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

Atherosclerosis 155 (2001) 477– 485 www.elsevier.com/locate/atherosclerosis

Human triglyceride-rich lipoprotein apo E kinetics and its relationship to LDL apo B-100 metabolism John S. Millar a, Alice H. Lichtenstein a, Jose M. Ordovas a, Gregory G. Dolnikowski b, Ernst J. Schaefer a,* a

Lipid Metabolism Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts Uni6ersity, 711 Washington Street, Boston, MA 02111, USA b Mass Spectrometry Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts Uni6ersity, 711 Washington Street, Boston, MA 02111, USA Received 9 September 1999; received in revised form 5 June 2000; accepted 18 July 2000

Abstract 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 (dB 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 91.0 vs. 2.9 92.1 h and 14.5 9 11.0 vs. 27.6 917.3 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. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lipoprotein apo E; Kinetics; Apo B-100 metabolism

1. Introduction Apolipoprotein (apo) E plays a number of roles in lipoprotein metabolism, the best known being a ligand for receptor-mediated clearance of triglyceride rich lipoproteins (TRL), chylomicrons and very low density lipoprotein (VLDL), from plasma [1,2]. Apo E has been * Corresponding author. Tel.: +1-617-5563100; fax: + 1-6175563103. E-mail address: [email protected] (E.J. Schaefer).

shown to bind to all known lipoprotein receptors [3] and is the primary mediator of chylomicron and VLDL remnant clearance from plasma [4,5]. The content of apo E on VLDL may be an important factor in determining whether VLDL are cleared from plasma or further metabolized to low density lipoprotein (LDL) [6,7]. Thus the concentration of apo E in VLDL may have a direct influence over LDL production rates. Factors known to influence the plasma concentration of apo E include sex hormones and, to a certain extent, diet [8,9]. A high-fat diet (\ 40% fat) increases plasma

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apo E levels although the kinetic mechanism(s) responsible for the increase is not known [9]. Kushwaha et al. demonstrated that progesterone increases plasma apo E levels in baboons by increasing the apo E production rate [10]. Estrogen lowered the plasma apo E concentration by decreasing its residence time in plasma [10]. Plasma apo E concentrations in type III hyperlipoproteinemic subjects are elevated due to an enhanced apo E production rate in combination with a relatively low clearance rate of apo E2 from plasma [11]. Considerable variation exists among normolipidemic individuals in plasma and VLDL apo E concentration [8,12]. However the kinetic mechanism responsible for this variability is unknown. Determination of the kinetic mechanism(s) that primarily influence VLDL apo E concentrations will ultimately enhance the understanding of factors that control the production of LDL. As the apo E content of VLDL is important for determining the metabolic fate of VLDL, we conducted this study (1) to assess the kinetic mechanisms responsible for determining TRL apo E concentrations and (2) to explore the relationships between TRL apo B-100 and apo E kinetics and subsequent LDL apo B-100 metabolism.

2. Methods

to that previously provided, the first representing 4/ 23rd (subjects 1, 3, 4, 5 and 9) [14] or 1/20th (all other subjects) [15] of the daily caloric intake and subsequent meals were equally divided among the remaining daily caloric intake. Five hours after the first meal, at 0 h of the infusion protocol, subjects were given a bolus injection (10 mmol/kg) immediately followed by a constant infusion of 2H3-leucine (10 mmol/kg/h). The duration of the infusion period was 15 h. Blood samples were drawn at the following timepoints: 0, 1, 2, 3, 4, 6, 8, 10, 12 and 15 h.

2.3. Plasma lipids and lipoprotein fraction preparation Blood was collected in tubes containing EDTA (0.1% final concentration). Plasma was separated by centrifugation at 3000 rpm for 20 min at 4°C. TRL (dB1.006 g/ml), that contains chylomicrons and VLDL, and LDL (d= 1.019 –1.063 g/ml) was isolated by ultracentrifugation [16]. Plasma total cholesterol and triglyceride concentrations were determined using enzymatic reagents as previously described [17]. High density lipoprotein (HDL) cholesterol was measured in the supernatant fraction after precipitation of apolipoprotein B-containing lipoproteins with a dextran-magnesium sulfate solution [18]. Lipid assays were standardized through the Lipid Standardization Program of the Centers for Disease Control, Atlanta, GA.

2.1. Subjects 2.4. Quantitation and isolation of apolipoproteins Nineteen male volunteers between the ages of 24 and 73 with total plasma cholesterol and triglyceride levels below the 90th percentile for age and sex norms [13] underwent a complete medical history and physical examination. The subjects were in good health and had normal hepatic, renal, and thyroid function. They did not smoke, and were not taking medications known to affect plasma lipid levels. The experimental protocol was approved by the Human Investigation Review Committee of the New England Medical Center and Tufts University. During a lead-in period prior to the kinetic studies all subjects were provided with a baseline diet containing 45– 49% carbohydrate, 15% protein, 36 – 40% fat (15 –17% saturated, 15 – 17% monounsaturated, and 6% polyunsaturated fatty acids) and 180 mg of cholesterol/1000 kcal per day for a minimum of 5 days. Caloric intake was adjusted, when necessary, to maintain body weight.

Apo B and apo E were assayed using enzyme-linked immunosorbent assays (ELISA) as described previously [19,20]. The apolipoprotein concentrations reported are the average of three or more timepoints taken during the infusion period. Apo E phenotyping was performed by isoelectric focusing of whole plasma followed by immunoblotting using purified anti-human apo E antiserum [21]. Apolipoproteins were isolated from TRL (apo B-100, E) and LDL (apo B-100) by preparative SDS polyacrylamide gel electrophoresis (4–22.5%) using a Tris –glycine buffer system as described previously [22]. The majority (\ 95%) of apo B in TRL is apo B-100 [19] and the majority of apo E in TRL is associated with VLDL [23,24]. Therefore, the assumptions were made that the TRL apo B-100 concentration was equivalent to the TRL apo B concentration and that the TRL apo E concentration was equivalent to VLDL apo E concentration.

2.2. Infusion protocol 2.5. Determination of isotopic enrichment At the end of the lead-in diet period, the metabolism of apo B-100 within TRL and LDL, and apo E in TRL was studied in the fed state using a primed-constant infusion of 2H3-leucine [14]. After a 12-h overnight fast, subjects consumed hourly meals similar in composition

Polyacrylamide gel bands containing apo B-100 or E were hydrolyzed in 12 N hydrochloric acid at 100°C for 24 h. Hydrolysates of apo B-100 were prepared as described previously [15]. Hydrolysates of apo E were

J.S. Millar et al. / Atherosclerosis 155 (2001) 477–485

prepared by one of two methods. Hydrolysates prepared using the first method were dried under nitrogen and resuspended in 1 N acetic acid. Free amino acids were then isolated using a Dowex AG-50W-X8 100 – 200 mesh cation exchange resin. Amino acids were eluted from the resin with 3 M ammonium hydroxide and dried. Hydrolysates prepared using the second method were dried under nitrogen without any further purification, resulting in an increased amino acid recovery. There were no differences in the results obtained from either method of sample preparation. The dried amino acids from the two preparative methods were then propylated, converted to the N-heptafluorobutyramide derivatives and extracted into ethyl acetate prior to analysis on a Hewlett – Packard 5890/5988A gas chromatograph/mass spectrometer [15].

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pressed on a molar basis assuming molecular weights for apo B-100 and apo E of 512 000 and 34 000 kDa, respectively.

2.7. Statistical analyses Statistical analyses were performed using Minitab (State College, PA). The TRL apo E production rate and the relative TRL apo E production rate were log transformed prior to data analysis to obtain a normal distribution. Pearson correlation coefficients were calculated to test for statistically significant correlations between parameters using PB 0.05 as a cutoff level of significance.

3. Results

2.6. Kinetic analysis Production and fractional clearance rates (FCRs) for TRL apo E were calculated from tracer/tracee data using a compartmental model for apo E as described previously [20]. The assumption is made that the kinetic behavior of apo E in TRL is representative of the VLDL apo E kinetic behavior since the majority of apo E in TRL is found on VLDL [23,24]. Briefly, the model accounts for a unique kinetic behavior that has been observed in a number of studies whereby there is removal and reintroduction of VLDL apo E from plasma. Newly secreted apo E either associates directly with VLDL or temporarily enters an extravascular compartment (hypothesized to be hepatic lymph) before association with VLDL. The coefficient of variation for TRL apo E FCR was B 10% with an average of 6%. Production and FCRs of TRL and LDL apo B-100 have been presented previously in a separate manuscript using a multicompartmental for apo B-100 as described previously [25]. Briefly, the model features secretion of newly synthesized apo B-100 in VLDL. The model structure includes a VLDL delipidation chain, a slow VLDL compartment, a fast and slow IDL compartment and a single LDL compartment. The mass of each apolipoprotein fraction (pool size) was calculated as the product of the plasma concentration of that fraction (mg/dl) and the estimated plasma volume (0.45 dl/kg body weight). Residence times for each apolipoprotein were calculated by taking the reciprocal of the FCR (1/FCR) and expressing the data in hours. Production rates were obtained by multiplying the FCR by the pool size and expressing the data per kilogram body weight per day. While the residence times and production rates for IDL apo B-100 were calculated previously [25], these data are not presented in the current manuscript. The relative TRL apo E production rate was calculated as the ratio of the TRL apo E production rate to the TRL apo B-100 production rate ex-

The subject characteristics are shown in Table 1. Subjects ranged in age from 24 to 73 years. Total cholesterol and triglyceride levels ranged between 135 – 233 and 33–182 mg/dl, respectively. HDL cholesterol levels ranged between 29–59 mg/dl. There were three subjects with an apo E 3/2 phenotype, eleven subjects with an apo E 3/3 phenotype and five subjects with an apo E 4/3 phenotype. Apo E and apo B-100 plasma concentrations and kinetic parameters are shown in Table 2. The kinetic data for TRL apo B-100 has been previously presented [7] but are presented again for comparison purposes. The apo E concentration in plasma and TRL averaged 5.19 1.7 mg/dl (range 2.3 –8.8 mg/dl) and 1.890.8 mg/dl (range1.0 –3.3 mg/dl), respectively. TRL apo B100 concentration averaged 7.19 5.9 mg/dl and ranged from 2.0 to 24.7 mg/dl. TRL apo B-100 residence time averaged 2.99 2.1 h (range 0.7 –7.4 h) and that for apo E averaged 1.891.0 h (range 0.7 –4.8 h). The TRL apo B-100 production rate averaged 27.69 17.3 mg/kg per day (range 5.3 –64.5 mg/kg per day) and the production rate of TRL apo E averaged 14.59 11.0 mg/kg per day (range 3.3 –49.2 mg/kg per day). The average relative TRL apo E produced 10.699.5 mol TRL apo E/mol TRL apo B100 (range 1.8 to 34.5 mol/mol). Pearson correlation coefficients between apo E and apo B-100 concentrations and kinetic parameters are shown in Table 3. The TRL apo E concentration was significantly correlated with the TRL apo B and plasma apo E concentrations (r=0.478, PB 0.04 and r= 0.721, P B0.001, respectively). There was no significant correlation between TRL apo E and apo B-100 residence times (r= 0.127, P= 0.60), while the correlation between TRL apo E and apo B-100 production rates was of borderline significance (r=0.424, P= 0.07). There were no significant relationships between the production rates of either TRL apo B-100 or E and LDL apo B-100 production rate (r=0.296, P =0.22;

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r = −0.216, P=0.38, respectively). There was a significant inverse correlation between the relative TRL apo E production rate and LDL production rate (r= − 0.591, P=0.008). There was no significant correlation between the residence time of TRL apo E and the LDL apo B-100 production rate (r = 0.273, P =0.26) while the correlation between the TRL apo B residence time and the LDL apo B-100 production rate was significant. (r = − 0.641, P =0.003). Multiple regression using the independent variables TRL apo E residence time, TRL apo E production rate, TRL apo B-100 residence time, TRL apo B-100 production rate, and the relative TRL apo E production rate to predict the LDL apo B-100 production rate resulted in the TRL apo B-100 residence time and the relative TRL apo E production rate being the best predictors (r2 = 0.65, P= 0.0002).

4. Discussion The results of this study provide insights into the kinetic determinants of the TRL apo E concentration and the influence that the TRL apo E content has on the production rate of LDL in normolipidemic men. The concentration of apo E in TRL significantly correlated to the production rate of TRL apo E. This result is similar to results obtained by Gregg et al., where the production rate of total plasma apo E correlated significantly with the plasma concentration [26]. Conditions

characterized by decreased clearance rates of TRL from plasma, such as type III hyperlipidemia, have also been associated with increased plasma apo E production rates [11]. The regulation of TRL apo E concentration by production rate could be a mechanism to control the receptor-mediated uptake of TRL. In instances where lipolysis of TRL is decreased, the receptor-mediated uptake of TRL can be increased by raising the production rate of TRL apo E, thus enhancing particle affinity for lipoprotein receptors. The data appear to suggest an effect of apo E phenotype on TRL apo E production rate, with E3/2 \E3/3 \ E4/3. However, in the current study, there is not sufficient statistical power to adequately address this question due to small subject numbers in the E3/2 and E4/3 groups. Previous investigators have shown that there is rapid exchange of apo E between VLDL and HDL in vitro [12,27,28]. Were this occurring in vivo it would undoubtedly complicate any attempt to calculate an apo E production rate in this study since there would be input of labeled and unlabeled apo E from HDL to TRL making the hepatic contribution to TRL apo E production difficult to assess. However, human kinetic studies on HDL apo E show that, in the steady state, there is a relatively minor contribution of HDL derived apo E (B 1%) to the total amount of apo E produced in TRL [20]. This is particularly evident in studies conducted by Gregg et al., where the tracer curve for HDL apo E demonstrates a slow clearance rate that is in disequilibrium with the much more rapid clearance rate evi-

Table 1 Subject characteristics Subject

Age (years)

TC (mg/dl)

TG (mg/dl)

HDL (mg/dl)

Apo E phenotype

Plasma apo E (mg/dl)

Plasma apo B (mg/dl)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

24 25 25 27 28 29 33 36 37 41 46 47 53 60 62 65 69 71 73

143 144 151 157 149 135 140 172 174 196 189 181 159 211 174 195 233 225 204

33 61 76 70 63 115 81 118 81 76 114 98 174 79 85 128 81 182 125

56 37 47 37 59 36 42 32 45 49 37 45 29 48 34 39 42 46 49

4/3 3/3 3/3 4/3 3/3 3/3 3/2 3/3 3/3 4/3 4/3 4/3 3/2 3/3 3/3 3/2 3/3 3/3 3/3

2.3 4.2 3.1 4.2 nd 4.4 8.2 6.3 4.5 5.8 3.1 3.3 6.1 5.5 4.0 5.9 6.2 6.0 8.8

37.6 54.2 62.6 97.1 61.8 60.5 54.0 87.6 68.1 104.1 111.6 111.9 87.3 96.0 77.8 103.1 139.1 127.6 110.0

Mean S.D.

45 17

175 30

97 38

43 8

5.1 1.7

86.9 27.7

1

nd, not determined.

Table 2 TRL apo E and apo B-100 concentrations and kinetic parameters TRL apo E (mg/dl)

TRL apo E production rate (mg/kg per day)

TRL apo E residence time (h)

TRL apo B-100 (mg/dl)

TRL apo B-100 production rate (mg/kg per day)

TRL apo B-100 residence time (h)

Relative TRL apo E production rate (mol/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1.4 1.0 1.2 1.2 1.0 2.1 2.7 2.8 1.4 1.4 1.0 1.6 3.0 2.0 1.1 3.3 1.1 2.6 3.2

6.4 10.9 18.5 5.0 4.5 21.6 24.3 13.3 16.5 3.3 7.8 14.0 18.9 12.2 4.8 26.7 5.7 11.3 49.2

2.3 1.0 0.7 2.6 2.4 1.0 1.2 2.3 0.9 4.8 1.4 1.2 1.7 1.7 2.4 1.4 2.0 2.5 0.7

3.0 2.8 2.0 10.1 5.1 3.3 2.6 4.6 5.4 3.7 3.9 6.3 15.9 24.7 4.1 12.3 2.2 11.1 11.6

5.3 15.1 12.2 40.0 12.7 10.3 10.6 48.2 46.1 27.6 15.3 31.5 23.6 35.1 10.4 55.0 22.7 37.8 64.5

6.0 1.9 1.4 2.6 4.3 3.1 2.6 1.0 1.2 1.4 1.9 0.7 7.2 7.4 4.1 2.4 1.0 3.1 0.7

18.2 10.8 22.8 1.9 5.3 31.6 34.5 4.2 5.4 1.8 7.7 6.7 12.0 5.2 6.9 7.3 3.8 4.5 11.5

Mean S.D.

1.8 0.8

14.5 11.0

1.8 1.0

7.1 5.9

27.6 17.3

2.9 2.1

10.6 9.5

J.S. Millar et al. / Atherosclerosis 155 (2001) 477–485

Subject

481

482

Table 3 Correlation between TRL apo E kinetic parameters and apo B-100 kinetic parametersa Plasma apo E TRL apo E

a

TRL apo B-100

TRL apo B-100 RT

TRL apo B-100 PR

Rel. TRL apo E PR

Plasma apo B

LDL apo B-100

LDL apo B-100 RT

0.721*

TRL apo E 0.010 RT TRL apo E 0.460 PR TRL apo 0.297 B-100 TRL apo −0.121 B-100 RT TRL apo 0.510* B-100 PR Rel. TRL apo −0.026 E PR Plasma apo B 0.314 LDL apo B-100 LDL apo B-100 RT LDL apo B-100 PR

TRL apo E PR

J.S. Millar et al. / Atherosclerosis 155 (2001) 477–485

TRL apo E

TRL apo E RT

−0.195 0.735*

−0.762*

0.478*

−0.038

0.246

0.127

−0.135

0.564*

0.572*

−0.063

0.424

0.495*

0.233

−0.670*

0.600*

0.181

0.221

0.253

0.069

0.032 0.179

0.092

−0.324

−0.214

0.088

−0.059

0.325

−0.298

0.525*

−0.597*

0.242

−0.132

0.099

−0.451

0.432

−0.580*

0.973*

0.257

0.103

−0.026

0.340

0.133

0.234

−0.191

0.657*

0.607*

−0.219

0.273

−0.216

−0.202

−0.641*

0.296

−0.591*

0.520*

0.598*

−0.438

−0.254

The values for TRL apo E and the relative TRL apo E production rate were log transformed for the analysis. Abbreviations: Rel., relative; PR, production rate; RT, residence time. * PB0.05.

J.S. Millar et al. / Atherosclerosis 155 (2001) 477–485

dent for VLDL apo E, suggestive of little or no apo E exchange between VLDL and HDL in vivo [26]. Rapid intravascular exchange of apo E between VLDL and HDL would have resulted in these curves being essentially superimposable. The results of this study also show that the production rate of apo E in TRL is weakly related to the production rate of TRL apo B-100. Fazio et al. reported that the production rate of apo E was independent of the production rate of apo B-100 in hepatocytes [29]. Mensenkamp et al. reported that there is no difference in VLDL apo B production rate in mice expressing apo E and apo E knockout mice [30], suggesting no relationship between VLDL apo B and apo E production. The results from the current study suggests that the production of these proteins may be mildly dependent but over a narrow range of production rates there is no apparent relationship. The finding that the residence time of apo E in TRL was not significantly related to the residence time of apo B-100 in TRL is consistent with the concept that apo E metabolism in TRL is dynamic, the particle acquiring apo E as it is metabolized. Davignon et al. have proposed a model whereby apo E-rich VLDL is cleared from plasma during its delipidation, resulting in a relative decrease in the production rate of LDL [7]. In support of this model, Schaefer et al. reported that subjects with familial defective apolipoprotein B-100 (FDB) had enhanced removal of VLDL leading to an LDL apo B-100 production rate that was 50% of the control value [31]. In their study FDB subjects had a relative VLDL apo E production rate that was 42% greater than controls [31]. Huang et al. found that adding exogenous apo E to TRL results in a decreased conversion of VLDL to LDL in vitro [32]. The results of the current study are consistent with previous work and demonstrate that the amount of apo E secreted per VLDL is a major determinant of LDL apo B100 production rates in normolipidemic humans. The finding that the amount of VLDL apo E secreted per apo B100 rather than the VLDL apo E content was related to LDL apo B100 production rates is consistent with the concept that circulating VLDL is a mixture of ‘normal’ and ‘remnant’ VLDL, the latter accumulating non-functional apo E that is not receptor active [33]. In contrast, results from studies in apo E knockout mice [34] and in human apo E deficiency [35] appear to contradict the current results. In these cases there is the formation and accumulation of VLDL remnants (IDL) rather than an accumulation of LDL as might be expected. There may be two reasons for this discrepancy. First, lack of apo E on VLDL should result in a decreased affinity of VLDL to heparan sulfate proteoglycans (HSPG) on the surface of the vascular endothelium, a process mediated by apo E [36]. Secondly, further inhibition of VLDL binding to HSPG in

483

apo E deficiency should occur as a result of an enrichment of VLDL with apo C-II and C-III. These apolipoproteins have been shown to inhibit the heparin binding of VLDL [37,38] but are normally displaced somewhat by apo E [32,39]. Lack of interaction of VLDL with HSPG of the hepatic vascular endothelium would impede its ability to interface with hepatic lipase which is required for the conversion of VLDL to LDL [40]. In the absence of this interaction VLDL and IDL remnants accumulate in plasma. IDL have been shown to strongly interact with the arterial wall matrix [41] and may explain the advanced rate of atherosclerosis observed in apo E deficiency [35]. The residence time of TRL apo B-100 and the relative TRL apo E production rate were the best predictors of the LDL apo B-100 production rate, accounting for 68% of its variability. VLDL with a long residence time are lipolyzed slowly and may have a greater likelihood of interacting with lipoprotein receptors prior to being converted to a denser particle. VLDL produced with a high content of apo E should also have a relatively enhanced affinity for lipoprotein receptors and would have a greater chance of being cleared from plasma rather than converted to LDL. Interestingly, Huang et al. found that apo E overexpression in mice resulted in hypertriglyceridemia, in part due to displacement of TRL apo C-II, the activator of lipoprotein lipase, by apo E [32]. Extrapolation of the current findings to individuals outside the ranges of apo E concentrations studied may not be warranted. As mentioned above, apo E deficiency is associated with low, rather than high, LDL production rates for reasons discussed previously. Overexpression of apo E is also associated with low LDL production rates due to increased removal of LDL precursors from plasma [32]. Thus, it seems that the highest LDL production rates occur when there is low, but existent, VLDL apo E production, possibly in conjunction with higher than normal concentrations of VLDL apo C-II, which would increase lipolysis of VLDL, and apo C-III, which would inhibit receptor binding of VLDL. The apo E knockout mouse and human apo E deficiencies are both associated with premature atherosclerosis while overexpression of apo E is associated with hypertriglyceridemia [32,34,35]. Normal expression of apo E is associated with considerable variability that can influence the risk of developing CHD. In the current study the data are consistent with the concepts that (1) TRL apo E concentrations are determined primarily by their production rate rather than their residence time and (2) LDL apo B-100 production rate is regulated by apo E content of newly secreted TRL produced and VLDL apo B-100 residence time. Apo E expression has a beneficial effect on apo B-100 metabolism, suggesting that moderate expression might lower the LDL apo B-100 production rate and thus decrease levels of LDL cholesterol.

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Acknowledgements The authors would like to thank the staff of the Metabolic Research Unit at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University for the expert care provided to the study subjects. We also acknowledge gratefully the cooperation of the study subjects who made this work possible. Supported by training grant T32-AG000209 and grant HL-39326 from the National Institutes of Health and contract 53-3K06-5-10 from the US Department of Agriculture. The contents of this publication do not necessarily reflect views or policies of the US Department of Agriculture, nor does the mention of trade names, commercial products, or organizations imply endorsement by the US Government. References [1] Carella M, Cooper AD. High affinity binding of chylomicron remnants to rat liver plasma membranes. Proc Natl Acad Sci USA 1979;76:338– 42. [2] Hui DY, Innerarity TL, Milne RW, Marcel YL, Mahley RW. Binding of chylomicron remnants and beta-very low density lipoproteins to hepatic and extrahepatic lipoprotein receptors. A process independent of apolipoprotein B48. J Biol Chem 1984;259:15060– 8. [3] Mahley RW, Ji ZS. Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E. J Lipid Res 1999;40:1–16. [4] Mahley RW, Hui DY, Innerarity TL, Beisiegel U. Chylomicron remnant metabolism. Role of hepatic lipoprotein receptors in mediating uptake. Arteriosclerosis (Suppl) 1989;9:I14–8. [5] Bradley WA, Gianturco SH. ApoE is necessary and sufficient for the binding of large triglyceride-rich lipoproteins to the LDL receptor; apoB is unnecessary. J Lipid Res 1986;27:40–8. [6] Hasty AH, Linton MF, Swift LL, Fazio S. Determination of the lower threshold of apolipoprotein E resulting in remnant lipoprotein clearance. J Lipid Res 1999;40:1529–38. [7] Davignon J, Gregg RE, Sing CF. Apolipoprotein E polymorphism and atherosclerosis. Arteriosclerosis 1988;8:1–21. [8] Phillipps NR, Havel RJ, Kane JP. Sex related differences in the concentration of apolipoprotein E in the blood plasma and plasma lipoproteins. J Lipid Res 1983;24:1525–31. [9] Cole TG, Patsch W, Kuisk I, Gonen B, Schonfeld G. Increases in dietary cholesterol and fat raise levels of apolipoprotein E-containing lipoproteins in the plasma of man. J Clin Endocrinol Metab 1983;56:1108–15. [10] Kushwaha RS, Foster DM, Barrett PH, Carey KD, Mernard MG. Metabolic regulation of plasma apolipoprotein E by estrogen and progesterone in the baboon (Papio sp). Metabolism 1991;40:93– 100. [11] Gregg RE, Zech LA, Schaefer EJ, Brewer HB. Type III hyperlipoproteinemia: defective metabolism of an abnormal apolipoprotein E. Science 1981;211:584–6. [12] Blum CB. Dynamics of apolipoprotein E metabolism in humans. J Lipid Res 1982;23:1308–16. [13] Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Summary of the Second Report of the National Cholesterol Education Program (NCEP) Expert Panel on the Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II). J Amer Med Assoc 1993;269:3015–23.

[14] Cohn JS, Wagner DA, Cohn SD, Millar JS, Schaefer EJ. Measurement of very low density lipoprotein apolipoprotein (apo) B-100 and high density lipoprotein apo A-I production in human subjects using deuterated leucine. Effect of fasting and feeding. J Clin Invest 1990;85:804– 11. [15] Lichtenstein AH, Hachey DL, Millar JS, Jenner JL, Booth L, Ordovas J, Schaefer EJ. Measurement of human apolipoprotein B-48 and B-100 kinetics in triglyceride-rich lipoproteins using [5,5,5-2H3] leucine. J Lipid Res 1992;33:907– 14. [16] Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:1345– 53. [17] McNamara JR, Schaefer EJ. Automatic enzymatic standardized lipid analyses for plasma and lipoprotein fractions. Clin Chim Acta 1987;166:1– 8. [18] Warnick GR, Benderson J, Albers JJ. Dextran sulfate–Mg2 + precipitation procedure for the quantitation of high density lipoproteins cholesterol. Clin Chem 1982;28:1379– 88. [19] Ordovas JM, Perterson J, Santaniello P, Cohn JS, Wilson PWF, Schaefer EJ. Enzyme linked immunosorbent assay for human apolipoprotein B. J Lipid Res 1987;28:1216– 24. [20] Millar JS, Lichtenstein AH, Dolnikowski GG, Ordovas JM, Schaefer EJ. Proposal of a multicompartmental model for use in the study of apolipoprotein E metabolism. Metabolism 1998;47:922– 8. [21] Havekes LM, de Kniff P, Beisiegel U, Havinga J, Smit M, Klasen E. A rapid micromethod for apolipoprotein E phenotyping directly in serum. J Lipid Res 1987;28:455– 63. [22] Cohn JS, McNamara JR, Cohn SD, Ordovas JM, Schaefer EJ. Plasma apolipoprotein changes in the triglyceride-rich lipoprotein fraction of human subjects fed a fat-rich meal. J Lipid Res 1988;29:925– 36. [23] Cohn JS, Johnson EJ, Millar JS, Cohn SD, Milne RW, Marcel YL, Russell RM, Schaefer EJ. Contribution of apoB-48 and apoB-100 triglyceride-rich lipoproteins (TRL) to postprandial increases in the plasma concentration of TRL triglycerides and retinyl esters. J Lipid Res 1993;34:2033– 40. [24] Bjorkegren J, Karpe F, Milne RW, Hamsten A. Differences in apolipoprotein and lipid composition between human chylomicron remnants and very low density lipoproteins isolated from fasting and postprandial plasma. J Lipid Res 1998;39:1412–20. [25] Millar JS, Lichtenstein AH, Cuchel M, Dolnikowski GG, Hachey DL, Cohn JS, Schaefer EJ. Impact of age on the metabolism of VLDL, IDL and LDL apolipoprotein B-100 in men. J Lipid Res 1995;36:1155– 67. [26] Gregg RE, Zech LA, Schaefer EJ, Brewer HB. Apolipoprotein E metabolism in normolipidemic human subjects. J Lipid Res 1984;25:1167– 76. [27] Rubinstein A, Gibson JC, Ginsberg HN, Brown WV. In vitro metabolism of apolipoprotein E. Biochim Biophys Acta 1986;879:355– 61. [28] Gibson JC, Brown WV. Effect of lipoprotein lipase and hepatic triglyceride lipase activity on the distribution of apolipoprotein E among the plasma lipoproteins. Atherosclerosis 1988;73:45–55. [29] Fazio S, Yao Z, McCarthy BJ, Rall SC, Jr. Synthesis and secretion of apolipoprotein E occur independently of synthesis and secretion of apolipoprotein B-containing lipoproteins in HepG2 cells. J Biol Chem 1992;267:6941– 5. [30] Mensenkamp AR, Jong MC, van Goor H, van Luyn, Bloks V, Havinga R, Voshol PJ, Hofker MH, van Dijk KW, Havekes LM, Kuipers F. Apolipoprotein E participates in the regulation of very low density lipoprotein-triglyceride secretion by the liver. J Biol Chem 1999;274:35711– 8. [31] Schaefer JR, Scharnagl H, Baumstark MW, Schweer H, Zech LA, Seyberth H, Winkler K, Steinmetz A, Marz W. Homozygous familial defective apolipoprotein B-100. Enhanced removal of apolipoprotein E-containing VLDLs and decreased

J.S. Millar et al. / Atherosclerosis 155 (2001) 477–485

[32]

[33]

[34]

[35]

[36]

[37]

production of LDLs. Arterioscler Thromb Vasc Biol 1997;17:348– 53. Huang Y, Liu XQ, Rall SC, Jr, Taylor JM, von Eckardstein A, Assman G, Mahley RW. Overexpression and accumulation of apolipoprotein E as a cause of hypertriglyceridemia. J Biol Chem 1998;273:26388– 93. Campos E, Kakajima K, Tanaka A, Havel RJ. Properties of an apolipoprotein E-enriched fraction of triglyceride-rich lipoproteins isolated from human blood plasma with a monoclonal antibody to apolipoprotein B-100. J Lipid Res 1992;33:369– 80. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 1992;258:468–71. Schaefer EJ, Gregg RE, Ghiselli G, Forte TM, Ordovas JM, Zech LA, Brewer HB, Jr. Familial apolipoprotein E deficiency. J Clin Invest 1986;78:1206–19. Weisgraber KH, Rall SC, Jr, Mahley RW, Milne RW, Marcel YL, Sparrow JT. Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3. J Biol Chem 1986;26:2068– 76. Shachter NS, Hayek T, Leff T, Smith JD, Rosenberg DW, Walsh A, Ramakrishnan R, Goldberg IJ, Ginsberg HN, Breslow

.

[38]

[39]

[40]

[41]

485

JL. Overexpression of apolipoprotein CII causes hypertriglyceridemia in transgenic mice. J Clin Invest 1994;93:1683–90. Aalto-Setala K, Weinstock PH, Bisgaier CL, Wu L, Smith JD, Breslow JL. Further characterization of the metabolic properties of triglyceride-rich lipoproteins from human and mouse apoCIII transgenic mice. J Lipid Res 1996;37:1802– 11. Weisgraber KH, Mahley RW, Kowal RC, Herz J, Goldstein JL, Brown MS. Apolipoprotein C-I modulates the interaction of apolipoprotein E with beta-migrating very low density lipoproteins (beta-VLDL) and inhibits binding of beta-VLDL to low density lipoprotein receptor-related protein. J Biol Chem 1990;265:22453– 9. Demant T, Carlson LA, Holmquist L, Karpe F, Nilsson-Ehle P, Packard CJ, Shepherd J. Lipoprotein metabolism in hepatic lipase deficiency: studies on the turnover of apolipoprotein B and on the effect of hepatic lipase on high density lipoprotein. J Lipid Res 1988;29:1603– 11. Anber V, Millar JS, McConnell M, Shepherd J, Packard CJ. Interaction of very-low-density, intermediate-density, and lowdensity lipoproteins with human arterial wall proteoglycans. Arterioscler Thromb Vasc Biol 1997;17:2507– 14.