apoB Production Rate Research Articles

In vivo turnover study demonstrates diminished clearance of lipoprotein(a) in hemodialysis patients

Lipoprotein(a) (Lp(a)) consists of a low-density lipoprotein-like particle and a covalently linked highly glycosylated protein, called apolipoprotein(a) (apo(a)). Lp(a) derives from the liver but its catabolism is still poorly understood. Plasma concentrations of this highly atherogenic lipoprotein are elevated in hemodialysis (HD) patients, suggesting the kidney to be involved in Lp(a) catabolism. We therefore compared the in vivo turnover rates of both protein components from Lp(a) (i.e. apo(a) and apoB) determined by stable-isotope technology in seven HD patients with those of nine healthy controls. The fractional catabolic rate (FCR) of Lp(a)-apo(a) was significantly lower in HD patients compared with controls (0.164+/-0.114 vs 0.246+/-0.067 days(-1), P=0.042). The same was true for the FCR of Lp(a)-apoB (0.129+/-0.097 vs 0.299+/-0.142 days(-1), P=0.005). This resulted in a much longer residence time of 8.9 days for Lp(a)-apo(a) and 12.9 days for Lp(a)-apoB in HD patients compared with controls (4.4 and 3.9 days, respectively). The production rates of apo(a) and apoB from Lp(a) did not differ significantly between patients and controls and were even lower for patients when compared with controls with similar Lp(a) plasma concentrations. This in vivo turnover study is a further crucial step in understanding the mechanism of Lp(a) catabolism: the loss of renal function in HD patients causes elevated Lp(a) plasma levels because of decreased clearance but not increased production of Lp(a). The prolonged retention time of Lp(a) in HD patients might importantly contribute to the high risk of atherosclerosis in these patients.

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.

Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited

We previously reported that liver-specific overexpression of ABCG5/G8 in mice is not atheroprotective, suggesting that increased biliary cholesterol secretion must be coupled with decreased intestinal cholesterol absorption to increase net sterol loss from the body and reduce atherosclerosis. To evaluate this hypothesis, we fed low density lipoprotein receptor-knockout (LDLr-KO) control and ABCG5/G8-transgenic (ABCG5/G8-Tg)xLDLr-KO mice, which overexpress ABCG5/G8 only in liver, a Western diet containing ezetimibe to reduce intestinal cholesterol absorption. On this dietary regimen, liver-specific ABCG5/G8 overexpression increased hepatobiliary cholesterol concentration and secretion rates (1.5-fold and 1.9-fold, respectively), resulting in 1.6-fold increased fecal cholesterol excretion, decreased hepatic cholesterol, and increased (4.4-fold) de novo hepatic cholesterol synthesis versus LDLr-KO mice. Plasma lipids decreased (total cholesterol, 32%; cholesteryl ester, 32%; free cholesterol, 30%), mostly as a result of reduced non-high density lipoprotein-cholesterol and apolipoprotein B (apoB; 36% and 25%, respectively). ApoB-containing lipoproteins were smaller and lipid-depleted in ABCG5/G8-TgxLDLr-KO mice. Kinetic studies revealed similar 125I-apoB intermediate density lipoprotein/LDL fractional catabolic rates, but apoB production rates were decreased 37% in ABCG5/G8-TgxLDLr-KO mice. Proximal aortic atherosclerosis decreased by 52% (male) and 59% (female) in ABCG5/G8-TgxLDLr-KO versus LDLr-KO mice fed the Western/ezetimibe diet. Thus, increased biliary secretion, resulting from hepatic ABCG5/G8 overexpression, reduces atherogenic risk in LDLr-KO mice fed a Western diet containing ezetimibe. These findings identify distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism and atherosclerosis.

Short-term overexpression of DGAT1 or DGAT2 increases hepatic triglyceride but not VLDL triglyceride or apoB production

Increased triglyceride synthesis resulting from enhanced flux of fatty acids into liver is frequently associated with VLDL overproduction. This has led to the common belief that hepatic triglyceride synthesis can directly modulate VLDL production. We used adenoviral vectors containing either murine acyl-coenzyme A:diacylglycerol transferase 1 (DGAT1) or DGAT2 cDNA to determine the effect of a short-term increase in hepatic triglyceride synthesis on VLDL triglyceride and apolipoprotein B (apoB) production in female wild-type mice. Hepatic DGAT1 and DGAT2 overexpression resulted in 2.0-fold and 2.4-fold increases in the triglyceride content of liver, respectively. However, the increase in hepatic triglyceride content had no effect on the production rate of VLDL triglyceride or apoB in either case. Liver subfractionation showed that DGAT1 and DGAT2 overexpression significantly increased the content of triglyceride within the cytoplasmic lipid fraction, with no change in the triglyceride content of the microsomal membrane or microsomal VLDL. The increased cytoplasmic triglyceride content was observed in electron micrographs of liver sections from mice overexpressing DGAT1 or DGAT2. Overexpression of DGAT1 or DGAT2 resulted in enhanced [(3)H]glycerol tracer incorporation into triglyceride within cytoplasmic lipids. These results suggest that increasing the cytoplasmic triglyceride pool in hepatocytes does not directly influence VLDL triglyceride or apoB production. In the presence of adequate cytoplasmic lipid stores, factors other than triglyceride synthesis are rate-limiting for VLDL production.

Low-density lipoprotein apolipoprotein B100 turnover in hypopituitary patients with GH deficiency: a stable isotope study

Epidemiological studies suggest that hypopituitary patients have an increased risk for cardiovascular mortality. The dyslipidaemia associated with this condition is often characterised by an increase in total cholesterol (TC) and low-density lipoprotein (LDL) cholesterol (LDL-C) and may contribute to these findings. The underlying mechanisms are not fully elucidated. LDL apolipoprotein B (apoB) production rate and metabolic clearance rate were measured in seven patients with hypopituitarism (including GH deficiency) under stable conventional replacement therapy (three males and four females; age 40-16.1 years; body mass index 29.0-6.1 kg/m(2) (means +/- s.d.)) and seven age-, gender- and body mass index-matched control subjects with an infusion of 1-(13)C-leucine. Fasting lipid profile and lipid composition of LDL were also measured. Fasting TC, triglycerides (TG), high-density lipoprotein-C, LDL-C and free fatty acid concentrations were not different between hypopituitary patients and control subjects. LDL-TG (P < 0.006) and LDL-TG/LDL apoB ratio (P < 0.02) were significantly increased in hypopituitary patients. LDL apoB pool size was not statistically different between patients and control subjects. In the hypopituitary patients, LDL apoB metabolic clearance rate (P < 0.05) and LDL apoB production rate (P < 0.02) were lower than in the control subjects. The present results suggest that LDL apoB turnover and LDL composition is altered in hypopituitary patients. Whether these findings explain the increased risk for cardiovascular disease in hypopituitary patients remains to be established.

Effect of Ezetimibe on the In Vivo Kinetics of ApoB-48 and ApoB-100 in Men With Primary Hypercholesterolemia

To examine the impact of ezetimibe, a selective inhibitor of intestinal cholesterol absorption, on the in vivo kinetics of apolipoproteins (apo) B-48 and B-100 in humans. Kinetics of triglyceride-rich lipoprotein (TRL) apoB-48 and very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) apoB-100 labeled with a stable isotope were assessed at baseline and at the end of 8 weeks of treatment with 10 mg/d of ezetimibe in 8 men with moderate primary hypercholesterolemia. Data were fit to a multicompartmental model using SAAMII to calculate fractional catabolic rate (FCR) and production rate (PR). Ezetimibe significantly decreased total and LDL cholesterol concentrations by -14.5% and -22.0% (P=0.004), respectively, with no significant change in plasma triglyceride and high-density lipoprotein (HDL) cholesterol levels. Ezetimibe had no significant effect on TRL apoB-48 kinetics and pool size (PS). However, VLDL and IDL apoB-100 FCRs were significantly increased (+31.2%, P=0.02 and +20.8%, P=0.04, respectively) with a concomitant elevation of VLDL apoB-100 PR (+20.9%, P=0.04). Furthermore, LDL apoB-100 PS was significantly reduced by -23.2% (P=0.004), caused by a significant increase in FCR of this lipoprotein fraction (+24.0%, P=0.04). These results indicate that reduction of plasma LDL cholesterol concentration after treatment with ezetimibe is associated with an increase in FCR of apoB-100-containing lipoproteins.

The ACAT inhibitor avasimibe increases the fractional clearance rate of postprandial triglyceride-rich lipoproteins in miniature pigs

Previously, we have shown, in vivo, that the acyl coenzyme A: cholesterol acyltransferase (ACAT) inhibitor avasimibe decreases hepatic apolipoprotein (apo) B secretion into plasma. To test the hypothesis that avasimibe modulates postprandial triglyceride-rich lipoprotein (TRL) metabolism in vivo, an oral fat load (2 g fat/kg) containing retinol was given to 9 control miniature pigs and to 9 animals after 28 days treatment with avasimibe (10 mg/kg/day, n = 5; 25 mg/kg/day, n = 4). The kinetic parameters for plasma retinyl palmitate (RP) metabolism were determined by multi-compartmental modeling using SAAM II. Avasimibe decreased the 2-h TRL ( d < 1.006 g/mL; S f > 20) triglyceride concentrations by 34%. The TRL triglyceride 0–12 h area under the curve (AUC) was decreased by 21%. In contrast, avasimibe had no effect on peak TRL RP concentrations, time to peak, or its rate of appearance into plasma, however, the TRL RP 0–12 h AUC was decreased by 17%. Analysis of the RP kinetic parameters revealed that the TRL fractional clearance rate (FCR) was increased 1.4-fold with avasimibe. The TRL RP FCR was negatively correlated with very low density lipoprotein (VLDL) apoB production rate measured in the fasting state ( r = −0.504). No significant changes in total intestinal lipid concentrations were observed. Thus, although avasimibe had no effect on intestinal TRL secretion, plasma TRL clearance was significantly increased; an effect that may relate to a decreased competition with hepatic VLDL for removal processes.

Microsomal triglyceride transfer protein −493T variant reduces IDL plus LDL apoB production and the plasma concentration of large LDL particles

The microsomal triglyceride transfer protein (MTP) is essential for the synthesis and secretion of apolipoprotein B (apoB)-containing lipoproteins. We investigated the role the MTP -493G/T gene polymorphism in determining the apoB-100 secretion pattern and LDL heterogeneity in healthy human subjects. Groups of carriers of the T and the G variants (n = 6 each) were recruited from a cohort of healthy 50-yr-old men. Kinetic studies were performed by endogenous [(2)H(3)]leucine labeling of apoB and subsequent quantification of the stable isotope incorporation. apoB production rates, metabolic conversions, and eliminations were calculated by multicompartmental modeling (SAAM-II). LDL subfraction distribution was analyzed in the entire cohort (n = 377). Carriers of the MTP -493T allele had lower plasma LDL apoB and lower concentration of large LDL particles [LDL-I: 136 +/- 57 (TT) vs. 175 +/- 55 (GG) mg/l, P < 0.01]. Kinetic modeling suggested that MTP -493T homozygotes had a 60% lower direct production rate of intermediate-density lipoprotein (IDL) plus LDL compared with homozygotes for the G allele (P < 0.05). No differences were seen in production rates of large and small VLDL, nor were there any differences in metabolic conversion or elimination rates of apoB between the genotype groups. This study shows that a polymorphism in the MTP gene affects the spectrum of endogenous apoB-containing lipoprotein particles produced in humans. Reduced direct production of LDL plus IDL appears to be related to lower plasma concentrations of large LDL particles.

Comparison of Apolipoprotein B100 Metabolism between Continuous Subcutaneous and Intraperitoneal Insulin Therapy in Type 1 Diabetes

In type 1 diabetic patients, the replacement of s.c. insulin infusion with i.p. insulin infusion restores the normal physiological gradient between the portal vein and the peripheral circulation, which is likely to modify lipoprotein metabolism. To check this hypothesis, we performed two apolipoprotein (apo) B100 kinetic studies in seven type 1 diabetic patients, first under s.c. insulin infusion and then 3 months after the beginning of i.p. insulin infusion. Glycemic control was similar under s.c. insulin infusion and i.p. insulin infusion, as assessed by glycated hemoglobin A1c and the capillary glycemic curve determined during the kinetic study. Very low-density and intermediate-density lipoprotein apoB100 pool size, production rate, and fractional catabolic rate (FCR) were similar under s.c. insulin infusion and i.p. insulin infusion. The low-density lipoprotein apoB100 FCR tended to decrease under ip insulin (0.45 +/- 0.06 vs. 0.55 +/- 0.11 pool/d), but the difference did not reach statistical significance (95% confidence interval for the difference, -0.33, 0.11). The low-density lipoprotein apoB100 pool size and production rate remained unchanged under i.p. insulin infusion compared with s.c. insulin infusion. In type 1 diabetic patients, the replacement of s.c. insulin infusion with i.p. insulin infusion does not induce profound modifications of apoB100-containing lipoprotein production and FCRs.

Normal metabolism of apolipoprotein B100-containing lipoproteins despite qualitative abnormalities in type 1 diabetic men

Type 1 diabetic subjects are at increased risk of cardiovascular disease and exhibit multiple qualitative abnormalities of apolipoprotein (apo) B100-containing lipoproteins. This stable isotope kinetic experiment was designed to study whether these abnormalities are associated with changes in the synthesis and fractional catabolic rates of VLDL-, IDL- and LDL-apoB100. Using a bolus followed by a 16-h constant infusion of 13C-leucine, we performed a kinetic study in eight men with type 1 diabetes treated with a continuous subcutaneous insulin infusion administered by an external pump and in seven healthy men, in the fed state. The mean HbA1c level in the type 1 diabetic patients was 8.00+/-1.48%. Plasma triglyceride, and total, LDL and HDL cholesterol levels were similar in patients and control subjects. VLDL were less triglyceride rich in type 1 diabetic patients than in control subjects (VLDL triglyceride : apoB 6.91+/-0.81 vs 8.29+/-1.24 mmol/g, p=0.05). Conversely, the IDL and LDL of the type 1 diabetic patients contained relatively higher levels of triglycerides (IDL triglycerides : apoB 2.16+/-0.36 vs 1.57+/-0.30 mmol/g, p<0.01; LDL triglycerides : apoB 0.27+/-0.06 vs 0.16+/-0.04 mmol/g, p<0.05). The apoB100 pool size, production and fractional catabolic rates in the two groups of subjects were similar for all lipoprotein fractions. Despite qualitative abnormalities, especially abnormalities of triglyceride content, the metabolism of apoB100-containing lipoproteins is not altered in type 1 diabetic men with fair glycaemic control with continuous subcutaneous insulin infusion. The high risk of atherosclerosis in these patients cannot be explained by kinetic abnormalities of apoB100-containing lipoproteins.

Effects of the PPARγ agonist pioglitazone on lipoprotein metabolism in patients with type 2 diabetes mellitus

Elevated plasma levels of VLDL triglycerides (TGs) are characteristic of patients with type 2 diabetes mellitus (T2DM) and are associated with increased production rates (PRs) of VLDL TGs and apoB. Lipoprotein lipase-mediated (LPL-mediated) lipolysis of VLDL TGs may also be reduced in T2DM if the level of LPL is decreased and/or the level of plasma apoC-III, an inhibitor of LPL-mediated lipolysis, is increased. We studied the effects of pioglitazone (Pio), a PPARgamma agonist that improves insulin sensitivity, on lipoprotein metabolism in patients with T2DM. Pio treatment reduced TG levels by increasing the fractional clearance rate (FCR) of VLDL TGs from the circulation, without changing direct removal of VLDL particles. This indicated increased lipolysis of VLDL TGs during Pio treatment, a mechanism supported by our finding of increased plasma LPL mass and decreased levels of plasma apoC-III. Lower apoC-III levels were due to reduced apoC-III PRs. We saw no effects of Pio on the PR of either VLDL TG or VLDL apoB. Thus, Pio, a PPARgamma agonist, reduced VLDL TG levels by increasing LPL mass and inhibiting apoC-III PR. These 2 changes were associated with an increased FCR of VLDL TGs, almost certainly due to increased LPL-mediated lipolysis.

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- d 3]-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.

Complete Deficiency of the Low-Density Lipoprotein Receptor Is Associated With Increased Apolipoprotein B-100 Production

We addressed the role of the low-density lipoprotein (LDL) receptor in determining clearance rates and production rate (PR) of apolipoprotein B (apoB) in humans. Kinetic studies using endogenous labeling of apoB with deuterated leucine were performed in 7 genetically defined patients with homozygous familial hypercholesterolemia (FH) and compared with 4 controls. The fractional catabolic rates (FCR) and PRs for apoB were determined by multicompartmental modeling. The FCRs of very-low-density lipoprotein 1 (VLDL1), VLDL2, intermediate-density lipoprotein (IDL), and LDL apoB were lower in FH than in controls, with the LDL apoB FCR being significantly lower (0.148+/-0.049 versus 0.499+/-0.099 pools x d(-1); P=0.008). Whereas receptor-defective FH patients had a total apoB PR similar to controls, receptor-null FH patients had a significantly greater total apoB PR than controls (35.97+/-10.51 versus 21.32+/-4.21 mg x kg(-1) x d(-1), respectively; P=0.02). This first study of apoB metabolism in homozygous FH using endogenous labeling with stable isotopes demonstrates that the LDL receptor contributes significantly to the clearance of LDL from plasma but plays a lesser role in the clearance of larger apoB-containing lipoproteins. Furthermore, these data also indicate that absence of a LDL receptor in humans substantially influences the apoB PR in vivo.

In vivo metabolism of LDL subfractions in patients with heterozygous FH on statin therapy: rebound analysis of LDL subfractions after LDL apheresis

LDL can be subfractionated into buoyant (1.020-1.029 g/ml(-1)), intermediate (1.030-1.040 g/ml(-1)), and dense (1.041-1.066 g/ml(-1)) LDLs. We studied the rebound of these LDL-subfractions after LDL apheresis in seven patients with heterozygous familial hypercholesterolemia (FH) regularly treated by apheresis (58 +/- 9 years, LDL-cholesterol = 342 +/- 87 mg/dl(-1), triglycerides = 109 +/- 39 mg/dl(-1)) and high-dose statins. Apolipoprotein B (apoB) concentrations were measured in LDL subfractions immediately after and on days 1, 2, 3, 5, and 7 after apheresis. Compartmental models were developed to test three hypotheses: 1) that dense LDLs are derived from the delipidation of buoyant and intermediate LDLs (model A); 2) that dense LDLs are generated directly from LDL-precursors (model B); or 3) that a model combining both pathways (model C) is necessary to describe the metabolism of dense LDLs. In all models, it was assumed that apoB production and fractional catabolic rate (FCR) did not change with apheresis. Apheresis decreased buoyant, intermediate, and dense LDL-apoB by 60 +/- 12%, 67 +/- 5%, and 69 +/- 11%, respectively. Models B and C, but not model A, described the rebound data. The model with the greatest biological plausibility (model C) was used to estimate metabolic parameters. FCR was 1.05 +/- 0.86 d(-1), 0.48 +/- 0.11 d(-1), and 0.69 +/- 0.24 d(-1) for buoyant, intermediate, and dense LDLs, respectively. Dense LDL production was 17.3 +/- 0.2 mg/kg(-1)/d(-1), 58% of which was derived directly from LDL precursors (VLDL, IDL, or direct secretion), while 42% was derived from buoyant and intermediate LDLs. Thus, our data indicate that in statin-treated patients with heterozygous FH dense LDLs originate from two sources. Whether this is also valid in other metabolic situations (with predominant small, dense LDLs) remains to be determined.

Intestinal lipoprotein production is stimulated by an acute elevation of plasma free fatty acids in the fasting state: studies in insulin-resistant and insulin-sensitized Syrian golden hamsters.

It is not known whether intestinal lipoprotein production is stimulated by an acute elevation of plasma free fatty acids (FFA). We examined the effect of an intralipid and heparin infusion on the intestinal lipoprotein production rate (PR) in insulin-sensitive [chow-fed (CHOW)], insulin-resistant [60% fructose (FRUC) or 60% fat-fed (FAT)], and insulin-sensitized [FRUC or FAT plus rosiglitazone (RSG)-treated] Syrian Golden hamsters. After 5 wk of treatment, overnight-fasted hamsters underwent in vivo Triton WR-1339 studies for measurement of apolipoprotein B48 (apoB48) PR in large (Svedberg unit, >400) and small (Svedberg unit, 100-400) lipoprotein fractions, with an antecedent 90-min infusion of 20% intralipid and heparin (IH) to raise plasma FFA levels approximately 5- to 8-fold vs. those in the saline control study. IH markedly increased apoB48 PR in CHOW by 3- to 5-fold, which was confirmed ex vivo in pulse-chase experiments in primary cultured hamster enterocytes. Oleate, but not glycerol, infusion was associated with a similar elevation of apoB48 PR as IH. In FRUC and FAT, basal (saline control) apoB48 PR was approximately 4-fold greater than that in CHOW; there was no additional stimulation with IH in vivo and only minimal additional stimulation ex vivo. RSG partially normalized basal apoB48 PR in FAT and FRUC, and PR was markedly stimulated with IH. We conclude that intestinal lipoprotein production is markedly stimulated by an acute elevation of plasma FFAs in insulin-sensitive hamsters, in which basal production is low, but minimally in insulin-resistant hamsters, in which basal production is already elevated. With RSG treatment, basal PR is partially normalized, and they become more susceptible to the acute FFA stimulatory effect.

Apolipoprotein B100 metabolism in autosomal-dominant hypercholesterolemia related to mutations in PCSK9.

We have reported further heterogeneity in familial autosomal-dominant hypercholesterolemia (FH) related to mutation in proprotein convertase subtilisin/kexin type 9 (PCSK9) gene previously named neural apoptosis regulated convertase 1 (Narc-1). Our aim was to define the metabolic bases of this new form of hypercholesterolemia. In vivo kinetics of apolipoprotein B100-containing lipoproteins using a 14-hour primed constant infusion of [2H3] leucine was conducted in 2 subjects carrying the mutation S127R in PCSK9, controls subjects, and FH subjects with known mutations on the low-density lipoprotein (LDL) receptor gene (LDL-R). Apo B100 production, catabolism, and transfer rates were estimated from very LDL (VLDL), intermediate-density lipoprotein (IDL), and LDL tracer enrichments by compartmental analysis. PCSK9 mutation dramatically increased the production rate of apolipoprotein B100 (3-fold) compared with controls or LDL-R mutated subjects, related to direct overproduction of VLDL (3-fold), IDL (3-fold), and LDL (5-fold). The 2 subjects also showed a decrease in VLDL and IDL conversion (10% to 30% of the controls). LDL fractional catabolic rate was slightly decreased (by 30%) compared with controls but still higher than LDL-R-mutated subjects. These results showed that the effect of the S127R mutation of PCSK9 on plasma cholesterol homeostasis is mainly related to an overproduction of apolipoprotein B100.

Increased production of VLDL apoB-100 in subjects with familial hypercholesterolemia carrying the same null LDL receptor gene mutation

Early radiokinetic studies revealed that the classical metabolic defect in patients with familial hypercholesterolemia (FH) is hypocatabolism of LDL due to decreased LDL receptor activity. However, recent studies have suggested that hepatic oversecretion of apolipoprotein B-100 (apoB-100)-containing lipoproteins could also contribute to the markedly elevated plasma concentrations of LDL-cholesterol found in FH. The aim of this study was to examine the kinetics of apoB-100 labeled with a stable isotope (l-[5,5,5-D(3)] leucine) in five normolipidemic controls and in seven well-characterized FH subjects that included six FH heterozygotes and one FH homozygote carrying the same null LDL receptor gene mutation. As compared with controls, the VLDL apoB-100 production rate was increased by 50% in the FH heterozygotes and by 109% in the FH homozygote. Furthermore, FH subjects had significantly higher LDL apoB-100 pool size and lower LDL apoB-100 fractional catabolic rate than controls. These results indicate that the elevation of plasma LDL-cholesterol found in FH is attributable to both decreased clearance of LDL and increased hepatic production of apoB-100-containing lipoproteins.

Hepatic secretion of apoB-100 is impaired in hypobetalipoproteinemic mice with an apoB-38.9-specifying allele

Apolipoprotein B (apoB) truncation-specifying mutations cause familial hypobetalipoproteinemia (FHBL). Lipoprotein kinetics studies have shown that production rates of apoB-100 are reduced by 70-80% in heterozygous FHBL humans, instead of the expected 50%. To develop suitable mouse models to study the underlying mechanism, apoB-38.9-only (Apob(38.9/38.9)) mice were crossbred with Apobec-1 knockout (Apobec-1(-/-)) mice or apoB-100-only (Apob(100/100)) mice to produce two lines of apoB-38.9 heterozygous mice that produce only apoB-38.9 and apoB-100, namely Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) mice. In vivo rates of apoB-100 secretion were measured using [35S]Met/Cys to label proteins and Triton WR-1339 to block apoB-100 VLDL lipolysis/uptake. Rates of secretion were reduced by 80%, rather than the expected 50%, in both Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) mice compared with those of the respective Apobec-1(-/-)/Apob(+/+) and Apob(100/100) control mice. Continuous labeling and pulse-chase experiments in primary hepatocyte cultures revealed that rates of apoB-100 synthesis by Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) hepatocytes were reduced to the expected 50% of those of the respective controls, but the efficiency of secretion of apoB-100 was significantly lower in apoB-38.9 heterozygous hepatocytes. The greater-than-expected decreases in apoB-100 production rates of FHBL heterozygous humans appear to be attributable to a defect in secretion rather than in the synthesis of apoB-100 from the unaffected apoB allele.

Effect of atorvastatin on apolipoprotein B100 containing lipoprotein metabolism in type-2 diabetes.

Seven hypertriglyceridemic patients with type-2 diabetes were treated with atorvastatin (40 mg/day) for 2 months. Kinetics of apolipoprotein B100 (apoB100)-containing lipoproteins were determined before and after atorvastatin treatment and compared with data obtained in five normolipidemic volunteers. ApoB100 metabolism was studied using stable isotopes and multicompartmental modeling. Compared with normolipidemic obese subjects, type-2 diabetic patients had a higher apoB100 concentration in very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), and low-density lipoproteins (LDL) (P < 0.005). Kinetic analysis showed an increase in the total apoB100 production rate (P < 0.005) related to VLDL apoB100 overproduction (P < 0.005). Patients were also characterized by a lower fractional catabolic rate (FCR) in VLDL (not significant) or IDL (P < 0.005) mainly related to a decrease in VLDL and IDL delipidation rate (P < 0.005). Catabolism of LDL was also lower in diabetic patients (P < 0.05). Atorvastatin treatment significantly decreased plasma triglycerides (P < 0.05), total and LDL cholesterol (P < 0.05), apoB100 in LDL, IDL, and VLDL (P < 0.05). Treatment significantly decreased total apoB100 production rate (P < 0.05), but only for VLDL (P < 0.05). Treatment normalized FCR in IDL and LDL (P < 0.05). We concluded that atorvastatin improved lipid abnormalities in type-2 diabetic patients not only by increasing the clearance of apoB100-containing lipoproteins but also by decreasing VLDL production.

Early kinetic abnormalities of apoB-containing lipoproteins in insulin-resistant women with abdominal obesity.

The kinetic abnormalities of apolipoprotein B (apoB)-containing lipoproteins in abdominally obese insulin-resistant individuals remain poorly understood. To determine the influence of insulin resistance, linked with abdominal obesity, on apoB metabolism at an early stage, we performed a stable isotope kinetic study of apoB in very low density lipoproteins (VLDLs), intermediate density lipoproteins (IDLs), and low density lipoproteins (LDLs) in 5 abdominally obese insulin-resistant women with normal fasting triglyceride levels and without impaired glucose tolerance and in 5 age-matched control women. Each subject received an intravenous injection of a 0.7 mg/kg bolus of L-[1-(13)C]leucine, immediately followed by a 16-hour constant infusion at 0.7 mg/kg per hour. Compared with control women, insulin-resistant women with abdominal obesity showed a significant 84% increase of the VLDL apoB production rate (27.18+/-11.53 versus 14.80+/-1.94 [control] mg/kg per day, P=0.009), a significant 54% increase of the IDL apoB production rate (20.63+/-3.66 versus 13.39+/-3.99 [control] mg/kg per day, P=0.009), and a significant 63% increase of the LDL apoB production rate (18.49+/-1.70 versus 11.33+/-3.79 [control] mg/kg per day, P=0.009), leading to significantly higher VLDL, IDL, and LDL apoB concentrations. The fractional catabolic rates of VLDL, IDL, and LDL apoB were not significantly different between abdominally obese insulin-resistant women and control women. Our study shows that patients at an early stage of insulin resistance linked with abdominal obesity (without glucose intolerance or fasting hypertriglyceridemia) already have an altered metabolism of the VLDL-IDL-LDL cascade (increased VLDL, IDL, and LDL apoB production rates), which is consistent with the augmented risk of atherosclerosis observed in this population.