Long-term safety and efficacy of anacetrapib in patients with atherosclerotic vascular disease.

AIMS: REVEAL was the first randomized controlled trial to demonstrate that adding cholesteryl ester transfer protein inhibitor therapy to intensive statin therapy reduced the risk of major coronary events. We now report results from extended follow-up beyond the scheduled study treatment period. METHODS AND RESULTS: A total of 30 449 adults with prior atherosclerotic vascular disease were randomly allocated to anacetrapib 100 mg daily or matching placebo, in addition to open-label atorvastatin therapy. After stopping the randomly allocated treatment, 26 129 survivors entered a post-trial follow-up period, blind to their original treatment allocation. The primary outcome was first post-randomization major coronary event (i.e. coronary death, myocardial infarction, or coronary revascularization) during the in-trial and post-trial treatment periods, with analysis by intention-to-treat. Allocation to anacetrapib conferred a 9% [95% confidence interval (CI) 3-15%; P = 0.004] proportional reduction in the incidence of major coronary events during the study treatment period (median 4.1 years). During extended follow-up (median 2.2 years), there was a further 20% (95% CI 10-29%; P < 0.001) reduction. Overall, there was a 12% (95% CI 7-17%, P < 0.001) proportional reduction in major coronary events during the overall follow-up period (median 6.3 years), corresponding to a 1.8% (95% CI 1.0-2.6%) absolute reduction. There were no significant effects on non-vascular mortality, site-specific cancer, or other serious adverse events. Morbidity follow-up was obtained for 25 784 (99%) participants. CONCLUSION: The beneficial effects of anacetrapib on major coronary events increased with longer follow-up, and no adverse effects emerged on non-vascular mortality or morbidity. These findings illustrate the importance of sufficiently long treatment and follow-up duration in randomized trials of lipid-modifying agents to assess their full benefits and potential harms. TRIAL REGISTRATION: International Standard Randomized Controlled Trial Number (ISRCTN) 48678192; ClinicalTrials.gov No. NCT01252953; EudraCT No. 2010-023467-18.

Visceral, subcutaneous abdominal adiposity and liver fat content distribution in normal glucose tolerance, impaired fasting glucose and/or impaired glucose tolerance.

OBJECTIVES: To examine the specific distribution of liver fat content, visceral and subcutaneous adiposity in normal glucose tolerance (NGT/NGT), isolated impaired fasting glucose (iIFG), isolated impaired glucose tolerance (iIGT) and combined conditions (IFG+IGT), as well as with newly diagnosed type 2 diabetes (nT2D). DESIGN: Multicenter, international observational study: cross-sectional analysis. SUBJECTS: Two thousand five hundred and fifteen patients (50.0% women, 54.5% non-Caucasian) without previously known diabetes were recruited from 29 countries. Abdominal fat distribution was measured by computed tomography (CT). Liver fat was estimated using the CT-liver mean attenuation. RESULTS: Compared with NGT/NGT patients, increased visceral adiposity was found in iIFG, iIGT, IFG+IGT and nT2D; estimated liver fat progressively increased across these conditions. A one-s.d. increase in visceral adiposity was associated with an increased risk of having iIFG (men: odds ratio (OR) 1.41 (95% confidence interval (CI) 1.15-1.74), women: OR 1.62 (1.29-2.04)), iIGT (men: OR 1.59 (1.15-2.01), women: OR 1.30 (0.96-1.76)), IFG+IGT (men: OR 1.64 (1.27-2.13), women: OR 1.83 (1.36-2.48)) and nT2D (men: OR 1.80 (1.35-2.42), women: OR 1.73 (1.25-2.41)). A one-s.d. increase in estimated liver fat was associated with iIGT (men: OR 1.46 (1.12-1.90), women: OR 1.81 (1.41-2.35)), IFG+IGT (men: OR 1.42 (1.14-1.77), women: OR 1.74 (1.35-2.26)) and nT2D (men: OR 1.77 (1.40-2.27), women: OR 2.38 (1.81-3.18)). Subcutaneous abdominal adipose tissue showed an inverse relationship with nT2D in women (OR 0.63 (0.45-0.88)). CONCLUSIONS: Liver fat was associated with iIGT but not with iIFG, whereas visceral adiposity was associated with both. Liver fat and visceral adiposity were associated with nT2D, whereas subcutaneous adiposity showed an inverse relationship with nT2D in women.

Clinical guidelines on hyperlipidaemia: recent developments, future challenges and the need for an Australian review.

Large reductions in cardiovascular disease (CVD) mortality have been achieved over the last 50 years in developed countries. The health policies that have contributed so much to this success have largely been coordinated by means of expert guidelines for the management of the classic modifiable risk factors such as blood pressure, diabetes and blood lipids. National and international guidelines for lipid management have demonstrated a high degree of consistency between numerous sets of recommendations. It has been argued that some important components of the consensus that has been established over the past decade have been challenged by the latest guidelines of the American Heart Association - American College of Cardiologists (AHA-ACC). Clinicians can be reassured that continued reliance on extensive scientific evidence has reaffirmed the importance of lipid metabolism as a modifiable risk factor for atherosclerotic cardiovascular disease. On the other hand, the recent AHA-ACC guidelines suggest changes in the strategies by which metabolic risk factors may be modified. This small number of important changes should not be sensationalised because these differences usefully reflect the need for guidelines to evolve to accommodate different contexts and changing perspectives as well as emerging issues and new information for which clinical trial evidence is incomplete. This article will consider the recent policies and responses of national and supranational organisations on topics including components of CVD risk assessment, sources of CVD risk information and re-appraisal of lipid-lowering interventions. Timely review of Australian lipid management guidelines will require consideration of these issues because they are creating a new context within which new guidelines must evolve.

Assessment of cardiometabolic risk and prevalence of meeting treatment guidelines among patients with type 2 diabetes stratified according to their use of insulin and/or other diabetic medications: results from INSPIRE ME IAA.

AIM: Visceral adipose tissue (VAT) and liver fat (LF) are strongly associated with type 2 diabetes. It is not known, however, how diabetes treatment and/or risk factor management modulates the association between VAT, LF and diabetes. The aim was to determine the level of VAT and LF in patients with type 2 diabetes according to their treatment status and achievement of the American Diabetes Association's (ADA) diabetes management goals. METHODS: We performed a cross-sectional analysis of the baseline data of the International Study of the Prediction of Intra-Abdominal Adiposity and its Relationship with Cardiometabolic risk/Intra-Abdominal Adiposity (INSPIRE ME IAA), a 3-year prospective cardiometabolic imaging study conducted in 29 countries. Patients (n = 3991) were divided into four groups: (i) those without type 2 diabetes (noT2D n = 1003 men, n = 1027 women); (ii) those with type 2 diabetes but not treated with diabetes medications (T2Dnomeds n = 248 men, n = 198 women); (iii) those with type 2 diabetes and treated with diabetes medications but not yet using insulin (T2Dmeds-ins n = 591 men, n = 484 women) and (iv) those with type 2 diabetes and treated with insulin (T2Dmeds+ins n = 233 men, n = 207 women). Abdominal and liver adiposity were measured by computed tomography. RESULTS: Fewer patients with high VAT or LF achieved the ADA's goals for high-density lipoprotein cholesterol (HDL-C) or triglycerides compared to patients with low VAT or LF. Visceral adiposity (p = 0.02 men, p = 0.003 women) and LF (p = 0.0002 men, p = 0.0004 women) increased among patients who met fewer of the ADA treatment criteria, regardless of type 2 diabetes treatment. CONCLUSION: Residual cardiometabolic risk exists among patients with type 2 diabetes characterized by elevated VAT and LF.

Comparison of the effects of different statins and doses on lipid levels in patients with diabetes: results from VOYAGER.

BACKGROUND AND AIMS: Diabetes mellitus is a well-known risk factor for cardiovascular disease, and brings an increased risk of vascular events and a higher mortality rate. Treatment guidelines recommend statins in patients with diabetes, with low-density lipoprotein cholesterol (LDL-C) targets of 100 mg dl(-1) (∼2.5 mmol l(-1)), and 80 (∼2.0 mmol l(-1)) or 70 mg dl(-1) (∼1.8 mmol l(-1)) in especially high-risk patients. The current study used the VOYAGER (an indiVidual patient data-meta-analysis Of statin therapY in At risk Groups: Effects of Rosuvastatin, atorvastatin, and simvastatin) database to characterise effects of rosuvastatin, atorvastatin and simvastatin in different doses on lipid levels in diabetes patients. METHODS AND RESULTS: The VOYAGER database included individual patient data from 37 studies involving comparisons of rosuvastatin with either atorvastatin or simvastatin. Of the 32 258 patients included, 8859 (27.5%) had diabetes. Rosuvastatin appeared to be the most efficacious of the three statins, both for lowering LDL-C and for reaching a target level of <70 mg dl(-1) for LDL-C. It was also more effective than atorvastatin at raising high-density lipoprotein cholesterol in the diabetes population. These results are consistent with the overall VOYAGER results. CONCLUSIONS: This meta-analysis of 8859 patients with diabetes mellitus shows favourable effects on lipids with the three statins studied, in line with results for the overall VOYAGER population. The importance of using an effective statin at an effective dose to reach treatment goals for such high-risk patients is evident.

Ability of traditional lipid ratios and apolipoprotein ratios to predict cardiovascular risk in people with type 2 diabetes.

AIMS/HYPOTHESIS: The apolipoprotein B (ApoB):apolipoprotein A (ApoA)-I ratio may be a better indicator of cardiovascular disease (CVD) risk in people with type 2 diabetes than traditional lipid risk markers (LDL-cholesterol, HDL-cholesterol and triacylglycerol), but whether the ApoB:ApoA-I ratio should be used to indicate lipid-lowering therapy is still debated. METHODS: The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study randomised 9,795 patients with type 2 diabetes to fenofibrate (200 mg daily) or placebo and followed them up for a median of 5 years. We compared ApoB, ApoA-I, ApoAII and the ApoB:ApoA-I ratio with traditional lipid variables as predictors of CVD risk. We estimated the HR of the effect of 1 SD difference in baseline concentrations of lipids, apolipoproteins and respective ratios on the risk of CVD events and also used receiver operating characteristic curve analysis. RESULTS: In the placebo group, the variables best predicting CVD events were non-HDL-cholesterol:HDL-cholesterol, total cholesterol:HDL-cholesterol (HR 1.21, p < 0.001 for both), ApoB:ApoA-I (HR 1.20, p < 0.001), LDL-cholesterol:HDL-cholesterol (HR 1.17, p < 0.001), HDL-cholesterol (HR 0.84, p < 0.001) and ApoA-I (HR 0.85, p < 0.001). In the fenofibrate group, the first four predictors were very similar (but ApoB:ApoA-I was fourth), followed by non-HDL-cholesterol and ApoB. Lipid ratios and ApoB:ApoA-I performed better than any single lipid or apolipoprotein in predicting CVD risk. CONCLUSIONS/INTERPRETATION: In patients with type 2 diabetes in the FIELD study, traditional lipid ratios were as strong as the ApoB:ApoA-I ratio in predicting CVD risk. The data provide little evidence for replacement of traditional lipids and their ratios with measures of ApoB, ApoA-I and their ratio.

Role of 3beta-hydroxysteroid-delta 24 reductase in mediating antiinflammatory effects of high-density lipoproteins in endothelial cells.

OBJECTIVE: The purpose of this study was to investigate the ability of high-density lipoproteins (HDLs) to upregulate genes with the potential to protect against inflammation in endothelial cells. METHODS AND RESULTS: Human coronary artery endothelial cells (HCAECs) were exposed to reconstituted HDLs (rHDLs) for 16 hours before being activated with tumor necrosis factor-alpha (TNF-alpha) for 5 hours. rHDLs decreased vascular cell adhesion molecule-1 (VCAM-1) promoter activity by 75% (P<0.05), via the nuclear factor-kappa B (NF-kappaB) binding site. rHDLs suppressed the canonical NF-kappaB pathway and decreased many NF-kappaB target genes. Suppression of NF-kappaB and VCAM-1 expression by rHDLs or native HDLs was dependent on an increase in 3beta-hydroxysteroid-Delta 24 reductase (DHCR24) levels (P<0.05). The effect of HDLs on DHCR24 is dependent on SR-BI but not ABCAI or ABCGI. Silencing DHCR24 expression increased NF-kappaB (1.2-fold, P<0.05), VCAM-1 (30-fold, P<0.05), and NF-kappaB p50 (4-fold, P<0.05) and p65 subunits (150-fold, P<0.05). TNF-alpha activation of siDHCR24-treated cells increased expression of VCAM-1 (550-fold, P<0.001) and NF-kappaB (9-fold, P<0.001) that could no longer be suppressed by rHDLs. CONCLUSIONS: Results suggest that antiinflammatory effects of rHDLs are mediated partly through an upregulation of DHCR24. These findings raise the possibility of considering DHCR24 as a target for therapeutic modulation.

Aggressive management of obesity in children and young adults: the known challenges and potential opportunities.

The world is confronted with an impending epidemic of premature cardiovascular disease (CVD) that is being fueled by an increase in the prevalence of central obesity.

Precursor-product relationship between pools of very low density lipoprotein triglyceride.

The process of removal of triglyceride from the plasma may involve a sequential conversion of larger to smaller glyceride-rich lipoproteins. This has been studied within the species of lipoproteins comprising the very low density lipoproteins (VLDL) which transport the bulk of endogenously formed triglyceride. Palmitic acid-(14)C which was used to label the plasma glycerides was administered either as a prolonged constant infusion or as a pulse label. The specific activity-time curves of triglyceride fatty acids (TGFA) were analyzed both in total VLDL and in two subfractions of VLDL. The nature of the curves for total VLDL that were observed during the constant infusions were consistent with slow isotopic equilibration of precursors of VLDL-TGFA or with the presence of a precursor-product relationship between different components of VLDL-TGFA. The curves did not indicate any detectable differences in (fractional) turnover rates of independently metabolized pools of VLDL-TGFA. Differences in the specific activity-time curves of TGFA in two subfractions of VLDL (Sf > 100 and Sf 20-100) were consistent with a precursor-product relationship between TGFA in the two subfractions; again there was no indication of significant differences in (fractional) turnover rates. The specific activity-time curves of TGFA in the two subfractions of VLDL that were obtained with single injections of radio-palmitate showed a consistent difference in the rates at which TGFA became labeled in the two subfractions, being slower in the Sf 20-100 fraction. The findings from all experiments when considered together, were compatible with a precursor-product relationship that suggested that larger VLDL were converted to progressively smaller species as triglyceride was being removed.

Diurnal fluctuations in triglyceride, free fatty acids, and insulin during sucrose consumption and insulin infusion in man.

Serial changes in circulating triglyceride, free fatty acids (FFA), insulin, and glucose have been measured in human subjects fed sucrose as the sole source of calories for 2- or 3-day periods. The sucrose was given either during the day with overnight fasting (19 subjects) or as continual 3-hour meals during the day and night (seven subjects). Insulin was infused overnight in five additional subjects on the day-feeding regimen to determine the effect on triglyceride concentration. The concentration of triglyceride increased during the study in all subjects, but there was a clear diurnal pattern in the response which was present even in the continual feeding studies. The rise in triglyceride occurred mainly overnight, and during the day there was frequently a fall in the concentration. The overnight increase was significantly less when insulin was infused. There were also diurnal fluctuations in FFA and insulin in both daytime and continual feeding regimens. The plasma FFA, like triglyceride, rose during the night and fell during the day while the insulin rose during the day and fell overnight. Separate statistical analysis of the daytime and overnight changes revealed that the changes in triglyceride were significantly but negatively correlated with changes in insulin during both periods. The changes in triglyceride and FFA were positively correlated during the day but not significantly related during the night. The data show that when sucrose is eaten for 2 or 3 days, there is a general increase in triglyceride concentration upon which are superimposed major diurnal fluctuations in the concentrations of triglyceride, insulin, and FFA. It is suggested that the highly significant inverse relationship between changes in triglyceride and insulin may be mediated through an effect of insulin on triglyceride removal.

Effect of inhibiting cholesteryl ester transfer protein on the kinetics of high-density lipoprotein cholesteryl ester transport in plasma: in vivo studies in rabbits.

OBJECTIVE: Inhibitors of cholesteryl ester transfer protein (CETP) have been developed as potential anti-atherogenic agents. Theoretically, however, they may be pro-atherogenic by blocking one of the pathways for removing high-density lipoprotein (HDL) cholesteryl esters (CE) from plasma in the final step of reverse cholesterol transport. Here we describe how CETP inhibition in rabbits impacts on the kinetics of HDL CE transport in plasma. METHODS AND RESULTS: Administration of a CETP inhibitor reduced CETP activity by 80% to 90% and doubled the HDL cholesteryl ester concentration. Multi-compartmental analysis was used to determine HDL CE kinetics in CETP-inhibited and control rabbits after injection of tracer amounts of both native and reconstituted HDL labeled with 3H in the CE moiety. In control rabbits, HDL CE was removed from plasma by both a direct pathway and an indirect pathway after transfer of HDL CE to the very-low-density lipoprotein/low-density lipoprotein fraction. In CETP-inhibited rabbits there was an almost complete block in removal via the indirect pathway. This did not compromise the overall removal of HDL CE from plasma, which was not different in control and inhibited animals. CONCLUSIONS: Inhibiting CETP in rabbits does not compromise the removal of HDL CE from plasma.

Effects of long-term fenofibrate therapy on cardiovascular events in 9795 people with type 2 diabetes mellitus (the FIELD study): randomised controlled trial.

BACKGROUND: Patients with type 2 diabetes mellitus are at increased risk of cardiovascular disease, partly owing to dyslipidaemia, which can be amenable to fibrate therapy. We designed the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study to assess the effect of fenofibrate on cardiovascular disease events in these patients. METHODS: We did a multinational, randomised controlled trial with 9795 participants aged 50-75 years, with type 2 diabetes mellitus, and not taking statin therapy at study entry. After a placebo and a fenofibrate run-in phase, we randomly assigned patients (2131 with previous cardiovascular disease and 7664 without) with a total-cholesterol concentration of 3.0-6.5 mmol/L and a total-cholesterol/HDL-cholesterol ratio of 4.0 or more or plasma triglyceride of 1.0-5.0 mmol/L to micronised fenofibrate 200 mg daily (n=4895) or matching placebo (n=4900). Our primary outcome was coronary events (coronary heart disease death or non-fatal myocardial infarction); the outcome for prespecified subgroup analyses was total cardiovascular events (the composite of cardiovascular death, myocardial infarction, stroke, and coronary and carotid revascularisation). Analysis was by intention to treat. The study was prospectively registered (number ISRCTN 64783481). FINDINGS: Vital status was confirmed on all but 22 patients. Averaged over the 5 years' study duration, similar proportions in each group discontinued study medication (10% placebo vs 11% fenofibrate) and more patients allocated placebo (17%) than fenofibrate (8%; p<0.0001) commenced other lipid treatments, predominantly statins. 5.9% (n=288) of patients on placebo and 5.2% (n=256) of those on fenofibrate had a coronary event (relative reduction of 11%; hazard ratio [HR] 0.89, 95% CI 0.75-1.05; p=0.16). This finding corresponds to a significant 24% reduction in non-fatal myocardial infarction (0.76, 0.62-0.94; p=0.010) and a non-significant increase in coronary heart disease mortality (1.19, 0.90-1.57; p=0.22). Total cardiovascular disease events were significantly reduced from 13.9% to 12.5% (0.89, 0.80-0.99; p=0.035). This finding included a 21% reduction in coronary revascularisation (0.79, 0.68-0.93; p=0.003). Total mortality was 6.6% in the placebo group and 7.3% in the fenofibrate group (p=0.18). Fenofibrate was associated with less albuminuria progression (p=0.002), and less retinopathy needing laser treatment (5.2%vs 3.6%, p=0.0003). There was a slight increase in pancreatitis (0.5%vs 0.8%, p=0.031) and pulmonary embolism (0.7%vs 1.1%, p=0.022), but no other significant adverse effects. INTERPRETATION: Fenofibrate did not significantly reduce the risk of the primary outcome of coronary events. It did reduce total cardiovascular events, mainly due to fewer non-fatal myocardial infarctions and revascularisations. The higher rate of starting statin therapy in patients allocated placebo might have masked a moderately larger treatment benefit.

Plaque stabilizing effects of apolipoprotein A-IV.

BACKGROUND AND AIMS: Apolipoprotein (apo) A-IV, the third most abundant HDL-associated protein, is atheroprotective and shares similar properties as apoA-I. We have reported previously that apoA-I, the most abundant apolipoprotein in HDL, inhibits plaque disruption in a mouse model. We aimed at examining the effects of apoA-IV on markers of plaque stability in vivo. METHODS: Plaques within brachiocephalic arteries of 16-week old apoE-knockout C57BL/6 mice were examined for changes in composition after 10 weeks on a high-fat diet (HFD). The animals received twice-weekly injections of human lipid-free apoA-IV (1 mg/kg, n = 31) or PBS (n = 32) during the 9th and 10th weeks of the HFD. RESULTS: In the apoA-IV treated mice, there were significantly fewer hemorrhagic plaque disruptions (9/31 vs. 18/32, p < 0.05), thicker fibrous caps, smaller lipid cores, a lower macrophage:SMC ratio, less MMP-9 protein, more collagen, and fewer proliferating cells. In the plaques of mice given apoA-IV, MCP-1, VCAM-1, and inducible NOS were also significantly lower. Based on the percentage of cleaved PARP-positive and TUNEL-positive plaque nuclei, apoA-IV reduced apoptosis. in HMDMs, apoA-IV reduced MMP-9 mRNA expression by half, doubled mRNA levels of TIMP1 and decreased MMP-9 activity. CONCLUSIONS: ApoA-IV treatment is associated with a more stable plaque phenotype and a reduced incidence of acute disruptions in this mouse model.

Evidence that lecithin:cholesterol acyltransferase acts on both high-density and low-density lipoproteins.

In incubations of plasma containing lipoproteins at physiological concentrations it has been confirmed that high-density lipoproteins (HDL) are the major initial recipients of the esterified cholesterol formed in the reaction catalysed by lecithin:cholesterol acyltransferase. It has also been confirmed, however, that a small proportion of the esterified cholesterol of lecithin:cholesterol acyltransferase origin is incorporated directly into low-density lipoproteins (LDL), via a pathway that bypasses the HDL. This direct incorporation of esterified cholesterol into LDL is compatible with either of two general models. Model A proposes that lecithin:cholesterol acyltransferase does not interact directly with LDL but rather that it acts only on lipoproteins outside the LDL fraction. According to model A, while most of the esterified cholesterol so formed is incorporated into HDL, a small proportion is transferred directly to LDL. Model B, by contrast, proposes that a direct incorporation of esterified cholesterol into LDL is the result of a direct action of lecithin:cholesterol acyltransferase on the free cholesterol associated with LDL. To differentiate between these two models, experiments have been performed in which incubation mixtures containing LDL, HDL and a source of lecithin:cholesterol acyltransferase were supplemented with free [3H]cholesterol which had previously been incorporated into either LDL or HDL. It was found that, of the esterified [3H]cholesterol which was subsequently formed, the proportion recovered in the LDL fraction was much greater in the incubations to which the free [3H]cholesterol had been added as a component of LDL than in those to which it had been added as a component of HDL. This essentially excluded model A but was consistent with model B. It has been concluded that, while most of the lecithin:cholesterol acyltransferase may interact with particles in the HDL fraction, a small proportion of the enzyme interacts directly with LDL.

Effects of various non-esterified fatty acids on the particle size redistribution of high density lipoproteins induced by the human cholesteryl ester transfer protein.

The effects of various non-esterified fatty acids on the CETP-mediated particle size redistribution of HDL were studied by incubating HDL3 and CETP for 24 h at 37 degrees C in the absence or in the presence of either saturated, monounsaturated or polyunsaturated non-esterified fatty acids. In the absence of non-esterified fatty acids, CETP induced a redistribution of the initial population of HDL3 (Stokes' radius 4.3 nm) by promoting the appearance of one larger (Stokes' radius 4.8 nm) and two smaller (Stokes' radii 3.9 and 3.7 nm) HDL subpopulations. Whereas the non-esterified fatty acids alone did not modify the HDL3 distribution profile, they were able to alter markedly the capacity of CETP to induce the particle size redistribution of HDL. All the saturated fatty acids with at least 10 carbons were able to increase the formation of the very small sized particles (Stokes' radius 3.7 nm) in a concentration dependent manner, the medium chain fatty acids (12 and 14 carbons) being the best activators. The potential effect of non-esterified fatty acids was also influenced by the presence of double bonds in their monomeric carbon chain. While at low concentrations of non-esterified fatty acids (0.1 mmol/l) the enhancement of the formation of very small HDL particles appeared to be greater with oleic and linoleic acids than with stearic acid, at higher concentrations (0.4 mmol/l), oleic, linoleic and arachidonic acids decreased the formation of the 3.7 nm radius particles. The inhibition of the process at high concentrations of unsaturated fatty acids was linked to the degree of unsaturation of their carbon chain, arachidonic acid being the strongest inhibitor. The present study has demonstrated that non-esterified fatty acids can modulate the particle size redistribution of HDL3 mediated by the cholesteryl ester transfer protein even in the absence of any other lipoprotein classes. The effect of non-esterified fatty acid is dependent on both the length and the degree of unsaturation of their monomeric carbon chain.

Effects of various non-esterified fatty acids on the transfer of cholesteryl esters from HDL to LDL induced by the cholesteryl ester transfer protein.

The effects of saturated, monounsaturated and polyunsaturated non-esterified fatty acids on the rate of transfer of radiolabeled cholesteryl esters from high density lipoproteins (HDL) to low density lipoproteins (LDL), induced by the cholesteryl ester transfer protein (CETP), have been studied. Human high-density lipoproteins-subfraction 3 (HDL3) containing radiolabeled cholesteryl esters were incubated with LDL at 37 degrees C with or without CETP and in the absence or in the presence of non-esterified fatty acids. Less than 6% of the total radioactivity was recovered in the LDL fraction after incubation of HDL3, and LDL for 3 h at 37 degrees C in the absence of CETP, regardless of whether or not non-esterified fatty acids were added. The addition of CETP to the incubation mixture induced a time-dependent redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. Non-esterified fatty acids were found to alter the rate of transfer of cholesteryl esters induced by CETP. While short chain saturated non-esterified fatty acids (caprylic and capric acids) had no effect on the rate of transfer of cholesteryl esters, the medium and long chain ones (lauric, myristic, palmitic and stearic acids) significantly increased the CETP-mediated transfers from HDL3 to LDL. At low concentrations, unsaturated fatty acids also stimulated the CETP-mediated redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. As the concentration of either oleic, linoleic or arachidonic acids increased to higher levels, a significant proportion of fatty acids remained unassociated with lipoprotein particles. Under these circumstances the transfer process was inhibited. These results show that non-esterified fatty acids can modulate the CETP-mediated transfer of cholesteryl esters from HDL to LDL and that this effect is dependent on both the length and the degree of unsaturation of their monomeric carbon chain.

Changes in the size and density of human high-density lipoproteins promoted by a plasma-conversion factor.

Incubation of human high-density lipoprotein subfraction-3 (HDL3) with rabbit lipoprotein-depleted plasma resulted in marked changes in the density and size of the HDL. After 24 h of incubation at 37 degrees C, the original HDL3 were converted into populations of larger (less dense) and smaller (more dense) particles. The degree of conversion increased with increasing concentrations of lipoprotein-depleted plasma and increasing incubation time. Furthermore, lecithin:cholesterol acyltransferase, lipoprotein lipase and lipid-transfer protein were shown not to be involved in the process. It was therefore proposed that a separate factor, the HDL-conversion factor, was responsible for the observed changes. Conversion-factor activity was assessed in the lipoprotein-depleted plasma of several species and found to be greater in rabbits and rats than in pigs and human subjects. It was also established that the conversion factor was able to be precipitated from rabbit lipoprotein-depleted plasma between 40 and 50% saturation of (NH4)2SO4. This information was used to partially purify the factor from human plasma. The proteins of human plasma which precipitated between 35 and 55% saturation of (NH4)2SO4 were recovered and subjected to ultracentrifugation to isolate the fraction of density 1.21-1.25 g/ml. This fraction, which was rich in HDL-conversion activity, was further purified by cation-exchange chromatography. In conclusion, a factor which promotes the conversion of HDL to populations of larger and smaller particles has been found to exist at various levels of activity in the plasma of several species. Partial purification of the factor from human plasma has been achieved.

Increases in the particle size of high-density lipoproteins induced by purified lecithin: cholesterol acyltransferase: effect of low-density lipoproteins.

Homogeneous subpopulations of human high-density lipoproteins subfraction-3 (HDL3) have been incubated at 37 degrees C with purified lecithin: cholesterol acyltransferase, human serum albumin and varying concentrations of human low-density lipoproteins (LDL). Changes in HDL particle size and composition during these incubations were monitored. Incubation of HDL3a (particle radius 4.3 nm) in the absence of LDL resulted in an esterification of more than 70% of the HDL free cholesterol after 24 h of incubation. This, however, was sufficient to increase the HDL cholesteryl ester by less than 10% and was not accompanied by any change in particle size. When this mixture was incubated in the presence of progressively increasing concentrations of LDL, which donated free cholesterol to the HDL, the molar rate of production of cholesteryl ester was much greater; at the highest LDL concentration HDL cholesteryl ester content was almost doubled after 24 h and there was an increase in the HDL particle size up to the HDL2 range. In the case of HDL3b (radius 3.9 nm), there were again only minimal changes in particle size in incubations not containing LDL. In the presence of the highest concentration of LDL tested, however, the particles were again enlarged into the HDL2 size range after 24 h incubation. These HDL2-like particles were markedly enriched with cholesteryl ester but depleted of phospholipid and free cholesterol when compared with native HDL2. Furthermore, the ratio of apolipoprotein A-I to apolipoprotein A-II resembled that in the parent-HDL3 and was very much lower than that in native HDL2. It has been concluded that purified lecithin: cholesterol acyltransferase is capable of increasing the size of HDL3 towards that of HDL2 but that other factors must operate in vivo to modulate the chemical composition of the enlarged particles.

Cholesteryl ester transfer protein promotes the association of HDL apolipoproteins A-I and A-II with LDL: potentiation by oleic acid.

The association of apolipoprotein (apo) A-I and apo A-II with apo-B-containing particles was measured after incubation at 37 degrees C of either total plasma or low-density lipoproteins (LDL) and high-density lipoproteins-3 (HDL3) in the presence of partially purified cholesteryl ester transfer protein (CETP). At the end of the incubation, apo-B-containing lipoproteins were separated by immunoprecipitation with an anti-apo B gamma-globulin fraction. In mixtures containing LDL and HDL3, either maintained at 4 degrees C or incubated at 37 degrees C, optimal concentrations of anti-apo B antibodies induced the precipitation of more than 95% of apo B without precipitation of apo A-I and apo A-II. When total plasma was incubated at 37 degrees C for 24 h, a significant proportion of apo A-I and apo A-II became associated with apo-B-containing lipoproteins. The fraction of HDL apoproteins associated with apo-B-containing lipoproteins was significantly reduced when plasma was supplemented with TP2 anti-CETP monoclonal antibodies, which are known to inhibit CETP activity. Incubation of LDL and HDL3 for 24 h at 37 degrees C in the presence of purified CETP also induced the association of a significant proportion of apo A-I and apo A-II with apo-B-containing particles. This effect was dependent on CETP concentration in the incubation mixtures and could be suppressed by the addition of anti-CETP monoclonal antibodies. While oleic acid alone, at a final concentration of 0.2 mmol/l, did not promote any association of HDL-apolipoproteins with LDL, it was able, at this concentration, to greatly enhance the CETP-mediated association of apo A-I and apo A-II with apo-B-containing particles. In the presence of both CETP and oleic acid, the association of apo A-I and apo A-II with apo-B-containing particles was apparent within 3 h of commencing the incubation. Approximately 3 mol of apo A-I and 1 mol of apo A-II co-precipitated with each mol of apo B after a 24 h incubation of LDL, HDL3 and CETP. When oleic acid was added to the incubation mixture in addition to CETP, up to 5.5 mol of apo A-I and 2.3 mol of apo A-II were associated with each mol of apo B.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in particle size of high density lipoproteins during incubation with very low density lipoproteins, cholesteryl ester transfer protein and lipoprotein lipase.

Previous reports have produced conflicting views of the effects of lipoprotein lipase (LPL) on the particle size distribution of high density lipoproteins (HDL). In this study we have investigated the changes in particle size of HDL promoted by the interaction of LPL, the cholesteryl ester transfer protein (CETP) and very low density lipoproteins (VLDL). When the plasma fraction of d less than 1.21 g/ml (containing all lipoprotein fractions) was incubated for 24 h with bovine milk LPL alone or with CETP alone, there was relatively little change in the particle size distribution of HDL. When both LPL and CETP were added to the lipoprotein mixture, there was a substantial reduction in the particle size of HDL. This reduction in HDL particle size was found to be a direct function of the concentration of CETP. It was also influenced by the concentrations of VLDL and LPL, although in these cases the relationships were complex. When mixtures of the plasma fraction of d = 1.006-1.21 g/ml (this fraction includes low density lipoproteins and HDL but not VLDL) were supplemented with both LPL and CETP and incubated in the presence of varying concentrations of added VLDL, there was a progressive increase in the conversion of HDL into very small HDL particles of radius 3.7 nm as the concentration of VLDL triacylglycerol increased up to about 400 nmol/nml. However, further increases in the concentration of VLDL were accompanied by a progressive reduction in the formation of small HDL particles until, at higher VLDL concentrations, the effect was all but abolished. There was a similar enhancement in the formation of small HDL when LPL was added at low but not at high concentrations. These findings are consistent with the existence of two opposing processes. On the one hand there is likely to be a synergism between CETP and the non-esterified fatty acids (NEFA) released by LPL; this will favour a reduction in HDL particle size. On the other hand, the transfer of lipolysis products from VLDL to HDL may mask any such particle size reduction. The fact that the reduction in HDL particle size promoted by LPL, CETP and VLDL was found to be all but abolished by adding fatty acid-poor albumin to the incubation mixture is consistent with the proposition that NEFA are involved in the process. It also suggests, however, that the phenomenon may have little if any physiological significance.