Overexpression of human lecithin:cholesterol acyltransferase in cholesterol-fed rabbits: LDL metabolism and HDL metabolism are affected in a gene dose-dependent manner

Lecithin:cholesterol acyltransferase (LCAT) is an enzyme well known for its involvement in the intravascular metabolism of high density lipoproteins; however, its role in the regulation of apolipoprotein (apo) B-containing lipoproteins remains elusive. The present study was designed to investigate the metabolic mechanisms responsible for the differential lipoprotein response observed between cholesterol-fed hLCAT transgenic and control rabbits. 131I-labeled HDL apoA-I and 125I-labeled LDL kinetics were assessed in age- and sex-matched groups of rabbits with high (HE), low (LE), or no hLCAT expression after 6 weeks on a 0.3% cholesterol diet. In HE, the mean total cholesterol concentration on this diet, mg/dl (230 +/- 50), was not significantly different from that of either LE (313 +/- 46) or controls (332 +/- 52) due to the elevated level of HDL-C observed in HE (127 +/- 19), as compared with both LE (100 +/- 33) and controls (31 +/- 4). In contrast, the mean nonHDL-C concentration for HE (103 +/- 33) was much lower than that for either LE (213 +/- 39) or controls (301 +/- 55). FPLC analysis of plasma confirmed that HDL was the predominant lipoprotein class in HE on the cholesterol diet, whereas cholesteryl ester-rich, apoB-containing lipoproteins characterized the plasma of LE and, most notably, of controls. In vivo kinetic experiments demonstrated that the differences in HDL levels noted between the three groups were attributable to distinctive rates of apoA-I catabolism, with the mean fractional catabolic rate (FCR, d-1) of apoA-I slowest in HE (0.282 +/- 0.03), followed by LE (0.340 +/- 0.01) and controls (0.496 +/- 0.04). A similar, but opposite, pattern was observed for nonHDL-C levels and LDL metabolism (h-1), such that HE had the lowest nonHDL-C levels with the fastest rate of clearance (0.131 +/- 0.027), followed by LE (0.057 +/- 0.009) and controls (0.031 +/- 0.001). Strong correlations were noted between LCAT activity and both apoA-I (r= -0.868, P < 0.01) and LDL (r = 0.670, P = 0.06) FCR, indicating that LCAT activity played a major role in the mediation of lipoprotein metabolism. In summary, these data are the first to show that LCAT overexpression can regulate both LDL and HDL metabolism in cholesterol-fed rabbits and provide a potential explanation for the prevention of diet-induced atherosclerosis observed in our previous study.     

Human plasma CETP deficiency: identification of a novel mutation in exon 9 of the CETP gene in a Caucasian subject from North America

Human plasma cholesteryl ester transfer protein (CETP) is a 476-residue hydrophobic glycoprotein that catalyzes the heterotransfer of cholesteryl esters and triacylglycerols among lipoproteins: Mutations in the CETP gene have been identified, mostly in the Japanese population. These mutations result in hypercholesterolemia due to the presence of large cholesteryl ester-rich HDL particles, elevated plasma apoA-I and apoE, and reduced apoB levels. Here we report the plasma lipoprotein phenotype and molecular defect in a 57-year-old female Nova Scotian subject lacking Japanese ancestry who is homozygous for a novel mutation in the CETP gene. Her total plasma cholesterol was 7.3 mmol/l with an LDL cholesterol of 2.9 mmol/l and HDL cholesterol of 4.4 mmol/l. She was mildly hypertriglyceridemic (1.6 mmol/l) and had markedly elevated apoA-I (256 mg/dl) and apoE (14.4 mg/dl) with only slightly reduced apo/B levels (94 mg/dl). Her VLDL and LDL were cholesteryl ester-poor (1.8 and 37.2% of lipids, respectively) and triacylglycerol-rich (67.3 and 18.9% of lipids, respectively) while her HDL was cholesteryl ester-rich (40.2-45.7% of lipids) and triacylglycerol-poor (3.3-2.5% of lipids). No plasma CETP activity or mass was detected. Bi-directional DNA sequence analysis of PCR products from all 16 exons showed a single base substitution (C-->T at nucleotide 836 in exon 9 resulting in 268 Arg-->STOP) in both alleles. No other mutation was detected. A single base mismatched, 26 bp reverse PCR primer that produced a single Mae III RFLP site upon amplification of the mutated DNA sequence was designed for rapid population screening. This subject is, we believe, the first Caucasian North American patient reported to have CETP deficiency.     

Cholesteryl ester transfer protein activity enhances plasma cholesteryl ester formation. Studies in CETP transgenic mice and human genetic CETP deficiency

The plasma cholesteryl ester transfer protein (CETP) promotes the removal of HDL cholesteryl esters and is thought to stimulate reverse cholesterol transport (RCT). However, mechanisms by which CETP may stimulate RCT are poorly understood. Thus, we examined the relationship between plasma CETP expression and plasma cholesteryl ester formation in CETP transgenic (Tg) mice, hamsters, and human subjects with genetic CETP deficiency. Incubation of CETP Tg mouse plasma showed a 20% to 40% increase in plasma cholesterol esterification rate (CER, P < .05) compared with control mice. Injection of a neutralizing CETP monoclonal antibody (MAb) (TP2) into natural flanking region CETP Tg mice resulted in an increase in plasma free cholesterol (FC) concentration, FC/CE ratio, FC/phosphatidylcholine ratio, and hepatic CETP mRNA. In hamsters, CETP inhibition also resulted in an increase in plasma FC/phosphatidylcholine ratio and increased CETP mRNA in adipose tissue. In humans with two common CETP gene mutations (an intron 14 splicing defect and a D442G missense mutation), mean plasma CERs were 39 and 60, respectively, compared with 89 nmol x mL-1 x h-1 in normal subjects. By contrast, lecithin:cholesterol acyltransferase (LCAT) mass was normal in CETP-deficient subjects. MAb neutralization of CETP activity in incubated human plasma did not alter the LCAT reaction, even after supplementation with discoidal HDL and VLDL. Thus, genetic alterations in CETP levels lead to secondary changes in the plasma LCAT reaction, possibly because of remodeling of HDL by CETP acting in concert with other factors in vivo. In human genetic CETP deficiency, a moderate impairment in the plasma LCAT reaction may contribute to a defect in RCT, providing a potential mechanism to explain the recently observed excess of coronary heart disease in these subjects.     

Production rate determines plasma concentration of large high density lipoprotein in non-human primates

Large LpAI HDL particles, containing only apoA-I without apoA-II, are reported to be the major anti-atherogenic portion of HDL and to be increased in individuals with low risk for coronary heart disease. To determine whether the plasma concentration of large LpAI is modulated by the rate of production or catabolism of apolipoprotein A-I (apoA-I) in large LpAI, kinetic studies of large LpAI were performed in African green monkeys consuming an atherogenic diet with either high plasma HDL concentration (120 +/- 36 mg/dl, mean +/- SD, n = 3) or low plasma HDL concentration (40 +/- 13 mg/dl, n = 3). Large LpAI was isolated, without ultracentrifugation, by immunoaffinity and gel filtration and radiolabeled. After injection, the specific activity of apoA-I in large HDL, consisting of both LpAI and LpAI:AII particles, was followed. A multicompartmental model was developed for the kinetics of apoA-I in large HDL, which indicated that a portion of large HDL is distributed to a sequestered pool, outside the circulating plasma, and reenters circulating plasma approximately 3 h after injection. There was no conversion of large LpAI to smaller HDL particles or transfer of radiolabeled apoA-I to smaller HDL particles. Although the mean fractional catabolic rate was not different comparing the high and low HDL group, the mean production rate of apoA-I in large HDL was 4-fold greater in the high HDL group compared with the low HDL group. These data support the hypothesis that the plasma concentration of large HDL is controlled primarily by the rate of production of apoA-I in large HDL.     

Decreased postprandial high density lipoprotein cholesterol and apolipoproteins A-I and E in normolipidemic smoking men: relations with lipid transfer proteins and LCAT activities

We have previously reported that normolipidemic smokers are lipid intolerant due to increased responses of triglyceride-rich lipoproteins (TRL) apolipoprotein B-48, triglyceride (TG), and retinyl esters to a mixed meal compared to non-smokers. To investigate whether postprandial high density lipoprotein (HDL), apolipoprotein A-I (apoA-I), apolipoprotein A-II (apoA-II), and apolipoprotein E (apoE) concentrations or lipid transfer protein activities are affected by cigarette smoking, we investigated 12 male smokers and 12 non-smokers with comparable fasting lipoprotein profile, BMI, and age. Plasma samples obtained after an overnight fast and postprandially were separated by density gradient ultracentrifugation. Postprandial apoA-I, lipoprotein AI-particles (LpA-I), HDL-cholesterol, and HDL apoE concentrations decreased in smokers, but remained unchanged in controls. Concomitantly, cholesterol and apoE concentrations increased significantly in TRL fractions in smokers. Fasting lecithin:cholesterol acyltransferase (LCAT) and phospholipid transfer protein (PLTP) activity levels, as well as esterification rates (EST) and phospholipid transfer rates were comparable between the groups. Cholesteryl ester transfer protein (CETP) activity levels were lower in the smokers. Postprandially EST increased, but CETP and PLTP activities deceased in smokers as compared to controls. We conclude, that even healthy, normolipidemic smokers have altered postprandial high density lipoprotein (HDL) cholesterol and apolipoprotein composition, as well as lipid transfer protein activities. The shift of cholesterol and apoE from HDL to the triglyceride-rich lipoprotein (TRL) fraction, together with decreased plasma apoA-I and LpA-I concentrations during alimentary lipemia may indicate impaired reverse cholesterol transport. Both the postprandial increase in TRL and the lowering of HDL may promote atherogenesis in smokers.     

Impact of gender on the metabolism of apolipoprotein A-I in HDL subclasses LpAI and LpAI:AII in older subjects

The behavior of apolipoprotein (apo) A-I in lipoprotein (Lp) AI and LpAI:AII was studied in 11 postmenopausal females and 11 males matched for plasma triglyceride and total cholesterol levels. Subjects consumed a baseline diet [35% fat (14% saturated, 15% monounsaturated, and 7% polyunsaturated), 15% protein, 49% carbohydrate, and 147 mg cholesterol/1000 kcal] for 6 weeks before the start of the kinetic study. At the end of the diet period, using a primed-constant infusion of [5,5,5-2H3]leucine, residence times (RT) and secretion rates (SR) of apoA-I were determined in 2 subpopulations of high-density lipoprotein (HDL) particles, LpAI and LpAI:AII. Plasma total cholesterol, low-density lipoprotein cholesterol, and triglyceride concentrations were similar in males and females. The mean plasma HDL cholesterol concentration in males (1.14 +/- 0.23 mmol/L; mean +/- SD) was lower than in females (1.42 +/- 0.18 mmol/L; P =. 0034). Similarly, the mean plasma concentration of apoA-I in males (130 +/- 21 mg/dL) was lower than that in females (150 +/- 19 mg/dL; P = .0421). The RT of apoA-I in either LpAI or LpAI:AII was similar between men and women. Despite the higher plasma apo A-I levels in female compared with male subjects, total apoA-I and apoA-I in LpAI and LpAI:AII pool sizes were similar between the two groups, attributable to the lower body weight of the female subjects. The mean SR of total apoA-I in males (8.5 +/- 2.7 mg.kg-1.d-1) was 22% lower than in females (10.9 +/- 2.3 mg.kg-1.d-1; P = .0389). The SR of both apoA-I in LpAI and LpAI:AII was lower in males than females, although the differences did not reach statistical significance. These data suggest that the difference observed in HDL cholesterol concentration between males and females is attributable to SR of apoA-I and not the catabolic rate.     

A randomized controlled trial of pelvic floor muscle exercises to treat postnatal urinary incontinence

BACKGROUND: The enzyme lecithin-cholesterol acyl transferase (LCAT) esterifies free cholesterol on high-density lipoprotein (HDL) and the cholesteryl ester transfer protein (CETP) transfers cholesteryl ester to very-low-density lipoprotein (VLDL) and low-density lipoproteins (LDL). Using statins, contradictory findings have been made regarding CETP activity in normolipidemic individuals and in those with familial dysbetalipoproteinemia. In contrast, LCAT activity appears to be unaffected by simvastatin. Antioxidants have also been proposed for the use of anti-atherosclerotic treatment, because the oxidation of LDL may have a key role in the pathophysiology of atherogenesis. OBJECTIVE: To investigate, in hypercholesterolemic patients, whether a combination of pravastatin with the antioxidant, vitamin E, has greater effects on the activity of CETP and of LCAT than does pravastatin alone. METHODS: This placebo-diet-controlled multicenter trial included 220 hypercholesterolemic patients who were assigned randomly to groups to receive: diet and 20-40 mg pravastatin (n = 52), diet and alpha-tocopherol (n = 60), or diet associated with placebo (n = 52). Plasma LCAT activity was determined using excess exogenous substrate, containing [3H]cholesterol. Plasma CETP activity was measured in the supernatant fraction after precipitation of endogenous apo B-containing lipoproteins with phosphotungstate-Mg2+. The exchange of cholesteryl esters between [14C]cholesteryl ester-labeled LDL and unlabeled HDL was measured during a 16-h incubation, while LCAT was inhibited. RESULTS: The addition of pravastatin to the diet induced a significant decrease in plasma CETP activity (P < 0.05); this effect was less evident in the group cotreated with vitamin E. For the first time, it was shown that CETP concentrations increased significantly after vitamin E alone (P < 0.05). No significant differences in the plasma activity of LCAT were observed among the groups. CONCLUSIONS: Pravastatin reduced CETP activity, but not that of LCAT. Addition of vitamin E prevented the decrease in CETP activity and had no effect on LCAT activity. The mechanism responsible for these effects is unknown, but could involve the prevention of radical-induced damage to CETP by vitamin E.     

Influence of plasma cholesteryl ester transfer activity on the LDL and HDL distribution profiles in normolipidemic subjects

The relations of cholesteryl ester transfer protein (CETP) activity to the distribution of low density lipoproteins (LDLs) and high density lipoproteins (HDLs) were investigated in fasting plasma samples from 27 normolipidemic subjects. LDL and HDL subfractions were separated by electrophoresis on 20-160 g/L and 40-300 g/L polyacrylamide gradient gels, respectively. Subjects were subdivided into two groups according to their LDL pattern. Monodisperse patterns were characterized by the presence of a single LDL band, whereas polydisperse patterns were characterized by the presence of several LDL bands of different sizes. To investigate the influence of lipid transfers on LDL patterns, total plasma was incubated at 37 degrees C in the absence of lecithin:cholesterol acyltransferase (LCAT) activity. The incubation induced a progressive transformation of polydisperse patterns into monodisperse patterns. Under the same conditions, initially monodisperse patterns remained unchanged. Measurements of the rate of radiolabeled cholesteryl esters transferred from HDL3s to very low density lipoproteins (VLDLs) and LDLs revealed that subjects with a monodisperse LDL pattern presented a significantly higher plasma CETP activity than subjects with a polydisperse LDL pattern (301 +/- 85%/hr per milliliter versus 216 +/- 47%/hr per milliliter, respectively; p < 0.02). In addition, when total plasma was incubated for 24 hours at 37 degrees C in the absence of LCAT activity, the relative mass of cholesteryl esters transferred from HDLs to apolipoprotein B-containing lipoproteins was greater in plasma with monodisperse LDL than in plasma with polydisperse LDL (0.23 +/- 0.06 versus 0.17 +/- 0.06, respectively; p < 0.02). These results indicated that in normolipidemic plasma, CETP could play an important role in determining the size distribution of LDL particles. The analysis of lipoprotein cholesterol distribution in the two groups of subjects sustained this hypothesis. Indeed, HDL cholesterol levels, the HDL:VLDL+LDL cholesterol ratio, and the esterified cholesterol:triglyceride ratio in HDL were significantly lower in plasma with the monodisperse LDL pattern than in plasma with the polydisperse LDL pattern (p < 0.01, p < 0.01, and p < 0.02, respectively). Plasma LCAT activity did not differ in the two groups. Plasma CETP activity correlated positively with the level of HDL3b (r = 0.542, p < 0.01) in the entire study population. Whereas plasma LCAT activity correlated negatively with the level of HDL2b (r = -0.455, p < 0.05) and positively with the levels of HDL2a (r = 0.475, p < 0.05) and HDL3a (r = 0.485, p < 0.05), no significant relation was observed with the level of HDL3b.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relationship between lipoprotein lipase and high density lipoprotein cholesterol in mice: modulation by cholesteryl ester transfer protein and dietary status

Plasma lipoprotein lipase (LPL) activity correlates with high density lipoprotein (HDL) cholesterol levels in humans. However, in several mouse models created either through transgenesis or targeted inactivation of LPL, no significant changes in HDL cholesterol values have been evident. One possible explanation for this species difference could be the absence of plasma cholesteryl ester transfer protein (CETP) activity in mice. To explore this possibility and further investigate interactions between LPL and CETP modulating HDL cholesterol levels in vivo, we examined the relationship between LPL activity and HDL levels in mice expressing the simian CETP transgene, compared with littermates not carrying the CETP gene. On a chow diet, increasing LPL activity was associated with a trend towards increased HDL levels (51 +/- 29 vs. 31 +/- 4 mg/dL highest vs. lowest tertiles of LPL activity, P = 0.07) in mice expressing CETP, while no such effects were seen in the absence of CETP (65 +/- 12 vs. 61 +/- 15 mg/ dL). Furthermore, in the presence of CETP, a significant positive correlation between LPL activity and HDL cholesterol was evident (r = 0.15, P = 0.006), while in the absence of CETP no such correlation was detected (r = 0.15, P = 0.36), highlighting the interactions between LPL and CETP in vivo. When mice were challenged with a high fat, high carbohydrate diet, strong correlations between LPL activity and HDL cholesterol were seen in both the presence (r = 0.45, P = 0.03) and absence (r = 0.73, P < 0.001) of CETP. Therefore, under altered metabolic contexts, such as those induced by dietary challenge, the relation between LPL activity and HDL cholesterol may also become evident. Here we have shown that both genetic and environmental factors may modulate the association between LPL activity and HDL cholesterol, and provide explanations for the absence of any changes in HDL values in mice either transgenic or with targeted disruption of the LPL gene.     

High density lipoprotein particle size restriction in apolipoprotein A-I(Milano) transgenic mice

Human carriers of apolipoprotein A-I(Milano) (Arg173 --> Cys substitution in apolipoprotein A-I) are characterized by an HDL deficiency in which small, dense HDL accumulate in plasma. Because affected individuals are heterozygous for this mutation, the full impact of apolipoprotein A-I(Milano) (apoA-I(Milano)) on HDL-cholesterol metabolism is unknown. In this study, apoA-I(Milano) transgenic mice were used to evaluate the extent of apoA-I(Milano) dimerization and HDL particle size restriction in the absence of wild-type apoA-I. Murine apoA-I knockout mice were utilized to express apoA-I(Milano) and human apoA-II in the presence of wild-type, human apoA-I (apoA-IMilano/A-Iwt/A-II) and in its absence (apoA-IMilano/A-II). Plasma HDL-cholesterol concentrations were similar (30 mg/dl) in both lines of apoA-I(Milano) transgenic mice. In the apoA-IMilano/A-Iwt/A-II phenotype, 14% of the apoA-I(Milano) formed homodimers and 33% formed heterodimers with apoA-II. ApoA-I(Milano) homodimers increased by 71% in the apoA-IMilano/A-II transgenics and was associated with an abundance of small, 7.6-nm HDL3-sized particles compared to the 9.5, 8.3, and 7.6-nm-sized particles in apoA-IMilano/A-Iwt/A-II mice. The unesterified cholesterol/cholesteryl ester mole ratio of HDL was elevated by 45% in apoA-IMilano/A-Iwt/A-II mice and by 90% in apoA-IMilano/A-II transgenics compared to wild-type (human apoA-I/A-II). Both apoA-I(Milano) transgenics possessed normal levels of plasma LCAT activity, but endogenous cholesterol esterification rates were reduced by 50% compared to controls. Thus, HDL particle size restriction was not the result of impaired LCAT activation; rather, dimerization of apoA-I(Milano) limited the esterification of cholesterol on endogenous HDL. In the absence of wild-type apoA-I, the more extensive dimerization of apoA-I(Milano) severely limited cholesteryl ester accumulation on plasma HDL accounting for the abundance of small, 7.6-nm HDL3 particles in apoA-IMilano/A-II mice.     

Emboli observed with use of transesophageal echocardiography immediately after tourniquet release during total knee arthroplasty with cement

We analyzed the genetic defect in a 67-year-old Japanese male patient with apolipoprotein (apo) A-I and high density lipoprotein (HDL) deficiencies, corneal opacities, and coronary artery disease. The plasma concentrations of apoA-I and HDL cholesterol were 2.9 to 7.3 mg/dL and 0.08 to 0.19 mmol/L, respectively. The lecithin:cholesterol acyltransferase (LCAT) activity and cholesterol esterification rate were <40% of normal control values. LCAT mass was 550% of normal control. Sequence analysis of polymerase chain reaction-amplified DNA of the proband's apoA-I gene showed a homozygous T-to-A transition resulting in the substitution of Val 156 with Glu (apoA-I Oita). Direct sequencing of samples obtained from other family members showed that the brother was homozygous, whereas the son was a heterozygous carrier of apoA-I Oita. The heterozygote for apo A-I Oita showed nearly 60% of normal apoA-I and normal HDL cholesterol levels. In vivo turnover studies in rabbits demonstrated that the variant apoA-I was rapidly cleared from plasma compared with normal human apoA-I. Our data suggest that the Val156Glu substitution is associated with apoA-I and HDL deficiency, partial LCAT deficiency, and corneal opacities and that Val156 of apoA-I may play an important role in apoA-I function.     

Elevated high density lipoprotein concentrations in heart transplant recipients are related to impaired plasma cholesteryl ester transfer and hepatic lipase activity

Accelerated atherosclerosis is a major complication of heart transplantation, and is frequently associated with a dyslipoproteinemia characterized by a paradoxical increase in HDL-cholesterol concentration. To define this abnormality, the lipoprotein profiles of 25 heart transplant recipients (HTR) were analyzed and compared with those of 26 control subjects. HDL, as separated on the basis of density in 3 subfractions, were increased in concentration: HDL2: +51%, HDL3a: +29%, HDL3b: +32%. HDL2 and HDL3a displayed an enrichment in surface components, phospholipids, unesterified cholesterol and apo E, leading to an increased size compared with subfractions of similar density in the controls. The major steps of plasma HDL metabolism were investigated: cholesterol esterification (LCAT activity), cholesteryl ester transfer to apo B-containing lipoproteins (CETP) and the hepatic hydrolysis of HDL components (HL activity). We demonstrated a partial deficiency in CETP (-28%) and hepatic lipase (-36%) activities with normal LCAT activity. Correlations in total study population (HTR plus controls) evidenced negative associations between CETP activity and HDL3a concentrations and between HL activity and HDL2-cholesterol as a percent of total HDL-cholesterol. Therapeutic agents used in post transplantation treatment such as glucocorticoids and/or cyclosporine may be speculated thus to affect both CETP and HL activities and, by arresting the HDL cycle in a CE-saturated state, do decrease the efficiency of reverse cholesterol extraction at the site of the graft.     

Targeted disruption of the mouse lecithin:cholesterol acyltransferase (LCAT) gene. Generation of a new animal model for human LCAT deficiency

We have established a mouse model for human LCAT deficiency by performing targeted disruption of the LCAT gene in mouse embryonic stem cells. Homozygous LCAT-deficient mice were healthy at birth and fertile. Compared with age-matched wild-type littermates, the LCAT activity in heterozygous and homozygous knockout mice was reduced by 30 and 99%, respectively. LCAT deficiency resulted in significant reductions in the plasma concentrations of total cholesterol, HDL cholesterol, and apoA-I in both LCAT -/- mice (25, 7, and 12%; p < 0. 001 of normal) and LCAT +/- mice (65 and 59%; p < 0.001 and 81%; not significant, p = 0.17 of normal). In addition, plasma triglycerides were significantly higher (212% of normal; p < 0.01) in male homozygous knockout mice compared with wild-type animals but remained normal in female knockout LCAT mice. Analyses of plasma lipoproteins by fast protein liquid chromatography and two-dimensional gel electrophoresis demonstrated the presence of heterogenous prebeta-migrating HDL, as well as triglyceride-enriched very low density lipoprotein. After 3 weeks on a high-fat high-cholesterol diet, LCAT -/- mice had significantly lower plasma concentrations of total cholesterol, reflecting reduced levels of both proatherogenic apoB-containing lipoproteins as well as HDL, compared with controls. Thus, we demonstrate for the first time that the absence of LCAT attenuates the rise of apoB-containing lipoproteins in response to dietary cholesterol. No evidence of corneal opacities or renal insufficiency was detected in 4-month-old homozygous knockout mice. The availability of a homozygous animal model for human LCAT deficiency states will permit further evaluation of the role that LCAT plays in atherosclerosis as well as the feasibility of performing gene transfer in human LCAT deficiency states.     

Minimally oxidized LDL is a potent inhibitor of lecithin:cholesterol acyltransferase activity

The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. As a variety of highly reactive lipid peroxidation products can transfer from oxidized LDL to HDL, we evaluated the potential deleterious effects of LDL oxidation on HDL-cholesterol metabolism. To address this issue, we exposed the HDL-containing d > 1.063 g/ml fraction of human plasma to copperoxidized LDL and assessed lecithin:cholesterol acyltransferase (LCAT) activity and apolipoproteinA-I (apoA-I) structure. To determine whether LCAT was directly affected by oxidized LDL, independent of crosslinking of apoA-I, we used an exogenous, [14C]cholesterol-labeled proteoliposome substrate to measure plasma LCAT activity. We observed an inhibition of LCAT activity where copper-oxidized LDL possessing only 2.3 +/- 0.1 and 7.3 +/- 1.4 TBARS produced 24 +/- 3% and 47 +/- 10% reductions in [14C]cholesterol esterification by 1 h, respectively. Copper-oxidized LDL that had been passed through a GF-5 desalting column, while retaining only one-third of its original TBARS, possessed nearly all of its LCAT inhibitory capacity suggesting that the LCAT inhibitory factor(s) was a lipophilic oxidation product. Analysis of polarlipids isolated from copper-oxidized LDL indicated that phospholipid and sterol fractions effectively inhibited LCAT. Copper-oxidized LDL, with as little as 6.3 TBARS, also produced intermolecular crosslinking of apoA-I molecules. Taken together, these data suggest that products of LDL oxidation may adversely affect HDL-cholesterol metabolism by two separate mechanisms: 1) a direct inhibitory effect on LCAT activity and 2) through crosslinking of apoA-I. If occurring in vivo, minimally oxidized LDL may impair cholesteryl ester formation on HDL thereby limiting the ability of HDL to function efficiently in the putative antiatherogenic reverse cholesterol transport pathway.     

Role of HDL phospholipid in efflux of cell cholesterol to whole serum: studies with human apoA-I transgenic rats

Sera of transgenic rats expressing human apoA-I were tested for their ability to stimulate efflux of radiolabeled cholesterol from Fu5AH rat hepatoma cells. Expression of human apoA-I resulted in a dose-dependent increase in HDL, as measured by both HDL-cholesterol and HDL-phospholipid, and produced a decrease in rat apoA-I. In rats expressing high concentrations of human apoA-I (TgR[hAI]high, human apoA-I > 250 mg/dl), the increase in HDL-phospholipid was not proportional to the increase in human apoA-I, as illustrated by a HDL-PL/total apoA-I ratio of 0.84 +/- 0.19 compared to a ratio of 1.28 +/- 0.29 for control rats and of 1.28 +/- 0.39 for rats expressing low levels of human apoA-I (TgR[hAI]low, human apoA-I < 250 mg/dl). Compared to sera from control animals, efflux of cell cholesterol was increased by 26% in the sera from TgR[hAI]low, and by 76% in the TgR[hAI]high. An examination of the relationships between efflux and HDL-related parameters demonstrated a hyperbolic relationship between efflux and either HDL-cholesterol or HDL-apoA-I. In contrast, there was a strong linear association (r2 = 0.84) between cholesterol efflux and HDL-phospholipid, indicating that this parameter is the component of HDL that best reflects the serum's efflux efficiency. The importance of phospholipids in modulating cholesterol efflux was further explored by measuring the effect of supplementation of serum with dimyristoylphosphatidylcholine (DMPC) vesicles, apoA-I, or both DMPC vesicles and apoA-I. Whereas addition of human apoA-I had no effect on efflux, supplementation with DMPC vesicles produced a substantial increase in efflux that was further stimulated by the combination of DMPC vesicles and apoA-I. These results demonstrate that a major component of HDL that modulates cell cholesterol efflux is phospholipid.     

Correction of hypoalphalipoproteinemia in LDL receptor-deficient rabbits by lecithin:cholesterol acyltransferase

Familial hypercholesterolemia (FH), a disease caused by a variety of mutations in the low density lipoprotein receptor (LDLr) gene, leads not only to elevated LDL-cholesterol (C) concentrations but to reduced high density lipoprotein (HDL)-C and apolipoprotein (apo) A-I concentrations as well. The reductions in HDL-C and apoA-I are the consequence of the combined metabolic defects of increased apoA-I catabolism and decreased apoA-I synthesis. The present studies were designed to test the hypothesis that overexpression of human lecithin:cholesterol acyltransferase (hLCAT), a pivotal enzyme involved in HDL metabolism, in LDLr defective rabbits would increase HDL-C and apoA-I concentrations. Two groups of hLCAT transgenic rabbits were established: 1) hLCAT+/LDLr heterozygotes (LDLr+/-) and 2) hLCAT+/LDLr homozygotes (LDLr-/-). Data for hLCAT+ rabbits were compared to those of nontransgenic (hLCAT-) rabbits of the same LDLr status. In LDLr+/- rabbits, HDL-C and apoA-I concentrations (mg/dl), respectively, were significantly greater in hLCAT+ (62 +/- 8, 59 +/- 4) relative to hLCAT- rabbits (21 +/- 1, 26 +/- 2). This was, likewise, the case when hLCAT+/ LDLr-/- (27 +/- 2, 19 +/- 6) and hLCAT-/LDLr-/- (5 +/- 1, 6 +/- 2) rabbits were compared. Kinetic experiments demonstrated that the fractional catabolic rate (FCR, d(-1)) of apoA-I was substantially delayed in hLCAT+ (0.376 +/- 0.025) versus hLCAT- (0.588) LDLr+/- rabbits, as well as in hLCAT+ (0.666 +/- 0.033) versus hLCAT- (1.194 +/- 0.138) LDLr-/- rabbits. ApoA-I production rate (PR, mg x kg x d(-1)) was greater in both hLCAT+/LDLr+/- (10 +/- 2 vs. 6) and hLCAT+/LDLr-/- (9 +/- 1 vs. 4 +/- 1) rabbits. Significant correlations (P < 0.02) were observed between plasma LCAT activity and HDL-C (r = 0.857), apoA-I FCR (r = -0.774), and apoA-I PR (r = 0.771), while HDL-C correlated with both apoA-I FCR (-0.812) and PR (0.751). In summary, these data indicate that hLCAT overexpression in LDLr defective rabbits increases HDL-C and apoA-I concentrations by both decreasing apoA-I catabolism and increasing apoA-I synthesis, thus correcting the metabolic defects responsible for the hypoalphalipoproteinemia observed in LDLr deficiency.     

Short-term effects of a high-sucrose diet on plasma lipid, lipoprotein cholesterol, tissue lipoprotein lipase and hepatic triglyceride lipase in rats

Short-term (2 weeks) effects of a high-sucrose diet on plasma lipids, lipoproteins, tissue lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activities were investigated in rats. Three days of sucrose feeding significantly increased plasma TG (42 +/- 3 mg/dl vs. 56 +/- 2 mg/dl, p = 0.032), while TC increased significantly after 10 days of the diet (50 +/- 2 mg/dl vs. 62 +/- 2 mg/dl, p = 0.0001). HDL-C increased significantly after 3 days of sucrose feeding (36.2 +/- 0.9 mg/dl vs. 42.4 +/- 2.7 mg/dl, p = 0.011). Although LDL-C tended to decrease on days 3, 7 and 10, these changes were not significant. The plasma glucose level did not change during the study. Increased LPL activity in adipose tissue and decreased enzyme activities in skeletal and heart muscles were observed. Adipose tissue LPL returned to the baseline value after 14 days of the diet treatment, while LPL in skeletal and heart muscles remained at the decreased level. HTGL and HTGL/total liver lipase activities were significantly increased after 14 days of the diet. The different responses of lipase activities in various tissues may help to regulate serum lipid and lipoprotein levels in sucrose-fed rats.     

Increased coronary heart disease in Japanese-American men with mutation in the cholesteryl ester transfer protein gene despite increased HDL levels

Plasma high density lipoprotein (HDL) levels are strongly genetically determined and show a general inverse relationship with coronary heart disease (CHD). The cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters from HDL to other lipoproteins and is a key participant in the reverse transport of cholesterol from the periphery to the liver. A high prevalence of two different CETP gene mutations (D442G, 5.1%; intron 14G:A, 0.5%), was found in 3,469 men of Japanese ancestry in the Honolulu Heart Program and mutations were associated with decreased CETP (-35%) and increased HDL chol levels (+10% for D442G). However, the overall prevalence of definite CHD was 21% in men with mutations and 16% in men without mutations. The relative risk (RR) of CHD was 1.43 in men with mutations (P < .05); after adjustment for CHD risk factors, the RR was 1.55 (P = .02); after additional adjustment for HDL levels, the RR was 1.68 (P = .008). Similar RR values were obtained for the D442G mutation alone. Increased CHD in men with mutations was primarily observed for HDL chol 41-60 mg/dl; for HDL chol > 60 mg/dl men with and without mutations had low CHD prevalence. Thus, genetic CETP deficiency appears to be an independent risk factor for CHD, primarily due to increased CHD prevalence in men with the D442G mutation and HDL cholesterol between 41 and 60 mg/dl. The findings suggest that both HDL concentration and the dynamics of cholesterol transport through HDL (i.e., reverse cholesterol transport) determine the anti-atherogenicity of the HDL fraction.     

Hepatic lipase deficiency increases plasma cholesterol but reduces susceptibility to atherosclerosis in apolipoprotein E-deficient mice

The effect of hepatic lipase (HL) deficiency on the susceptibility to atherosclerosis was tested using mice with combined deficiencies in HL and apoE. Mice lacking both HL and apoE (hhee) have a plasma total cholesterol of 917 +/- 252 mg/dl (n = 24), which is 184% that of mice lacking only apoE (HHee; 497 +/- 161 mg/dl, n = 20, p < 0. 001). The increase in cholesterol was mainly in beta-migrating very low density lipoproteins, although high density lipoprotein cholesterol (HDLc) was also increased (53 +/- 37 versus 20 +/- 13 mg/dl, p < 0.01). Despite the increase in plasma cholesterol, we found that HL deficiency significantly decreased aortic plaque sizes in female mice fed normal chow (31 x 10(3) +/- 22 x 10(3) microm2 in hhee versus 115 x 10(3) +/- 69 x 10(3) microm2 in HHee, p < 0.001). Reduction of plaque sizes was also observed in female heterozygous apoE-deficient mice fed an atherogenic diet (2 x 10(3) +/- 2.5 x 10(3) microm2 in hhEe versus 56 x 10(3) +/- 49 x 10(3) microm2 in HHEe, p < 0.01). Changes in aortic lesion size were not apparent in the small number of male mice studied. In HHee females, both HDLc and the capacity of high density lipoprotein (HDL) particles to promote cholesterol efflux from cultured cells were 26% of the wild type. The absence of HL in hhee females partially restored HDLc levels to 57% and cholesterol efflux to 55% of the wild type. Circulating pre-beta1-migrating HDL were present in all mutants, suggesting that there are alternative pathways in the formation of these pre-beta-HDL not involving apoE, HL, or cholesteryl ester transfer protein. The improved capacity to promote cholesterol efflux, together with increased HDL, may explain why these animals can overcome the increase in atherogenic lipoproteins.     

Apolipoprotein A-I metabolism in cholesteryl ester transfer protein transgenic mice. Insights into the mechanisms responsible for low plasma high density lipoprotein levels

Expression of simian cholesteryl ester transfer protein (CETP) in C57BL/6 mice causes the animals' high density lipoprotein (HDL) levels to decrease. The purpose of these studies was to determine how CETP expression caused that reduction. Chemical analysis showed that the HDL of the CETP transgenic mice had about twice as much triglyceride and only about 60% as much cholesteryl ester as the HDL from the C57BL/6 mice. Both strains of mouse had high levels of a circulating lipase. When plasma from the mice was incubated at 37 degrees C for 5 h, the triglycerides in the HDL were hydrolyzed, and apoA-I was shed from the particle. However, apoA-I was shed from the CETP HDL more rapidly than it was shed from the C57BL/6 HDL. Because "free" apoA-I is rapidly cleared by the kidney, increased production of free apoA-I would be expected to shorten the average life span of apoA-I in the mouse. Kinetic analyses indicated that the life span of apoA-I was significantly reduced in the CETP transgenic mice. It was concluded that CETP expression enriched the core of the HDL with triglyceride, which rendered it vulnerable to lipolysis, causing apoA-I to be shed from the particle. That shortened the life span of apoA-I in the CETP mice, which led to lower plasma levels of the protein.