Lipoprotein distribution of apolipoprotein C-III and its relationship to the presence in plasma of triglyceride-rich remnant lipoproteins

The distribution of apolipoprotein C-III (apoC-III) between high-density lipoprotein (HDL) and apoB-containing lipoproteins has been used in lipid-lowering angiographic trials to establish a link between impaired triglyceride (TG)-rich lipoprotein (TRL) metabolism and the progression of coronary artery disease. To investigate the extent to which plasma lipoprotein apoC-III levels reflect the presence in plasma of potentially atherogenic remnant lipoproteins, we studied 4 groups of subjects: (1) normolipidemic (NL, n = 10), (2) hypercholesterolemic (HC, type IIa, low-density lipoprotein cholesterol [LDL-C] > 3.4 mmol/L, n = 10), (3) hypertriglyceridemic (HTG, type IV, TG > 2.3 mmol/L, n = 10), and (4) combined hyperlipidemic (CHL, type IIb, TG > 2.3 mmol/L, LDL-C > 3.4 mmol/L, n = 10). The apoC-III level was measured in plasma lipoproteins separated either by density (ultracentrifugation) or by size (fast protein liquid chromatography [FPLC]), and was compared with 4 parameters reflecting remnant lipoprotein levels (ie, very-low-density lipoprotein cholesterol [VLDL-C], intermediate-density lipoprotein cholesterol [IDL-C], remnant-like particle cholesterol [RLP-C], and intermediate-sized lipoprotein [ISL] apoE). Our results demonstrate that (1) increased amounts of apoC-III associated with plasma VLDL, TRL, or apoB-containing lipoproteins (LpB), as well as increased levels of TRL remnant lipoproteins, are a characteristic of HTG patients rather than patients with increased LDL, and (2) plasma levels of apoC-III in VLDL, TRL, or LpB, as well as the HDL apoC-III to LpB apoC-III ratios, are strongly correlated with circulating levels of TRL, although these apoC-II parameters more closely reflect the balance between TRL TG production and lipolysis than the extent of plasma TRL remnant accumulation.     

Influence of atorvastatin and simvastatin on apolipoprotein B metabolism in moderate combined hyperlipidemic subjects with low VLDL and LDL fractional clearance rates

Subjects with moderate combined hyperlipidemia (n=11) were assessed in an investigation of the effects of atorvastatin and simvastatin (both 40 mg per day) on apolipoprotein B (apoB) metabolism. The objective of the study was to examine the mechanism by which statins lower plasma triglyceride levels. Patients were studied on three occasions, in the basal state, after 8 weeks on atorvastatin or simvastatin and then again on the alternate treatment. Atorvastatin produced significantly greater reductions than simvastatin in low density lipoprotein (LDL) cholesterol (49.7 vs. 44.1% decrease on simvastatin) and plasma triglyceride (46.4 vs. 39.4% decrease on simvastatin). ApoB metabolism was followed using a tracer of deuterated leucine. Both drugs stimulated direct catabolism of large very low density lipoprotein (VLDL(1)) apoB (4.52+/-3.06 pools per day on atorvastatin; 5.48+/-4.76 pools per day on simvastatin versus 2.26+/-1.65 pools per day at baseline (both P<0.05)) and this was the basis of the 50% reduction in plasma VLDL(1) concentration; apoB production in this fraction was not significantly altered. On atorvastatin and simvastatin the fractional transfer rates (FTR) of VLDL(1) to VLDL(2) and of VLDL(2) to intermediate density lipoprotein (IDL) were increased significantly, in the latter instance nearly twofold. IDL apoB direct catabolism rose from 0.54+/-0.30 pools per day at baseline to 1.17+/-0.87 pools per day on atorvastatin and to 0.95+/-0.43 pools per day on simvastatin (both P<0.05). Similarly the fractional transfer rate for IDL to LDL conversion was enhanced 58-84% by statin treatment (P<0.01) LDL apoB fractional catabolic rate (FCR) which was low at baseline in these subjects (0.22+/-0.04 pools per day) increased to 0.44+/-0.11 pools per day on atorvastatin and 0.38+/-0.11 pools per day on simvastatin (both P<0.01). ApoB-containing lipoproteins were more triglyceride-rich and contained less free cholesterol and cholesteryl ester on statin therapy. Further, patients on both treatments showed marked decreases in all LDL subfractions. In particular the concentration of small dense LDL (LDL-III) fell 64% on atorvastatin and 45% on simvastatin. We conclude that in patients with moderate combined hyperlipidemia who initially have a low FCR for VLDL and LDL apoB, the principal action of atorvastatin and simvastatin is to stimulate receptor-mediated catabolism across the spectrum of apoB-containing lipoproteins. This leads to a substantial, and approximately equivalent, percentage reduction in plasma triglyceride and LDL cholesterol.     

Overexpression of apolipoprotein E3 in transgenic rabbits causes combined hyperlipidemia by stimulating hepatic VLDL production and impairing VLDL lipolysis

The differential effects of overexpression of human apolipoprotein (apo) E3 on plasma cholesterol and triglyceride metabolism were investigated in transgenic rabbits expressing low (<10 mg/dL), medium (10 to 20 mg/dL), or high (>20 mg/dL) levels of apoE3. Cholesterol levels increased progressively with increasing levels of apoE3, whereas triglyceride levels were not significantly affected at apoE3 levels up to 20 mg/dL but were markedly increased at levels of apoE3 >20 mg/dL. The medium expressers had marked hypercholesterolemia (up to 3- to 4-fold over nontransgenics), characterized by an increase in low density lipoprotein (LDL) cholesterol, while the low expressers had only slightly increased plasma cholesterol levels. The medium expressers displayed an 18-fold increase in LDL but also had a 2-fold increase in hepatic very low density lipoprotein (VLDL) triglyceride production, an 8-fold increase in VLDL apoB, and a moderate decrease in the ability of the VLDL to be lipolyzed. However, plasma clearance of VLDL was increased, likely because of the increased apoE3 content. The increase in LDL appears to be due to an enhanced competition of VLDL for LDL receptor binding and uptake, resulting in the accumulation of LDL. The combined hyperlipidemia of the apoE3 high expressers (>20 mg/dL) was characterized by a 19-fold increase in LDL cholesterol but also a 4-fold increase in hepatic VLDL triglyceride production associated with a marked elevation of plasma VLDL triglycerides, cholesterol, and apoB100 (4-, 9-, and 25-fold over nontransgenics, respectively). The VLDL from the high expressers was much more enriched in apoE3 and markedly depleted in apoC-II, which contributed to a >60% inhibition of VLDL lipolysis. The combined effects of stimulated VLDL production and impaired VLDL lipolysis accounted for the increases in plasma triglyceride and VLDL concentrations in the apoE3 high expressers. The hyperlipidemic apoE3 rabbits have phenotypes similar to those of familial combined hyperlipidemia, in which VLDL overproduction is a major biochemical feature. Overall, elevated expression of apoE3 appears to determine plasma lipid levels by stimulating hepatic VLDL production, enhancing VLDL clearance, and inhibiting VLDL lipolysis. Thus, the differential expression of apoE may, within a rather narrow range of concentrations, play a critical role in modulating plasma cholesterol and triglyceride levels and may represent an important determinant of specific types of hyperlipoproteinemia.     

Overexpression of apolipoprotein E in transgenic mice: marked reduction in plasma lipoproteins except high density lipoprotein and resistance against diet-induced hypercholesterolemia.

Apolipoprotein E (apoE) has a high affinity to cell-surface low density lipoprotein (LDL) receptor. To determine the role of apoE in plasma lipoprotein metabolism, transgenic mouse lines with integrated rat apoE gene under the control of the metallothionein promoter were established. We found that a high expressor line produced rat apoE mainly in the liver, and the gene product was almost entirely associated with plasma lipoproteins. The plasma level of rat apoE in homozygotes for the transgene was 17.4 mg/dl after zinc induction (vs. 4.56 mg/dl of mouse apoE in controls). In this group, plasma cholesterol and triglyceride levels were 43% and 68% reduced as compared with controls, respectively. Heterozygotes showed decreases in both lipids to a lesser extent. Gel filtration chromatography showed that lipid reduction was mainly due to decreased very low density lipoproteins (VLDL) and LDL. Especially in zinc-treated homozygotes, VLDL had almost disappeared, and a remarkable decrease in LDL and a slight decrease in high density lipoprotein were also observed. Consistently, the plasma level of apoB, a structural protein of VLDL and LDL, was 78% lower than that of controls, indicating a marked reduction in lipoproteins containing apoB. Furthermore, the transgenic mice, in contrast to controls, did not develop hypercholesterolemia when fed a high cholesterol diet. These results demonstrated that overexpression of apoE reduces plasma cholesterol and triglyceride levels and prevents diet-induced hypercholesterolemia. From dramatic and dose-related decreases in plasma lipoproteins in transgenic mice, we conclude that apoE plays a key role in plasma lipoprotein metabolism.     

Rate of production of plasma and very-low-density lipoprotein (VLDL) apolipoprotein C-III is strongly related to the concentration and level of production of VLDL triglyceride in male subjects with different body weights and levels of insulin sensitivity

Overweight individuals with reduced insulin sensitivity often have mild to moderate hypertriglyceridemia. To investigate the role of apolipoprotein (apo)C-III metabolism in the etiology of hypertriglyceridemia in these individuals, we investigated 10 male subjects with different body weights (body mass index, 24-34 kg/m(2)) and insulin sensitivity (homeostasis model assessment, 4.7-35.0). Total plasma and very-low-density lipoprotein (VLDL) apoC-III kinetics, as well as VLDL triglyceride (TG) and VLDL apoB kinetics, were measured with iv injected stable isotopes. The apoC-III, TG, and apoB levels in VLDL ranged from 2.9-18.2 mg/dl, 0.49-2.89 mmol/liter, and 6.7-29.3 mg/dl, respectively. Mean production rates (PRs) were: VLDL apoC-III, 20.2 +/- 4.1 micromol/d (range, 8.0-44.8); VLDL TG, 26.9 +/- 4.6 mmol/d (range, 10.2-51.1); and VLDL apoB, 4.4 +/- 0.8 micromol/d (range, 1.5-9.1). VLDL apoC-III PRs were significantly correlated with body mass index, homeostasis model assessment, and plasma TG (r = 0.66, P < 0.05; r = 0.80, P < 0.01; r = 0.95, P < 0.001, respectively). Similar correlations were found for plasma apoC-III PRs (r = 0.70, P < 0.05; r = 0.67, P < 0.05; r = 0.80, P < 0.01, respectively). Fractional catabolic rates (FCRs) were not significantly related to metabolic variables. VLDL TG levels were strongly related to VLDL apoC-III levels (r = 0.99, P < 0.001) and VLDL apoC-III PRs (r = 0.94, P < 0.001). VLDL apoC-III levels were more strongly correlated with VLDL TG PRs (r = 0.81, P < 0.01) than with VLDL TG FCRs or VLDL apoB FCRs (r = -0.53, P = 0.12; r = -0.37, P = 0.29). These results suggest that increased hepatic production of VLDL apoC-III is characteristic of subjects with higher body weights and lower levels of insulin sensitivity and is strongly related to the plasma concentration and level of production of VLDL TG.     

Lipid transfer inhibitor protein defines the participation of lipoproteins in lipid transfer reactions: CETP has no preference for cholesteryl esters in HDL versus LDL

Cholesteryl ester transfer protein (CETP) catalyzes the net transfer of cholesteryl ester (CE) between lipoproteins in exchange for triglyceride (heteroexchange). It is generally held that CETP primarily associates with HDL and preferentially transfers lipids from this lipoprotein fraction. This is illustrated in normal plasma where HDL is the primary donor of the CE transferred to VLDL by CETP. However, in plasma deficient in lipid transfer inhibitor protein (LTIP) activity, HDL and LDL are equivalent donors of CE to VLDL (Arterioscler Thromb Vasc Biol. 1997;17:1716-1724). Thus, we have hypothesized that the preferential transfer of CE from HDL in normal plasma is a consequence of LTIP activity and not caused by a preferential CETP-HDL interaction. We have tested this hypothesis in lipid mass transfer assays with partially purified CETP and LTIP, and isolated lipoproteins. With a physiological mixture of lipoproteins, the preference ratio (PR, ratio of CE mass transferred from a lipoprotein to VLDL versus its CE content) for HDL and LDL in the presence of CETP alone was approximately 1 (ie, no preference). Fourfold variations in the LDL/HDL ratio or in the levels of HDL in the assay did not result in significant preferential transfer from any lipoprotein. On addition of LTIP, the PR for HDL was increased up to 2-fold and that for LDL decreased in a concentration-dependent manner. Under all conditions where LDL and HDL levels were varied, LTIP consistently resulted in a PR >1 for CE transfer from HDL. Short-term experiments with radiolabeled lipoproteins and either partially purified or homogenous CETP confirmed these observations and further demonstrated that CETP has a strong predilection to mediate homoexchange (bidirectional transfer of the same lipid) rather than heteroexchange (CE for TG); LTIP had no effect on the selection of CE or TG by CETP or its mechanism of action. We conclude, in contrast to current opinion, that CETP has no preference for CE in HDL versus LDL, suggesting that the previously reported stable binding of CETP to HDL does not result in selective transfer from this lipoprotein. These data suggest that LTIP is responsible for the preferential transfer of CE from HDL that occurs in plasma. CETP and LTIP cooperatively determine the extent of CETP-mediated remodeling of individual lipoprotein fractions.     

Action of atorvastatin in combined hyperlipidemia : preferential reduction of cholesteryl ester transfer from HDL to VLDL1 particles

Combined hyperlipidemia (CHL) is characterized by a concomitant elevation of plasma levels of triglyceride-rich, very low density lipoproteins (VLDLs) and cholesterol-rich, low density lipoproteins (LDLs). The predominance of small, dense LDLs contributes significantly to the premature development of coronary artery disease in patients with this atherogenic dyslipoproteinemia. In the present study, we evaluated the impact of atorvastatin, a newly developed inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase, on the cholesteryl ester transfer protein (CETP)-mediated remodeling of apolipoprotein (apo) B-containing lipoprotein subspecies, and more specifically, the particle subpopulations of VLDL and LDL in CHL. In parallel, we evaluated the atorvastatin-induced modulation of the quantitative and qualitative features of atherogenic apo B-containing and cardioprotective apo AI-containing lipoprotein subspecies. Atorvastatin therapy (10 mg/d for a 6-week period) in patients with a lipid phenotype typical of CHL (n=18) induced reductions of 31% (P<0.0001) and 36% (P<0.0001) in plasma total cholesterol and LDL cholesterol, respectively. In addition, atorvastatin significantly reduced VLDL cholesterol, triglycerides, and apo B levels by 43% (P<0.0001), 27% (P=0.0006), and 31% (P<0.0001), respectively. The plasma concentrations of triglyceride-rich lipoproteins (VLDL1, Sf 60 to 400; VLDL2, Sf 20 to 60; and intermediate density lipoproteins, Sf 12 to 20) and of LDL, as determined by chemical analysis, were markedly diminished after drug therapy (-30% and -28%, respectively; P<0.0007). Atorvastatin significantly reduced circulating levels of all major LDL subspecies, ie, light (-28%, P<0.0008), intermediate (-27%, P<0.0008), and dense (-32%, P<0.0008) LDL; moreover, in terms of absolute lipoprotein mass, the reduction in dense LDL levels (mean -62 mg/dL) was preponderant. In addition, the reduction in plasma dense LDL concentration after therapy was significantly correlated with a reduction in plasma VLDL1 levels (r=0.429, P=0.0218). Atorvastatin induced a significant reduction (-7%, P=0.0039) in total CETP-dependent CET activity, which accurately reflects a reduction in plasma CETP mass concentration. Total CETP-mediated CET from high density lipoproteins to apo B-containing lipoproteins was significantly reduced (-26%, P<0.0001) with drug therapy. Furthermore, CETP activity was significantly correlated with the atorvastatin-induced reduction in plasma VLDL1 levels (r=0.456, P=0. 0138). Indeed, atorvastatin significantly and preferentially decreased CET from HDL to the VLDL1 subfraction (-37%, P=0.0064), thereby reducing both the levels (-37%, P=0.0001) and the CE content (-20%, P<0.005) of VLDL1. We interpret our data to indicate that 2 independent but complementary mechanisms may be operative in the atorvastatin-induced reduction of atherogenic LDL levels in CHL: first, a significant degree of normalization of both the circulating levels and the quality of their key precursors, ie, VLDL1, and second, enhanced catabolism of the major LDL particle subclasses (ie, light, intermediate, and dense LDL) due to upregulation of hepatic LDL receptors.     

Effects of ApoE genotype on ApoB-48 and ApoB-100 kinetics with stable isotopes in humans

Subjects with the apolipoprotein (apo) E4 allele have been shown to have higher low density lipoprotein (LDL) cholesterol and apoB levels than do subjects with the other alleles. To elucidate the metabolic mechanisms responsible for this finding, we examined the kinetics of apoB-48 within triglyceride-rich lipoproteins (TRLs) and of apoB-100 within very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and LDL by using a primed constant infusion of [5,5,5-(2)H(3)]leucine in the fed state (hourly feeding) during consumption of an average American diet in 18 normolipidemic subjects, 12 of whom had the apoE3/E3 genotype and 6, the apoE3/E4 genotype. Lipoproteins were isolated by ultracentrifugation and apolipoproteins, by sodium dodecyl sulfate gels; isotope enrichment was assessed by gas chromatography-mass spectrometry. Kinetic parameters were calculated by multicompartmental modeling of the data with SAAM II software. Compared with the apoE3/E3 subjects, the apoE3/E4 subjects had significantly higher levels of total apoB, 100. 1+/-17.8 versus 135.4+/-34.0 mg/dL (P=0.009), and significantly higher levels of LDL apoB-100, 88.1+/-19.2 versus 127.5+/-32.7 mg/dL (P=0.005), respectively. The pool size of TRL apoB-48 was 17.4% lower for apoE3/E4 subjects compared with apoE3/E3 subjects due to a 33.3% lower production rate (P=0.28). There was no significant difference in the TRL apoB-48 fractional catabolic rate (5.1+/-2.2 versus 5.0+/-2.1 pools per day). The pool size for VLDL apoB-100 was 36% lower for apoE3/E4 subjects compared with apoE3/E3 subjects due entirely to a 30% lower production rate (P=0.04). The LDL apoB-100 pool size was 57.8% higher (P=0.003) for apoE3/E4 subjects compared with apoE3/E3 subjects due to a 35.5% lower fractional catabolic rate of LDL apoB-100 (P=0.003), with no significant difference in production rate. In addition, 77% of VLDL apoB-100 was converted to LDL apoB-100 in apoE3/E4 subjects compared with 58% in apoE3/E3 subjects (P=0.05). In conclusion, the presence of 1 E4 allele was associated with higher LDL apoB-100 levels owing to lower fractional catabolism of LDL apoB-100 and a 33% increase in the conversion of VLDL apoB-100 to LDL apoB-100.     

The magnitude of decrease in hepatic very low density lipoprotein apolipoprotein B secretion is determined by the extent of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition in miniature pigs.

It has been postulated that the rate of hepatic very low density lipoprotein (VLDL) apolipoprotein (apo) B secretion is dependent upon the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. To test this hypothesis in vivo, apoB kinetic studies were carried out in miniature pigs before and after 21 days treatment with high-dose (10 mg/kg/day), atorvastatin (A) or simvastatin (S) (n = 5). Pigs were fed a diet containing fat (34% of calories) and cholesterol (400 mg/day; 0.1%). Statin treatment decreased plasma total cholesterol [31 (A) vs. 20% (S)] and low density lipoprotein (LDL) cholesterol concentrations [42 (A) vs. 24% (S)]. Significant reductions in plasma total triglyceride (46%) and VLDL triglyceride (50%) concentrations were only observed with (A). Autologous [131I]VLDL, [125I]LDL, and [3H]leucine were injected simultaneously, and apoB kinetic parameters were determined by triple-isotope multicompartmental analysis using SAAM II. Statin treatment decreased the VLDL apoB pool size [49 (A) vs. 24% (S)] and the hepatic VLDL apoB secretion rate [50 (A) vs. 33% (S)], with no change in the fractional catabolic rate (FCR). LDL apoB pool size decreased [39 (A) vs. 26% (S)], due to reductions in both the total LDL apoB production rate [30 (A) vs. 21% (S)] and LDL direct synthesis [32 (A) vs. 23% (S)]. A significant increase in the LDL apoB FCR (15%) was only seen with (A). Neither plasma VLDL nor LDL lipoprotein compositions were significantly altered. Hepatic HMG-CoA reductase was inhibited to a greater extent with (A), when compared with (S), as evidenced by 1) a greater induction in hepatic mRNA abundances for HMG-CoA reductase (105%) and the LDL receptor (40%) (both P < 0.05); and 2) a greater decrease in hepatic free (9%) and esterified cholesterol (25%) (both P < 0.05). We conclude that both (A) and (S) decrease hepatic VLDL apoB secretion, in vivo, but that the magnitude is determined by the extent of HMG-CoA reductase inhibition.     

Decreased post-prandial triglyceride response and diminished remnant lipoprotein formation in cholesteryl ester transfer protein (CETP) deficiency

Plasma cholesteryl ester transfer protein (CETP) mediates CE/TG exchange among various lipoproteins. CETP deficiency results in low LDL and high HDL phenotype including apoE-rich large HDL. Large HDL could provide apoE to chylomicron/VLDL during lipolysis in post-prandial state, accelerating remnant lipoprotein uptake in the liver. To determine the effects of low CETP levels on post-prandial lipoprotein metabolism, lipid levels of plasma remnant-like lipoprotein particles (RLP) fraction were determined in one homozygous and three heterozygous CETP deficiency and controls with apoE3/3 phenotype. After oral fat-load, the area under curve (AUC) of TG levels were remarkably decreased in CETP deficiency as compared to controls (423+/-187 [S.D.] mg/dl x h in three heterozygous CETP deficiency and 926+/-268 [S.D.] in 10 controls, P=0.012). Similarly, the homozygote had a low AUC of TG levels (416 mg/dl x h). Plasma RLP-cholesterol levels were decreased in heterozygotes, but not significantly as compared to controls (P=0.14). HPLC analysis showed that increased RLP-cholesterol level was not due to conventional VLDL-LDL size RLP, but to those in large HDL size in the homozygote. In heterozygotes, bimodal distribution of RLP-cholesterol level was found in lipoprotein sizes of conventional VLDL-LDL and large HDL. Subjects with CETP deficiency appeared to have low levels of TG response and diminished remnant lipoprotein formation after fat-load.     

Effects of unopposed conjugated equine estrogen on lipoprotein composition and apolipoprotein-E distribution.

Administration of conjugated equine estrogen to 31 postmenopausal women for 3 months produced 14.6% and 9.4% decreases in low density lipoprotein cholesterol (LDL-C) and apolipoprotein-B (apoB), and 11.5%, 12.7%, and 9.6% increases in high density lipoprotein cholesterol (HDL-C), apoA-I and apoA-II, respectively. Phospholipids of HDL2 and HDL3 were increased 57.9% and 19.3%, respectively, while relatively small increases in cholesterol of the two subfractions were not significant. Compositions of LDL and HDL and its subfractions were altered substantially with estrogen treatment. The proportion of LDL triglyceride to LDL-C was increased. The phospholipid content in both the HDL2 and HDL3 subfractions (compared to cholesterol) was increased significantly (34.8% and 10.7%, respectively), while the triglyceride content was increased only in the HDL2 subfraction (43.6%). Estrogen use also caused a 9.1% reduction in total apoE levels and a redistribution of apoE to the very low density lipoprotein (VLDL) from the LDL plus HDL fraction, resulting in a significant 19.5% decrease in apoE in the LDL plus HDL fraction. Changes in apoE in the VLDL fraction were associated positively with changes in the cholesterol levels of the VLDL fraction and inversely with changes in LDL-C and apoB levels, while changes in apoE in the LDL plus HDL fraction were associated positively with changes in the levels of HDL-C. Thus, estrogen causes alterations in lipoproteins that could potentially affect their metabolism and/or function.     

Dose-dependent effect of rosuvastatin on apolipoprotein B-100 kinetics in the metabolic syndrome

In a randomized, double-blind, crossover trial of 5-week treatment period with placebo or rosuvastatin (10 or 40 mg/day) with 2-week placebo wash-outs between treatments, the dose-dependent effect of rosuvastatin on apolipoprotein (apo) B-100 kinetics in metabolic syndrome subjects were studied. Compared with placebo, there was a significant dose-dependent decrease with rosuvastatin in plasma cholesterol, triglycerides, LDL cholesterol, apoB and apoC-III concentrations and in the apoB/apoA-I ratio, lathosterol:cholesterol ratio, HDL cholesterol concentration and campesterol:cholesterol ratio also increased significantly. Rosuvastatin significantly increased the fractional catabolic rates (FCR) of very-low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and LDL-apoB and decreased the corresponding pool sizes, with evidence of a dose-related effect. LDL apoB production rate (PR) fell significantly with rosuvastatin 40 mg/day with no change in VLDL and IDL-apoB PR. Changes in triglycerides were significantly correlated with changes in VLDL apoB FCR and apoC-III concentration, and changes in lathosterol:cholesterol ratio were correlated with changes in LDL apoB FCR, the associations being more significant with the higher dose of rosuvastatin. In the metabolic syndrome, rosuvastatin decreases the plasma concentration of apoB-containing lipoproteins by a dose-dependent mechanism that increases their rates of catabolism. Higher dose rosuvastatin may also decrease LDL apoB production. The findings provide a dose-related mechanism for the benefits of rosuvastatin on cardiovascular disease in the metabolic syndrome.     

Apolipoprotein B-100 kinetics in visceral obesity: associations with plasma apolipoprotein C-III concentration

Obesity is strongly associated with dyslipidemia, which may account for the associated increased risk of atherosclerosis and coronary disease. We aimed to test the hypothesis that kinetics of hepatic apolipoprotein B-100 (apoB) metabolism are disturbed in men with visceral obesity and to examine whether these kinetic defects are associated with elevated plasma concentration of apolipoprotein C-III (apoC-III). Very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) apoB kinetics were measured in 48 viscerally obese men and 10 age-matched normolipidemic lean men using an intravenous bolus injection of d(3)-leucine. ApoB isotopic enrichment was measured using gas chromatography-mass spectrometry (GCMS). Kinetic parameters were derived using a multicompartmental model (Simulation, Analysis, and Modeling Software II [SAAM-II]). Compared with controls, obese subjects had significantly elevated plasma concentrations of plasma triglycerides, cholesterol, LDL-cholesterol, VLDL-apoB, IDL-apoB, LDL-apoB, apoC-III, insulin, and lathosterol (P <.01). VLDL-apoB secretion rate was significantly higher (P =.034) in obese than control subjects; the fractional catabolic rates (FCRs) of IDL-apoB and LDL-apoB (P <.01) and percent conversion of VLDL-apoB to LDL-apoB (P <.02) were also significantly lower in obese subjects. However, the decreased VLDL-apoB FCR was not significantly different from the lean group. In the obese group, plasma concentration of apoC-III was significantly and positively associated with VLDL-apoB secretion rate and inversely with VLDL-apoB FCR and percent conversion of VLDL to LDL. In multiple regression analysis, plasma apoC-III concentration was independently and significantly correlated with the secretion rate of VLDL-apoB (regression coefficient [SE] 0.511 [0.03], P =.001) and with the percent conversion of VLDL-apoB to LDL-apoB (-0.408 [0.01], P =.004). Our findings suggest that plasma lipid and lipoprotein abnormalities in visceral obesity may be due to a combination of overproduction of VLDL-apoB particles and decreased catabolism of apoB containing particles. Elevated plasma apoC-III concentration is also a feature of dyslipidemia in obesity that contributes to the kinetic defects in apoB metabolism.     

Overexpression of human hepatic lipase and ApoE in transgenic rabbits attenuates response to dietary cholesterol and alters lipoprotein subclass distributions

The effect of the expression of human hepatic lipase (HL) or human apoE on plasma lipoproteins in transgenic rabbits in response to dietary cholesterol was compared with the response of nontransgenic control rabbits. Supplementation of a chow diet with 0.3% cholesterol and 3.0% soybean oil for 10 weeks resulted in markedly increased levels of plasma cholesterol and VLDL and IDL in control rabbits as expected. Expression of either HL or apoE reduced plasma cholesterol response by 75% and 60%, respectively. The HL transgenic rabbits had substantial reductions in medium and small VLDL and IDL fractions but not in larger VLDL. LDL levels were also reduced, with a shift from larger, more buoyant to smaller, denser particles. In contrast, apoE transgenic rabbits had a marked reduction in the levels of large VLDLs, with a selective accumulation of IDLs and large buoyant LDLs. Combined expression of apoE and HL led to dramatic reductions of total cholesterol (85% versus controls) and of total VLDL+IDL+LDL (87% versus controls). HDL subclasses were remodeled by the expression of either transgene and accompanied by a decrease in HDL cholesterol compared with controls. HL expression reduced all subclasses except for HDL2b and HDL2a, and expression of apoE reduced large HDL1 and HDL2b. Extreme HDL reductions (92% versus controls) were observed in the combined HL+apoE transgenic rabbits. These results demonstrate that human HL and apoE have complementary and synergistic functions in plasma cholesterol and lipoprotein metabolism.     

Platelet function in patients with familial hypertriglyceridemia: evidence that platelet reactivity is modulated by apolipoprotein E content of very-low-density lipoprotein particles

We evaluated platelet function in patients with familial hypertriglyceridemia (FHTG). Compared with healthy gender-matched controls, platelets from patients showed lower aggregation (P < .01) and thromboxane formation (P < .01) in response to collagen. Very-low-density lipoprotein (VLDL) particles obtained from the patients inhibited collagen-induced aggregation, whereas VLDL particles from controls had opposite effects. The VLDL-induced effect was regulated by its apolipoprotein E (apoE) content. Indeed, apoE-VLDL-rich fractions caused antiaggregative effects, whereas apoE-VLDL-poor fractions produced a strong proaggregative response. Since we have recently demonstrated that VLDL particles may regulate the activity of platelet low-density lipoprotein (LDL) receptor by a phenomenon of downregulation and desensitization, in this study, we have investigated the effect of prolonged exposure to circulating VLDL levels on the activity of platelet LDL receptor by a double-blind controlled study with gemfibrozil (600 mg twice daily) in 18 subjects with FHTG. Platelets from patients exhibited fewer platelet LDL receptors and 125I-LDL binding was saturable at a lower protein concentration. After 6 months, gemfibrozil therapy versus placebo had a marked lipid-lowering effect, significantly decreased triglycerides (61%), VLDL cholesterol (72%), apoB (28%), and apoE (55%), and increased high-density lipoprotein (44%) and apoA-I (18%). Furthermore, gemfibrozil affected the apoprotein composition of VLDL: total protein increased by 28%, the molar ratio of apoE to apoB decreased 64%, and apoE content decreased 55%. However, no differences in phospholipid, triglyceride, or total cholesterol were detected. Moreover, platelet function was markedly altered by gemfibrozil therapy. Collagen-induced aggregation and thromboxane formation were significantly enhanced (P < .01). The initial antiaggregative pattern of VLDL particles was changed to a significant proaggregative effect (P < .01), and the number of LDL binding sites was markedly upregulated (P < .001). Both receptor upregulation and the change in the aggregative effect of VLDL particles were associated with the reduction of apoE content in VLDL particles (P < .05). The overall results indicate that in the regulation of platelet reactivity in hypertriglyceridemic patients, apoE content of VLDL particles and their interaction with the platelet LDL receptor are involved.     

Combining beta-adrenergic and peroxisome proliferator-activated receptor gamma stimulation improves lipoprotein composition in healthy moderately obese subjects

Current pharmacological regimens for hypertriglyceridemia and low high-density lipoprotein (HDL) are limited to the peroxisome proliferator-activated receptor (PPAR) alpha activating fibrates, niacin, and statins. This pilot study examined the impact of simultaneous stimulation of cyclic adenosine monophosphate with a beta-adrenergic agonist and PPARgamma with pioglitazone (PIO) on lipoprotein composition in moderately obese, healthy subjects. Subjects were treated with PIO (45 mg) to stimulate PPARgamma or a combination of ephedrine (25 mg TID), a beta-agonist, with caffeine (200 mg TID), a phosphodiesterase inhibitor (ephedrine plus caffeine), or both for 16 weeks. Lipoproteins were separated by gradient ultracentrifugation into very low-density lipoprotein (VLDL), intermediate-density lipoprotein, low-density lipoprotein (LDL), and 3 HDL (L, M, and D) subfractions. Apolipoproteins were measured by high-performance liquid chromatography. PIO alone reduced the core triglyceride (TG) content relative to cholesterol ester (CE) in VLDL (-40%), IDL (-25%), and HDL-M (-38%). Ephedrine plus caffeine alone reduced LDL CE (-13%), phospholipids (-9%), and apolipoprotein (apo) B (-13%); increased HDL-M LpA-I (HDL containing apoA-I without apoA-II, 28%), CE/TG (23%), and CE/apoA-I (8%) while reducing apoA-II (-10%); and increased HDL-L LpA-I (29%). Combination therapy reduced total plasma TG (-28%), LDL cholesterol (LDL-C, -10%), apoB (-16%), apoB/apoA-I ratio (-21%) while increasing HDL cholesterol (HDL-C, 21%), total plasma apoA-I (12%), LpA-I (43%), and apoC-I (26%). It also reduced VLDL total mass (-34%) and apoC-III (-39%), LDL CE (-13%), apoB (-13%), and total mass (-11%). Combination therapy increased HDL-L CE/TG (32%), apoC-I (30%), apoA-I (56%), and LpA-I (70%), as well as HDL-M CE (35%), phospholipids (24%), total mass (19%), apoC-I (25%), apoA-I (18%), and LpA-I (56%). In conclusion, simultaneous beta-adrenergic and PPARgamma activation produced beneficial effects on VLDL, LDL, HDL-L, and HDL-M. Perhaps the most important impact of combination therapy was dramatic increases in LpA-I and apoC-I in HDL-L and HDL-M, which were much greater than the sum of the monotherapies. Because LpA-I appears to be the most efficient mediator of reverse-cholesterol transport and a major negative risk factor for cardiovascular disease, this combination therapy may provide very effective treatment of atherosclerosis.     

Effect of the combination of methyltestosterone and esterified estrogens compared with esterified estrogens alone on apolipoprotein CIII and other apolipoproteins in very low density, low density, and high density lipoproteins in surgically postmenopausal women

Androgens are known to lower plasma triglycerides, an independent risk factor for coronary heart disease (CHD). Triglycerides are carried in plasma on very low density (VLDL) and low density (LDL) lipoprotein particles. Apolipoprotein CIII (apoCIII), a strong predictor of CHD, impairs the metabolism of VLDL and LDL, contributing to increased triglycerides. The objective of this study was to assess the effect of oral methyltestosterone (2.5 mg/d), added to esterified estrogens (1.25 mg/d), on concentrations of apolipoproteins and lipoproteins, specifically those containing apoCIII, compared with esterified estrogens alone in surgically postmenopausal women. The women in the methyltestosterone plus esterified estrogen group had significant decreases in total triglycerides, apoCI, apoCII, apoCIII, apoE, and high density lipoprotein (HDL) cholesterol compared with those in the esterified estrogen group. The decreases in apoCIII concentrations occurred in VLDL (62%; P = 0.02), LDL (35%; P = 0.001), and HDL (17%; P < 0.0001). There were also decreases in cholesterol and triglycerides concentrations of apoCIII containing LDL, and apoCI concentration of apoCIII containing VLDL. There was no effect on VLDL and LDL particles that did not contain apoCIII or on apoB concentrations. In conclusion, methyltestosterone, when administered to surgically postmenopausal women taking esterified estrogen, has a selective effect to reduce the apoCIII concentration in VLDL and LDL, a predictor of CHD. Methyltestosterone may lower plasma triglycerides through a reduction in apoCIII.     

Differential effects of apolipoprotein E isoforms on lipolysis of very low-density lipoprotein triglycerides

Apolipoprotein (apo) E plays a key role in lipoprotein metabolism and has been proposed to modulate triglyceride (TG) lipolysis. However, no systematic investigation on lipolysis using all 3 isoforms of apoE has been performed. To clarify the role of common human apoE isoforms in the lipolysis of very low-density lipoprotein (VLDL) TGs, we overexpressed human apoE isoforms in apoE and low-density lipoprotein receptor-deficient mice using adenoviral-mediated gene transfer and used VLDL particles obtained from these mice for in vitro lipolysis assay. Overexpression of apoE, regardless of its isoforms, increased the TG content of VLDL in mice in vivo. In vitro analysis of the effect of apoE on lipolysis revealed that irrespective of its isoforms, apoE did inhibit TG lipolysis at every concentration of apoE examined, and this inhibitory effect became more pronounced as the apoE content of VLDL increased. No difference was observed in TG lipolysis activity among isoforms at low apoE/TG ratio; however, intermediate ratios of apoE/TG, which reflect physiologic VLDL apoE/TG ratios, demonstrated a significantly greater level of lipolysis inhibition in apoE2, but less so in apoE4 compared with other isoforms. This differential effect by apoE isoforms on lipolysis was attenuated at higher apoE/TG ratios; nevertheless, apoE2 still inhibited lipolysis significantly more than did apoE4. Enrichment of VLDL with apoE decreased both the apoC contents and apoC-II/C-III ratios of VLDL, contributing, at least in part, to the inhibitory function of apoE on lipolysis. The present study clarifies the differential lipolysis-modulating effect of apoE isoforms, which would help explain the difference in pre- and postprandial TG levels among humans carrying different apoE isoforms.     

Abnormalities in lipoprotein metabolism in Gaucher type 1 disease

We have previously described an association between Gaucher type 1 disease and reduced levels of total, low density lipoprotein (LDL), and high density lipoprotein (HDL) cholesterol. Plasma concentrations of apolipoprotein B and apolipoprotein AI were reduced in these subjects, while plasma apolipoprotein E (apoE), which can be synthesized and secreted by macrophages, was increased. To study the pathophysiologic basis for these changes in lipoprotein and apolipoprotein levels, we studied very low density lipoprotein (VLDL), LDL, and HDL metabolism in-depth in four subjects with Gaucher disease. Gel filtration of their plasma revealed that apoE was present in essentially a single population of lipoproteins in the large HDL range. In subject no. 4, studied presplenectomy and post-splenectomy, plasma apoE levels fell after surgery in association with a redistribution of apoE among the plasma lipoproteins to a pattern seen in normal subjects. Determination of the rates of secretion and catabolism of VLDL apoB and triglyceride were within normal limits. The reduced plasma levels of LDL and HDL cholesterol, and of both plasma apoB and apoAI, were associated with increased fractional catabolic rates of these apolipoproteins in LDL and HDL. These results indicate that the hypocholesterolemia present in subjects with Gaucher type 1 disease is associated with increased fractional catabolism of LDL and HDL. These findings, together with the evidence for alternations in plasma apoE metabolism in this disorder, suggest a role for the macrophage as the basis for these abnormalities.     

Effects of estrogen therapy on apolipoprotein E in type II hyperlipoproteinemia.

Type III hyperlipoproteinemia (HLP) is characterized by the accumulation in plasma of an abnormal cholesterol-enriched lipoprotein ($\text{VLDL-Chol}\text{VLDL-TG}\text{ratio > 0.42}$, VLDL-Chol/total TG ratio > 0.30) that floats in the S_f 12–100 range and migrates as β-VLDL on lipoprotein electrophoresis. Type III patients have increased levels of apolipoprotein E and the apoE-III subspecies is deficient, while apoE-I and apoE-II are increased. The role of the apoE abnormalities in the production of abnormal lipoprotein compositions is unknown. Since estrogens exert a hypolipidemic effect, correct the VLDL composition, and improve rates of remnant removal in type III HLP, we investigated whether the administration of estrogen would also correct the apoE-III deficiency. Three type III subjects (two females, one male) were given ethinyl estradiol 1 μg/kg/day for 6–8 wk and fasting plasmas were tested at 2-wk intervals. Lipoprotein lipids were determined chemically, apoA-I, apoC-II, and apoC-III by radioimmunoassay, and the subspecies of apoC and apoE in VLDL by analytic gel isoelectric focusing (IEF). In the women total TG, total Chol, VLDL-Chol, and LDL-Chol decreased whereas HDL-Chol and apoA-I increased. The abnormal lipid ratios reverted to normal and the broad beta lipoprotein decreased or disappeared. Furthermore, the apoE/apoC area ratio (by IEF) in apoVLDL decreased in response to estrogen. This occurred because of a decrease in apoE levels in VLDL. Despite these changes, the apoE-III deficiency remained. In contrast to the females, the male type III exhibited a progressive hyperlipidemia. By the 8th wk, total-TG had risen to 2387 mg/dl. His apoE/apoC area ratio in apoVLDL increased because of an increase in apoE levels in VLDL. Despite the great increases in lipid levels, the relative proportions of the apoE subspecies and apoE deficiency remained unaltered. The dissociation between apoE composition and lipoprotein physiology suggests either that apoE plays no important role in the pathophysiology of the dysbetalipoproteinemia and hyperlipoproteinemia, or if apoE does play an important role in the untreated patient, treatment bypasses any “metabolic blocks” due to the apoE abnormalities.