL particles. The quartile-based odds ratios for decreased HDL particle and apolipoprotein AI levels in patients compared with controls were 6.5 and 6.0 (95% CI, 2.3 to 19 and 2.1 to 17), respectively. Odds ratios for apolipoprotein B/apolipoprotein AI ratio and LDL cholesterol/HDL cholesterol ratio were 6.3 and 2.7 (95% CI, 1.9 to 21 and 1.1 to 6.5), respectively. When polymorphisms in genes for hepatic lipase, endothelial lipase, and cholesteryl ester transfer protein were analyzed, patients differed significantly from controls in the allelic frequency for the TaqI B1/B2 polymorphism in cholesteryl ester transfer protein, consistent with the observed pattern of lower HDL and higher LDL. Conclusions-Venous thrombosis in men aged <55 years old is associated with dyslipoproteinemia involving lower levels of HDL particles, elevated levels of small LDL particles, and an elevated ratio of apolipoprotein B/apolipoprotein AI. This dyslipoproteinemia seems associated with a related cholesteryl ester transfer protein genotype difference. © 2005 American Heart Association, Inc.

The putative role of some conserved water molecules in the structure and function of human transthyretin

Human transthyretin (hTTR) is a multifunctional protein that is involved in several neurodegenerative diseases. Besides the transportation of thyroxin and vitamin A, it is also involved in the proteolysis of apolipoprotein A1 and A beta peptide. Extensive analyses of 32 high-resolution X-ray and neutron diffraction structures of hTTR followed by molecular-dynamics simulation studies using a set of 15 selected structures affirmed the presence of 44 conserved water molecules in its dimeric structure. They are found to play several important roles in the structure and function of the protein. Eight water molecules stabilize the dimeric structure through an extensive hydrogen-bonding network. The absence of some of these water molecules in highly acidic conditions (pH <= 4.0) severely affects the interfacial hydrogen-bond network, which may destabilize the native tetrameric structure, leading to its dissociation. Three pairs of conserved water molecules contribute to maintaining the geometry of the ligand-binding cavities. Some other water molecules control the orientation and dynamics of different structural elements of hTTR. This systematic study of the location, absence, networking and interactions of the conserved water molecules may shed some light on various structural and functional aspects of the protein. The present study may also provide some rational clues about the conserved water-mediated architecture and stability of hTTR.

Effect of changing the amount and type of fat and carbohydrate on insulin sensitivity and cardiovascular risk: the RISCK (Reading, Imperial, Surrey, Cambridge, and Kings) trial

Background: Insulin sensitivity (Si) is improved by weight loss and exercise, but the effects of the replacement of saturated fatty acids (SFAs) with monounsaturated fatty acids (MUFAs) or carbohydrates of high glycemic index (HGI) or low glycemic index (LGI) are uncertain. Objective: We conducted a dietary intervention trial to study these effects in participants at risk of developing metabolic syndrome. Design: We conducted a 5-center, parallel design, randomized controlled trial [RISCK (Reading, Imperial, Surrey, Cambridge, and Kings)]. The primary and secondary outcomes were changes in Si (measured by using an intravenous glucose tolerance test) and cardiovascular risk factors. Measurements were made after 4 wk of a high-SFA and HGI (HS/HGI) diet and after a 24-wk intervention with HS/HGI (reference), high-MUFA and HGI (HM/HGI), HM and LGI (HM/LGI), low-fat and HGI (LF/HGI), and LF and LGI (LF/LGI) diets. Results: We analyzed data for 548 of 720 participants who were randomly assigned to treatment. The median Si was 2.7 × 10−4 mL · μU−1 · min−1 (interquartile range: 2.0, 4.2 × 10−4 mL · μU−1 · min−1), and unadjusted mean percentage changes (95% CIs) after 24 wk treatment (P = 0.13) were as follows: for the HS/HGI group, −4% (−12.7%, 5.3%); for the HM/HGI group, 2.1% (−5.8%, 10.7%); for the HM/LGI group, −3.5% (−10.6%, 4.3%); for the LF/HGI group, −8.6% (−15.4%, −1.1%); and for the LF/LGI group, 9.9% (2.4%, 18.0%). Total cholesterol (TC), LDL cholesterol, and apolipoprotein B concentrations decreased with SFA reduction. Decreases in TC and LDL-cholesterol concentrations were greater with LGI. Fat reduction lowered HDL cholesterol and apolipoprotein A1 and B concentrations. Conclusions: This study did not support the hypothesis that isoenergetic replacement of SFAs with MUFAs or carbohydrates has a favorable effect on Si. Lowering GI enhanced reductions in TC and LDL-cholesterol concentrations in subjects, with tentative evidence of improvements in Si in the LF-treatment group. This trial was registered at clinicaltrials.gov as ISRCTN29111298.

High cholesterol intake modifies chylomicron metabolism in normolipidemic young men

Whether the consumption of egg yolk, which has a very high cholesterol content without excess saturated fats, has deleterious effects on lipid metabolism is controversial. Absorbed dietary cholesterol enters the bloodstream as chylomicrons, but the effects of regular consumption of large amounts of cholesterol on the metabolism of this lipoprotein have not been explored even though the accumulation of chylomicron remnants is associated with coronary artery disease (CAD). We investigated the effects of high dietary cholesterol on chylomicron metabolism in normolipidemic, healthy young men. The plasma kinetics of a chylomicron-like emulsion, doubly-labeled with 14C-cholesteryl ester ( 14C-CE) and 3H-triolein ( 3H-TG) were assessed in 25 men (17-22 y old, BMI 24.1 ± 3.4 kg/m 2). One group (n = 13) consumed 174 ± 41 mg cholesterol/d and no egg yolk. The other group (n = 12) consumed 3 whole eggs/d for a total cholesterol intake of 804 ± 40 mg/d. The nutritional composition of diets was the same for both groups, including total lipids and saturated fat, which comprised 25 and 7%, respectively, of energy intake. Serum LDL and HDL cholesterol and apoprotein B concentrations were higher in the group consuming the high-cholesterol diet (P < 0.05), but serum triacylglycerol, apo AI, and lipoprotein (a) did not differ between the 2 groups. The fractional clearance rate (FCR) of the 14C-CE emulsion, obtained by compartmental analysis, was 52% slower in the high-cholesterol than in the low-cholesterol group (P < 0.001); the 3H-TG FCR did not differ between the groups. Finally, we concluded that high cholesterol intakes increase the residence time of chylomicron remnants, as indicated by the 14C-CE kinetics, which may have undesirable effects related to the development of CAD. © 2006 American Society for Nutrition.

Acute-phase protein behavior in dairy cattle herd naturally infected with Trypanosoma vivax

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

GENETIC EVENTS PREDISPOSING TO WILMS' TUMOR

Wilms' tumor (WT) is a childhood embryonic tumor of the kidney. In some cases, WT has been associated with a chromosome deletion in the region 11p13. The majority of WT cases, however, have normal karyotypes with no discernable deletions or rearrangements of chromosome 11.^ To study the genetic events predisposing to the development of WT, I have used a number of gene markers specific for chromosome 11. Gene probes for human catalase and apolipoprotein A1 were localized to chromosome 11 by in situ hybridization. A number of other probes previously mapped to chromosome 11 were also used. Nine WT patients who were heterozygous for at least one 11p marker were shown to lose heterozygosity in their tumor DNA. Gene dosage experiments demonstrated that two chromosomes 11 were present although loss of heterozygosity had occurred in all but two cases. By using gene probes from the short and long arms of chromosome 11, I discerned that loss of heterozygosity was due to somatic recombination in four cases, chromosome deletion in two cases, and chromosome loss and reduplication or somatic recombination in these cases. Examination of DNAs from the parents of six of these patients indicated that the alleles that were lost in tumor tissues were alleles inherited from the mother. In sporadic WT cases one would expect the loss of alleles to be random. These data suggest that the loss of alleles resulting in the development of WT is not a random event, however, the significance of this is not known. ^

Cholesterol acceptor capacity is preserved by different mechanisms in preterm and term fetuses.

Fetal serum cholesterol and lipoprotein concentrations differ between preterm and term born neonates. An imbalance of the flow of cholesterol from the sites of synthesis or efflux from cells of peripheral organs to the liver, the reverse cholesterol transport (RCT), is linked to atherosclerosis and cardiovascular disease (CVD). Preterm delivery is a risk factor for the development of CVD. Thus, we hypothesized that RCT is affected by a diminished cholesterol acceptor capacity in preterm as compared to term fetuses. Cholesterol efflux assays were performed in RAW264.7, HepG2, and HUVEC cell lines. In the presence and absence of ABC transporter overexpression by TO-901317, umbilical cord sera of preterm and term born neonates (n = 28 in both groups) were added. Lipid components including high density lipoprotein (HDL), low density lipoprotein (LDL), apolipoprotein A1, and apolipoprotein E were measured and related to fractional cholesterol efflux values. We found overall, fractional cholesterol efflux to remain constant between the study groups, and over gestational ages at delivery, respectively. However, correlation analysis revealed cholesterol efflux values to be predominantly related to HDL concentration at term, while in preterm neonates, cholesterol efflux was mainly associated with LDL In conclusion cholesterol acceptor capacity during fetal development is kept in a steady state with different mechanisms and lipid fractions involved at distinct stages during the second half of fetal development. However, RCT mechanisms in preterm neonates seem not to be involved in the development of CVD later in life suggesting rather changes in the lipoprotein pattern causative.