Effects of fenofibrate on apolipoprotein kinetics in patients with coexisting dysbetalipoproteinemia and heterozygous familial hypercholesterolemia

Dysbetalipoproteinemia (dysb) and familial hypercholesterolemia (FH) are two genetic disorders giving rise to severe disturbances of lipid homeostasis and premature atherosclerosis. The co-occurrence of both metabolic abnormalities is very rare and is estimated to affect 1 individual per 2,500,000 in the general population. However, the relative contribution of these two dyslipidemias to the combined lipoprotein phenotype is unknown. The two objectives of this study were (1) to compare the in vivo kinetics of triglyceride-rich lipoprotein (TRL) apolipoprotein (apo) B48, VLDL, IDL and LDL apo B100 as well as plasma apo A-l labelled with a stable isotope (l-(5,5,5-D3) leucine) in two subjects presenting both heterozygous FH and dysbetalipoproteinemia (FH+/dysb+), in six FH heterozygotes and in five normolipidemic controls, and (2) to examine the impact of a 6-week treatment with micronized fenofibrate 200 mg/d on apolipoprotein kinetics in FH+/dysb+. As compared with FH heterozygotes and controls, the two FH+/dysb+ subjects showed elevated TRL apo B48 and VLDL, IDL apo B100 pool sizes (PS) mainly due to lower fractional catabolic rates (FCR). Moreover, as compared with FH heterozygotes, FH+/dysb+ subjects presented lower LDL apo B100 PS due to a higher FCR. Pool size, FCR and production rate (PR) of apo A-l were higher in FH+/dysb+ subjects than in FH heterozygotes. In FH+/dysb+ subjects, fenofibrate treatment was associated with a decreased TRL apo B48 PS (-52 and -61%), VLDL apo B100 (-61 and -63%) and IDL apo B100 (-37 and -16%) and an increased FCR of TRL apo B48 (10 and 67%), VLDL apo B100 (123 and 57%) and IDL apo B100 (29 and 10%). Fenofibrate also increased LDL apo B100 PS (3 and 57%) due to an increase in PR (80 and 26%) but had divergent effects on LDL apo B100 FCR. These results indicate that the coexistence of dysbetalipoproteinemia and heterozygous FH results in a mixed lipoprotein phenotype that is intermediate between the two pure phenotypes and that fenofibrate treatment partially reverses lipoprotein abnormalities, mostly through changes in PR and FCR of apo B48- and B100-containing lipoproteins.     

Human apolipoprotein (Apo) B-48 and ApoB-100 kinetics with stable isotopes

The kinetics of apolipoprotein (apo) B-100 and apoB-48 within triglyceride-rich lipoproteins (TRLs) and of apoB-100 within IDL and LDL were examined with a primed-constant infusion of (5,5,5-(2)H(3)) leucine in the fed state (hourly feeding) in 19 subjects after consumption of an average American diet (36% fat). Lipoproteins were isolated by ultracentrifugation and apolipoproteins by SDS gels, and isotope enrichment was assessed by gas chromatography/mass spectrometry. Kinetic parameters were calculated by multicompartmental modeling of the data with SAAM II. The pool sizes (PS) of TRL apoB-48, VLDL apoB-100, and LDL apoB-100 were 17+/-10, 273+/-167, and 3325+/-1146 mg, respectively. There was a trend toward a faster fractional catabolic rate (FCR) for VLDL apoB-100 than for TRL apoB-48 (6.73+/-3.48 versus 5.02+/-2.07 pools/d, respectively, P=0.06). The mean FCRs for IDL and LDL apoB-100 were 10.07+/-7.28 and 0.27+/-0.08 pools/d, respectively. The mean production rate (PR) of TRL apoB-48 was 6.5% of VLDL apoB-100 (1. 3+/-0.90 versus 20.06+/-6.53 mg. kg(-1). d(-1), P<0.0001). TRL apoB-48 PS was correlated with apoB-48 PR (r=0.780, P<0.0001) but not FCR (r=-0.1810, P=0.458). VLDL apoB-100 PS was correlated with both PR (r=0.713, P=0.0006) and FCR (r=-0.692, P=0.001) of VLDL apoB-100 and by apoB-48 PR (r=0.728, P=0.0004). LDL apoB-100 PS was correlated with FCR (r=-0.549, P=0.015). These data indicate that (1) the FCRs of TRL apoB-48 and VLDL apoB-100 are similar in the fed state, (2) TRL apoB-48 PS is correlated with TRL apoB-48 PR, (3) VLDL apoB-100 PS is correlated with both PR and FCR of VLDL apoB-100 and PR of TRL apoB-48, and (4) LDL apoB-100 PS is correlated with LDL FCR.     

Apolipoprotein A-I, B-100, and B-48 metabolism in subjects with chronic kidney disease, obesity, and the metabolic syndrome

The metabolism of apolipoproteins (apo)B-48, B-100, and A-I was studied with a primed constant infusion of deuterium-labeled leucine in the fed state in 3 male individuals with chronic kidney disease (CKD), a glomerular filtration rate (GFR) of 28 to 57 mL/min/1.73 m2, obesity (body mass index [BMI] 33.1), and the metabolic syndrome. Compared to 5 obese controls (BMI 30.1) and 13 non-obese controls (BMI 25.2), these CKD subjects had high plasma levels of triglycerides (TG) (343 +/- 27.5 mg/dL v 144 +/- 34.4 in the obese controls, P < .001) and low apoA-I (86.7 +/- 3.9 mg/dL). An abnormal high-density lipoprotein (HDL) particle subpopulation pattern was found, with low levels of pre beta-1 and alpha1. Compared to the obese controls, very-low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL) apoB-100 levels were elevated 2- to 3-fold, while LDL apoB-100 levels were slightly lower (-7 %) and apoB-48 levels were comparable. The high TG levels were not associated with statistically significant changes in VLDL apoB-100 kinetics, although the production rate (PR) was higher and the fractional catabolic rate (FCR) was lower. The slightly lower LDL apoB-100 levels were accompanied by a significant 3-fold increase in the FCR and a 2.7-fold increase in the PR. The lower apoA-I levels were accompanied by a 1.6-fold increase in the FCR. Compared to the non-obese controls, the PR of apoA-I was increased by 61% and 38%, respectively (P < .001) in CKD and in obese control subjects. In the control subjects, the PR of apoA-I was significantly correlated with the BMI (r = 0.81, P < .0001). The kinetic results are consistent with these hypotheses: (1) CKD is associated with decreased clearance of the TG-rich lipoproteins (TRLs) and increased catabolism of LDL; (2) obesity increases apoB-100 and apoA-I production; and (3) in CKD, TG transfer to HDL, making HDL more susceptible to catabolism, accounts for the low apoA-I levels.     

Human triglyceride-rich lipoprotein apo E kinetics and its relationship to LDL apo B-100 metabolism

Apolipoprotein (apo) E is a multifunctional protein that can act as a ligand for lipoprotein receptors. The receptor-mediated clearance of the triglyceride-rich lipoproteins (TRL) chylomicrons and VLDL from plasma is, in part, dependent on apo E. Enrichment of VLDL with apo E is thought to enhance receptor-mediated clearance of VLDL resulting in a low rate of conversion of VLDL to LDL. However, the kinetic mechanism controlling the concentration of apo E in VLDL is not known. We conducted kinetic studies on apo E in the TRL fraction (d < 1.006 g/ml) and apo B-100 in the TRL and LDL (d = 1.019-1.063 g/ml) fractions to assess the kinetic determinants of apo E concentration in TRL and to determine the effects that TRL apo E production and clearance rates have on the production rate of LDL apo B-100. Nineteen males between the ages of 24 and 73 underwent a primed-constant infusion with deuterated leucine tracer in the constantly-fed state. Apo B-100 from TRL and LDL, and apo E from TRL were isolated and their tracer incorporation measured by gas chromatography/mass spectrometry. The residence time and production rates of each protein were determined from the kinetic data using the SAAM II modeling program. The residence time and production rate of TRL apo E were about one-half that of TRL apo B-100 (1.8 +/- 1.0 vs. 2.9 +/- 2.1 h and 14.5 +/- 11.0 vs. 27.6 +/- 17.3 mg/kg per day, respectively). The production rate of TRL apo E was weakly correlated with the production rate of TRL apo B-100 (r = 0.424, P = 0.07). Multiple regression analysis showed that the residence time of TRL apo B-100 and the relative TRL apo E production rate (relative to the TRL apo B100 production rate) were negatively associated with LDL apo B-100 production rate, accounting for 68% of its variability. We conclude that (1) the concentration of apo E in TRL is highly correlated to its production rate, suggesting that production rate regulates the TRL apo E concentration, and (2) individuals with a relatively short TRL apo B-100 residence time and those producing TRL with a relatively low apo E content have the highest LDL apo B-100 production rates.     

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 synthesis in humans: liver synthesizes only apolipoprotein B-100

Apolipoprotein (apo) B-100 and B-48 are prominent apolipoproteins in VLDL, IDL, and chylomicrons. Organ cultures of normal adult human liver were established to ascertain the form of apo B synthesized by hepatocytes in humans. Human liver was minced and incubated in 15 mL methionine-free RPMI-1640 medium with 10% dialyzed fetal calf serum plus 250 microCi 35S-methionine for eight hours at 37 degrees C. Lipoproteins secreted by the liver were isolated by ultracentrifugation and the content of newly synthesized apo B determined by quantitation of radioactivity in the apoB-100 and apoB-48 bands after separation by 3% NaDodSO4 gel electrophoresis. In the eight-hour period, 2.5% to 3.2% of added 35S-methionine was secreted in TCA-precipitable protein of which 0.34% was apo B. Ninety-nine percent of the apo B in VLDL, IDL, and LDL was in the apo B-100 electrophoretic band. No significant radioactivity was detected in the apo B-48 electrophoretic band. Eighty-nine percent of the total radioactivity of apo B-100 was in VLDL with 3% and 8% in IDL and LDL, respectively. These results establish that adult human liver in organ culture synthesizes apo B-100 but not apo B-48.     

Lipoprotein metabolism in subjects with hepatic lipase deficiency

A heritable deficiency of hepatic lipase (HL) provides insights into the physiologic function of HL in vivo. The metabolism of apolipoprotein B (apoB)-100 in very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) and of apoA-I and apoA-II in high-density lipoprotein (HDL) particles lipoprotein (Lp)(AI) and Lp(AI:AII) was assessed in 2 heterozygous males for compound mutations L334F/T383M or L334F/R186H, with 18% and 22% of HL activity, respectively, compared with 6 control males. Subjects were provided with a standard Western diet for a minimum of 3 weeks. At the end of the diet period, apo kinetics was assessed using a primed-constant infusion of [5,5,5-(2)H(3)] leucine. Mean plasma triglyceride (TG) and HDL cholesterol levels were 55% and 12% higher and LDL cholesterol levels 19% lower in the HL patients than control subjects. A higher proportion of apoB-100 was in the VLDL than IDL and LDL fractions of HL patients than control subjects due to a lower VLDL apoB-100 fractional catabolic rate (FCR) (4.63 v 9.38 pools/d, respectively) and higher hepatic production rate (PR) (33.24 v 10.87 mg/kg/d). Delayed FCR of IDL (2.78 and 6.31 pools/d) and LDL (0.128 and 0.205 pools/d) and lower PR of IDL (3.67 and 6.68 mg/kd/d) and LDL 4.57 and 13.07 mg/kg/d) was observed in HL patients relative to control subjects, respectively. ApoA-I FCR (0.09 and 0.13 pools/d) and PR (4.01 and 6.50 mg/kg/d) were slower in Lp(AI:AII) particles of HL patients relative to control subjects, respectively, accounting for the somewhat higher HDL cholesterol levels. HL deficiency may result in a lipoprotein pattern associated with low heart disease risk.     

Metabolism of apolipoproteins B-48 and B-100 of triglyceride-rich lipoproteins in normal and lipoprotein lipase-deficient humans.

The metabolism of apolipoproteins B-48 and B-100 (apo B-48 and B-100) in large triglyceride-rich lipoproteins (300 to 1500 A in diameter) has been compared in three normal subjects and two subjects with genetically determined deficiency of lipoprotein lipase. The triglyceride-rich lipoproteins were obtained from a lipoprotein lipase-deficient donor 4 hr after a fat-rich meal in order to obtain chylomicrons (containing apo B-48) and very low density lipoproteins (VLDL) (containing apo B-100), whose properties had not been modified by the action of this enzyme. The triglyceride-rich lipoproteins were labeled with 125I and injected intravenously into recipients who had fasted overnight. In normal recipients, most of the apo B-48 was removed from the blood within 15 min, and most of the apo B-100 was removed within 30 min. In the lipoprotein lipase-deficient recipients, most of the injected apo B-100 remained in the blood for more than 8 hr; removal of apo B-48 was only slightly more rapid. In all subjects, only trace amounts of either protein were found in lipoproteins more dense than 1.006 g/ml. The results indicate that (i) the removal of the apo B of both chylomicrons and large VLDL from the blood is dependent upon the hydrolysis of their component triglycerides by lipoprotein lipase, and (ii) little or no apo B-48 of chylomicrons or apo B-100 of large VLDL is converted appreciably to low density lipoproteins (LDL). Our results suggest that the reported variability of the conversion of VLDL to LDL may be related to the size and composition of the particles secreted from the liver. The rapid production of remnant particles that are removed efficiently by the liver may minimize the opportunity for further reactions leading to the formation of LDL.     

Effects of atorvastatin on postprandial plasma lipoproteins in postinfarction patients with combined hyperlipidaemia

Enhanced and prolonged postprandial lipaemia is implicated in coronary and carotid artery disease. This study assessed the effects of atorvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, on postprandial plasma concentrations of triglyceride-rich lipoproteins (TRLs). Sixteen middle-aged men with combined hyperlipidaemia (baseline low density lipoprotein (LDL) cholesterol and plasma triglyceride concentrations (median (interquartile range) of 4.54 (4.17-5.26)) and 2.66 (2.04-3.20) mmol/l, respectively) and previous myocardial infarction were randomised to atorvastatin 40 mg or placebo once daily for 8 weeks in a double-blind, cross-over design. The apolipoprotein (apo) B-48 and B-100 contents were determined in subfractions of TRLs as a measure of chylomicron remnant and very low density lipoprotein (VLDL) particle concentrations (expressed as mg apo B-48 or apo B-100 per litre of plasma), in the fasting state and after intake of a mixed meal. Atorvastatin treatment reduced significantly the fasting plasma concentrations of VLDL cholesterol, LDL cholesterol and VLDL triglycerides (median% change) by 29, 44 and 27%, respectively, and increased high density lipoprotein (HDL) cholesterol by 19%, compared with baseline. The postprandial plasma concentrations of large (Svedberg flotation rate (Sf) 60-400) and small (Sf 20-60) VLDLs and chylomicron remnants were almost halved compared with baseline (mean 0-6 h plasma concentrations were reduced by 48% for Sf 60-400 apo B-100, by 46% for Sf 60-400 apo B-48, by 46% for Sf 20-60 apo B-100 and by 27% for Sf 20-60 apo B-48), and the postprandial triglyceridaemia was reduced by 23% during active treatment. In conclusion, atorvastatin 40 mg once daily causes profound reductions of postprandial plasma concentrations of all TRLs in combined hyperlipidaemic patients with premature coronary artery disease.     

Plasma metabolism of apoB-containing lipoproteins in patients with hepatic lipase deficiency

The metabolism of apoB-containing lipoproteins was investigated in the fasted state in three complete and three partial hepatic lipase (HL)-deficient subjects as well as in seven normotriglyceridemic (NTG) and two hypertriglyceridemic (HTG) controls using a 12 h primed-constant infusion of l-[5,5,5-d₃]-leucine. Two males with complete HL deficiency had increased plasma pool sizes of VLDL and IDL apoB-100 due to substantial reductions in fractional catabolic rate (FCR) of VLDL and IDL apoB-100 compared with both NTG and HTG controls. Reductions in LDL apoB-100 production rate (PR) were also observed in these two patients compared with NTG and HTG controls. Complete HL deficiency in the female proband was associated with normal VLDL apoB-100 kinetics, while plasma IDL apoB-100 pool size was increased by 124% due to an 82% decrease in the FCR of IDL apoB-100. The FCR and PR of LDL apoB-100 were reduced by 64 and 51%, respectively, in the proband compared with sex-matched controls. Partial HL-deficient patients were characterized by apoB-containing lipoprotein metabolism similar to that of controls. These results indicate that complete HL deficiency is associated with a potentially atherogenic apoB-containing lipoprotein metabolism that can be modulated considerably by secondary factors such as gender and abdominal obesity.     

Apolipoprotein B-100 and ApoA-II Kinetics as Determinants of Cellular Cholesterol Efflux

Context: Cellular cholesterol efflux is a key step in reverse cholesterol transport and may depend on the metabolism of apolipoprotein (apo) B-100, apoA-I, and apoA-II. Objective: We examined the associations between cholesterol efflux and plasma concentrations and kinetics of very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL)-apoB-100, high-density lipoprotein (HDL)-apoA-I, and HDL-apoA-II in men. Design, Subjects, and Methods: Thirty men were recruited from the community with a wide range of body mass index. The capacity of plasma and HDL to efflux cholesterol was measured ex vivo. Apolipoprotein kinetics were measured using stable isotope techniques and multicompartmental modeling. Results: Cholesterol efflux to whole plasma was correlated with plasma levels of cholesterol, triglyceride, apoB-100, insulin, cholesteryl ester transfer protein, and lecithin-cholesterol acyltransferase, body mass index and waist circumference (P < 0.05 in all). Cholesterol efflux was inversely correlated with the fractional catabolic rate (FCR) of VLDL (r = -0.728), IDL (r = -0.662), and LDL-apoB-100 (r = -0.479) but positively correlated with the FCR (r = 0.438) and production rate (r = 0.468) of HDL-apoA-II. In multiple regression analysis, the concentration and FCR of VLDL-apoB-100 (β-coefficient = 0.708 and -0.518, respectively) and IDL-apoB-100 (β-coefficient = 0.354 and -0.447, respectively) were independent predictors of cholesterol efflux. The association of cholesterol efflux with apoB-100 metabolism was diminished after removal of apoB-100-containing lipoproteins from plasma prior to efflux. All associations, except for cholesteryl ester transfer protein, were lost when cholesterol efflux to isolated HDL was tested. Conclusions: The plasma concentration and kinetics of apoB-100-containing lipoproteins are significant predictors of the capacity of whole plasma to effect cellular cholesterol efflux.     

Lack of evidence for reduced plasma apo B48 catabolism in patients with heterozygous familial hypercholesterolemia carrying the same null LDL receptor gene mutation

Increasing evidence suggests that remnants of chylomicrons and very low density lipoprotein (VLDL) also known as triglyceride-rich lipoproteins (TRL) are directly related to the pathogenesis of atherosclerosis. While studies in animals suggest that low density lipoprotein (LDL) receptor deficiency delays clearance of chylomicron remnants, human data supporting this hypothesis are conflicting. The objective of this study was to compare the fractional catabolic rate (FCR) and production rate (PR) of TRL apolipoprotein B48, the principal structural protein of intestinally derived chylomicron remnants, between familial hypercholesterolemic (FH) heterozygotes and non-FH controls. This was achieved by examining the kinetics of TRL apo B48 labelled with a stable isotope (L-(5,5,5-D3)leucine) in five normolipidemic males (age: 24.7 +/- 1.3 years; body mass index (BMI): 23.9 +/- 1.4 kg/m2) and six genetically defined FH heterozygous males (age: 29.7 +/- 9.9 years; (BMI): 22.0 +/- 4.3 kg/m2) carrying the same null LDL receptor gene mutation. All participants were apo E3 homozygotes. During the kinetic study, the subjects consumed 1/30 of their daily food intake every 30 min over a 15 h period. No significant difference was observed between FH heterozygotes and controls for FCR of TRL apo B48 (7.9 +/- 2.1 versus 7.9 +/- 2.6 pools per day, P = 0.99) while the TRL apo B48 pool size (10.5 +/- 5.4 versus 5.7 +/- 2.4 mg, P = 0.03) and PR (1.1 +/- 0.3 versus 0.6 +/- 0.3 mg kg(-1) per day, P = 0.02) were significantly higher among FH than in controls. In conclusion, this study shows no evidence for reduced plasma apo B48 catabolism in patients with heterozygous FH carrying the same null LDL receptor gene mutation and suggests that the plasma levels of intestinally derived TRL are elevated in FH due to an increased production rate.     

Changes in the composition of plasma lipoproteins in the chronic uremic rat

The effect of chronic renal failure on the lipid and apolipoprotein concentrations of plasma, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) was studied in an experimental uremic rat model. Control rats were sham-operated and were divided into adlibitum-fed and pair-fed groups. The rats were studied (after an overnight fast) 32 days after the onset of uremia. The uremic rats had a 4-fold increase in plasma urea nitrogen and creatinine. The pair-fed and ad-lib-fed controls had similar levels of plasma urea nitrogen and lipid profiles. In the uremic rats, plasma triglyceride (TG) levels were increased 3.8-fold due to increased TG in the VLDL, IDL and HDL fractions. Their 2-3-fold increase in plasma free cholesterol (FC), esterified cholesterol (EC) and phospholipids (PL) were due to FC, EC and PL increases in VLDL, IDL, LDL and HDL. Their increase in plasma apo B (x 2.4) and apo E (x 1.5) were due to increases in VLDL, IDL and LDL. Their plasma apo A-I increased 2.4 fold due to increases in the LDL and HDL fractions. Uremic rats also had increases in the FC/PL molar ratio in VLDL, IDL and LDL. In their LDL, the apo B/total cholesterol (TC), apo B/PL and apo B/apo E molar ratios were decreased. In their HDL, the apo E/TC and apo E/PL molar ratios were decreased and the apo A-I/apo E molar ratio was increased. In conclusion, chronic uremia causes both quantitative changes in the levels and qualitative changes in the composition of the plasma lipoprotein particles. These results are compatible with the decreased hepatic lipase activities and impairment of remnant clearance observed in human chronic renal failure.     

Preferential reduction of very low density lipoprotein-1 particle number by fenofibrate in type IIB hyperlipidemia: consequences for lipid accumulation in human monocyte-derived macrophages

The combined (mixed) type IIB phenotype is typically associated with premature atherosclerosis and characterised by concomitant elevation of plasma levels of atherogenic triglyceride-rich lipoproteins, consisting of very low density lipoprotein (VLDL)-1 (Sf 60-400), VLDL-2 (Sf 20-60), and intermediate density lipoprotein (IDL) (Sf 12-20), as well as small dense LDL. After dietary stabilisation, type IIB patients received micronised fenofibrate (267 mg/day) for up to 12 months. At baseline (T0), patients (n=11) displayed fasting triglyceride, cholesterol and apoB levels of 308+/-13, 350+/-17 and 187+/-9 mg/dl, respectively. Micronised fenofibrate (M-fenofibrate) induced marked reductions in plasma triglyceride (TG) (-61%, P<0.0001), total cholesterol (-32%, P=0.0005) and apolipoprotein (apo) B (-33%, P<0.001) at 12 months (T12); similar effects were seen after 3 months (T3) of treatment. These changes resulted from significant reductions in VLDL-1 (-75%, P=0.00001), VLDL-2 (-46%, P=0.002) and LDL (-33%, P<0.0003); IDL concentrations were unchanged. At baseline, VLDL-1 constituted the major TG-rich lipoprotein (TRL) fraction (50% of total mass), but only 25% at T12. These drug effects were accompanied by marked increase in HDL-C (+20%, P=0.018). Quantitative changes in triglyceride-rich lipoproteins were accompanied by significant qualitative modifications in particle size and chemical composition (VLDL-1: TG, -10.7%, P<0.001; FC, +59%, P=0.0002; PL, +19%, P=0.033; VLDL-2: FC, +11%, P=0.027; IDL: FC, +14%, P=0.0004; PL, +12%, P=0.002). Reduction in the TG content of VLDL-1 was reflected in a shift of particle size distribution to smaller diameters (mean 45.4 and 42.3 nm, respectively, at T0 and T12). We evaluated the relative atherogenicity of TRL subfractions by determining their capacity, when normalised to equal particle numbers (as apoB 100 content), to induce lipid accumulation in human monocyte-derived macrophages. Among TRL subfractions, VLDL-1 (100 microg apoB/ml) possessed the highest capacity to induce macrophage lipid loading (up to sevenfold increase in TG content, P<0.001; free cholesterol, up to 1.7-fold; P<0.05). At 100 microg apoB/ml, cellular TG loading from VLDL-1 was twofold greater than that for VLDL-2 (P<0.01), and fivefold greater than for IDL (P<0.01). Despite drug-induced changes in the qualitative properties of TRL subfractions, the activity of VLDL-1, VLDL-2 and IDL as ligands which lead to induction of macrophage lipid accumulation, at equivalent particle numbers, was not detectably altered. By contrast, the fibrate-mediated reduction in the number of circulating VLDL-1 and VLDL-2 particles (four and twofold, respectively) resulted in marked decrease in cellular lipid loading. Considered together, these findings suggest that fenofibrate may act at systemic and arterial levels to reduce the cardiovascular risk associated with VLDL subfractions in patients with a combined hyperlipidemic (type IIB) phenotype. Indeed, we speculate that reductions in circulating levels of VLDL-1 and VLDL-2 may diminish intimal penetration of these particles and thus their propensity to enhance arterial macrophage lipid accumulation and foam cell formation. Finally, fenofibrate further attenuated the atherogenic lipid profile in these patients by inducing marked reduction in LDL and elevation in cardioprotective HDL.     

The hyperenergetic-fed obese dog, a model of disturbance of apolipoprotein B-100 metabolism associated with insulin resistance: kinetic study using stable isotopes

The hyperenergetic-fed beagle dog model of obesity-associated insulin resistance has previously demonstrated lipoprotein abnormalities similar to those of obese insulin-resistant humans. The aim of this study was to check, in the insulin-resistant dog, the mechanism leading to abnormalities in the mass of apolipoprotein B-100 (apo B-100) containing lipoproteins. Six healthy male beagle dogs were overfed with a high-fat diet for 28 +/- 2.5 weeks. Obesity was associated with insulin resistance as assessed by the euglycemic hyperinsulinemic clamp technique. The kinetics of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) apo B-100 were recorded in dogs, at healthy and insulin-resistant states, using a primed constant infusion of [5,5,5-D(3)]leucine. Isotopic enrichment was measured by gas chromatography-mass spectrometry (GC-MS). A multicompartmental model was used for the analysis of tracer kinetics data. Apolipoprotein B-100 concentration was higher in VLDL (2.8-fold, P < .05) but lower in LDL (2-fold, P < .05) in the insulin-resistant compared to the healthy state. Kinetic analysis showed a higher VLDL apo B-100 production (1.7-fold, P < .05). The fractional catabolic rate of VLDL did not change significantly, but the lipolysis was decreased significantly (3-fold, P < .05). The lower LDL apo B-100 level in insulin-resistant dogs was explained by a higher LDL fractional catabolic rate (2.5-fold, P < .05). The mechanisms leading to hypertriglyceridemia (higher production rate and lower lipolysis of VLDL) in insulin-resistant dogs were similar to those described in the insulin-resistant humans.     

Effects of insulin on hypercholesterolemia in the streptozotocin-induced diabetic rats fed a high cholesterol diet

The effect of dietary cholesterol (Ch) on plasma lipoprotein and apolipoproteins (apo) in diabetic rats was investigated. Ch-fed diabetic rats were severely hypercholesterolemic and hypertriglyceridemic. They had higher concentrations of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL). Concentration of high density lipoprotein (HDL) was decreased. beta-VLDL increased predominantly in Ch-fed diabetic rats, whereas IDL increased in the Ch and propylthiouracil-fed control rats. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis, VLDL and IDL from Ch-fed diabetic rats were unusual in that they contained more apo E, A-I and A-IV. Concentrations of plasma apo A-I and apo E were measured by radioimmunoassay. The diabetic rats fed a labo chow showed a significantly lower concentration of plasma apo E than control rats. Plasma apo E was extremely higher in the diabetic rats fed a cholesterol diet. Plasma apo A-I was significantly increased in the diabetic rats fed a labo chow and those fed a cholesterol. Insulin treatment significantly decreased the concentrations of VLDL, IDL and LDL and plasma concentration and distribution of apolipoproteins in lipoprotein subfractions changed toward normal. However, decreased HDL in the Ch-fed diabetic rats was not recovered by insulin treatment.     

Prolonged inhibition of cholesterol synthesis by atorvastatin inhibits apo B-100 and triglyceride secretion from HepG2 cells.

Atorvastatin is a new HMG-CoA reductase inhibitor that strongly lowers plasma cholesterol and triglyceride (TG) levels in humans and animals. Since previous data indicated that atorvastatin has prolonged inhibition of hepatic cholesterol synthesis, we tested whether this longer duration of inhibitory effect on cholesterol synthesis decreased hepatic lipoprotein secretion in vitro. We used the HepG2 hepatoma cell line to: (1) determine the time required until levels of secreted apo B-100 and TG declined significantly, (2) examine the relation to the mass of cellular cholesteryl ester (CE) and (3) test microsomal triglyceride transfer protein (MTP) activity which leads to decreased apo B-100 production. Although atorvastatin significantly inhibited cholesterol synthesis in HepG2 cells regardless of treatment duration (1, 14 or 24 h), it did not inhibit TG synthesis. Apo B-100 and TG secretion were unchanged after 1-h atorvastatin treatment, but declined significantly after 24-h treatment. Atorvastatin treatment also reduced cellular CE mass, exhibiting both time- and dose-dependency. Mevalonolactone, a product of HMG-CoA reductase, attenuated the inhibitory effects of atorvastatin. Atorvastatin strongly reduced mRNA levels of MTP, whereas it did not inhibit MTP activity as measured by TG transfer assay between liposomes. Simvastatin also induced treatment- and time-dependent reductions in apo B-100, whereas the MTP inhibitor BMS-201038 exhibited no time dependency, instead inhibiting this variable even on 1-h treatment. These results indicate that reduced apo B-100 secretion caused by atorvastatin is a secondary result owing to decreased lipid availability, and that atorvastatin's efficacy depends on the duration of cholesterol synthesis inhibition in the liver.     

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.     

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

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

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.