©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chylomicron Metabolism in Normal, Cholesterol-fed, and Watanabe Heritable Hyperlipidemic Rabbits
SATURATION OF THE SEQUESTRATION STEP OF THE REMNANT CLEARANCE PATHWAY (*)

M. Mahmood Hussain (§) , Thomas L. Innerarity (1) (2), Walter J. Brecht , Robert W. Mahley (1) (2) (3)(¶)

From the (1) Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, Departments of (2) Pathology and (3) Medicine, University of California, San Francisco, California 94110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The plasma clearance of radiolabeled chylomicrons was compared in normal, cholesterol-fed, and Watanabe heritable hyperlipidemic (WHHL) rabbits. Chylomicron clearance was rapid in normal rabbits but was significantly retarded in cholesterol-fed and WHHL rabbits. At 40 min after the injection of chylomicrons, 14-17% of the injected dose remained in the plasma of normal rabbits, whereas 40-50% of the injected dose remained in the plasma of cholesterol-fed and WHHL rabbits. The differences were reflected in the reduced plasma clearance by the liver and bone marrow of the cholesterol-fed and WHHL rabbits. The hyperlipidemic rabbits expressed normal levels of low density lipoprotein (LDL) receptor-related protein/-macroglobulin receptor in the liver. In contrast, the hepatic levels of LDL receptors were lower in hyperlipidemic rabbits; as expected, they were significantly lower in WHHL rabbits compared with normal and cholesterol-fed rabbits. Furthermore, it was demonstrated that lipoproteins accumulating in the plasma of the hyperlipidemic rabbits competed for and retarded the clearance of chylomicrons from the plasma. Competition was demonstrated by cross-circulation of normal and cholesterol-fed or normal and WHHL rabbits, in which the rapid influx of plasma containing the accumulated plasma lipoproteins from cholesterol-fed or WHHL rabbits was shown to impair the uptake of chylomicrons by the liver and bone marrow of normal rabbits. These observations were extended by infusing isolated lipoproteins into normal rabbits. The rabbit d < 1.02 g/ml (remnant) fraction and the canine cholesterol-rich high density lipoproteins (HDL) with apolipoprotein E (HDL) inhibited chylomicron clearance, whereas human LDL and HDL from humans and rabbits did not. We conclude that the low LDL receptor activity in the cholesterol-fed and WHHL rabbits may contribute, at least in part, to the impaired clearance by decreasing remnant uptake and causing the accumulation of chylomicron and/or very low density lipoprotein remnants. The accumulated remnant lipoproteins then compete for and saturate the mechanism responsible for the initial rapid clearance of chylomicrons from the plasma. We speculate that saturation of the initial rapid clearance may occur at the sequestration step, which involves the binding of remnants to heparan sulfate proteoglycans in the space of Disse.


INTRODUCTION

Chylomicrons are the major lipoproteins responsible for the transport of dietary lipids to various tissues. Lipoprotein lipase hydrolyzes triglycerides present in the core of chylomicrons, converting them to remnants. Chylomicron remnants are rapidly cleared from the plasma, and this process is mediated primarily by apolipoprotein E (apoE)() (for review, see Refs. 1-5).

It has been postulated that several steps are involved in chylomicron remnant clearance from the plasma and ultimate uptake of these lipoproteins by hepatocytes (1, 3, 5) . The initial process responsible for the normal rapid clearance of remnants from the plasma is thought to involve a sequestration of the particles in the space of Disse. This process may involve the binding of apoE on the remnants to heparan sulfate proteoglycans (HSPG) and the interaction of remnants with hepatic lipase (6, 7) . Several lines of evidence have now established that remnant lipoproteins bind initially to cell-surface HSPG and that, in the absence of HSPG, remnant binding and uptake are decreased even in the presence of a functional low density lipoprotein (LDL) receptor-related protein (LRP) (8, 9) . Presumably, remnant binding to HSPG is necessary for the LRP to mediate uptake. Further lipolytic processing in the space of Disse may occur before recognition of the lipoprotein particles by the lipoprotein receptors. It has been shown that lipoprotein lipase facilitates the binding of remnants to the LRP (10) and that hepatic lipase can stimulate the binding of remnants to HSPG and facilitate uptake of these particles presumably through the LRP (11) . Finally, uptake of the remnants via an apoE-mediated process appears to involve both the LDL receptor and the LRP (1, 2, 3, 4, 5) .

The LDL receptor has been shown in a variety of in vitro systems to mediate the uptake of apoE-containing remnant lipoproteins. Choi et al. (12) have inhibited uptake of chylomicrons using antibodies that block lipoprotein uptake by LDL receptors. Recently, several lines of evidence have demonstrated that the LRP also can be involved in chylomicron remnant clearance (13, 14, 15, 16, 17, 18, 19, 20) . The LRP has been shown to bind remnants and remnant-like lipoproteins containing excess added apoE. The space of Disse is an environment rich in this apolipoprotein (21) . Thus, remnants could acquire excess apoE and then be recognized by the LRP. Physiological evidence that the LRP is involved in chylomicron remnant clearance in vivo was suggested by the partial inhibition of the clearance of radioactive chylomicrons from the plasma of mice that had been injected with activated -macroglobulin (16, 19) . Although both the LDL receptor and the LRP have been implicated in the uptake of chylomicron remnants, it has been difficult to determine which, if either, plays the dominant role in this process.

The present studies of chylomicron metabolism were conducted in rabbits, since this species has been used extensively in lipoprotein studies. It was previously shown that the liver is responsible for about two-thirds of the uptake of intravenously injected chylomicrons and that the bone marrow also takes up significant amounts (22, 23) . Furthermore, chylomicron catabolism by the liver increased in rabbits when the chylomicrons were incubated with apoE (22) . In addition, plasma cholesterol levels decreased when apoE was infused into cholesterol-fed and Watanabe heritable hyperlipidemic (WHHL) rabbits (24) .

The hyperlipidemia of cholesterol-fed rabbits is quite different from that of WHHL rabbits. High cholesterol diets cause a marked accumulation of -migrating very low density lipoproteins (-VLDL), which consist of VLDL remnants and chylomicron remnants (25, 26) , and a down-regulation of LDL receptors (27, 28, 29, 30, 31) . The hyperlipidemia induced by dietary cholesterol is due to overproduction of lipoproteins and impaired plasma clearance secondary to the down-regulation of LDL receptor expression (27, 32, 33, 34) . In contrast, a genetic deficiency of LDL receptors in WHHL rabbits causes the accumulation of LDL and lesser amounts of VLDL and intermediate density lipoprotein remnant particles (for review, see Refs. 35 and 36).

There are conflicting reports concerning the effect of cholesterol-induced hyperlipidemia on chylomicron catabolism in different species. Chylomicron clearance has been shown to be retarded in cholesterol-fed rabbits and hypothyroid rats (32, 33, 37, 38, 39) but not in cholesterol-fed rats (32) and dogs (40, 41) . It has been reported that chylomicron clearance was normal in WHHL rabbits (42, 43) . However, other studies suggest that chylomicron clearance is retarded in these rabbits (44, 45) .

The present study employs the novel technique of cross-circulation of plasma lipoproteins either between normal and WHHL rabbits or normal and cholesterol-fed rabbits to examine the steps involved in the remnant pathway and to understand the mechanism involved in the development of hyperlipidemia in these rabbits. Cross-circulation eliminates the potential problem of denaturing the fragile chylomicrons and their subsequent uptake by liver Kupffer cells (46) . We demonstrate that chylomicron clearance is impaired in WHHL rabbits and in cholesterol-fed rabbits. In addition, using this technique, we demonstrate that lipoproteins present in the plasma of hyperlipidemic rabbits impair chylomicron clearance. The impairment was due, in part, to competition with endogenously derived lipoproteins containing apoE. We postulate that these lipoproteins inhibit the initial sequestration step in remnant clearance, i.e. the binding of chylomicron remnants to hepatic HSPG.


MATERIALS AND METHODS

Animals and Diets

Male New Zealand White rabbits (Animal West, Soquel, CA or Nitabell, Hayward, CA) were maintained on a normal chow diet (Purina Mills, St. Louis, MO). The plasma cholesterol levels in these rabbits ranged between 30 and 80 mg/dl. Rabbits kept on a 0.5% cholesterol diet (Zeigler Brothers, Gardners, PA) for 2-3 weeks prior to use (24, 47) showed hypercholesterolemia that ranged between 220 and 830 mg/dl. The homozygous WHHL rabbits used in the study were maintained on a normal chow diet (Purina Mills) and had cholesterol levels between 200 and 450 mg/dl. The weights of normal, cholesterol-fed, and WHHL rabbits ranged from 2 to 3.5 kg. All animals were fasted overnight.

Preparation of Lipoproteins

[H]Retinol- and [C]cholesterol-labeled chylomicrons were obtained from the thoracic duct of fat-fed dogs (22, 23) , and all lipoproteins used for infusion were isolated by ultracentrifugation as described (48) . The cholesterol-rich high density lipoproteins with apoE (HDL) from cholesterol-fed dogs were isolated by Pevikon-block electrophoresis (49) .

Iodination of 9D9 and LRP-515 Immunoglobulins

Monoclonal antibodies 9D9 and LRP-515 recognize the rabbit LDL receptor and the LRP, respectively, and have been shown to be useful in determining the activity of cell-surface receptors expressed in vivo (18, 50) . The 9D9 and LRP-515 immunoglobulins were purified from ascites fluid using protein A-Sepharose (Pharmacia Biotech, Inc.) column chromatography. Purified 9D9 immunoglobulin or LRP-515 (1 mg of protein) was radiolabeled using Iodobeads (Pierce) with I (0.5 mCi) in 1 ml of phosphate-buffered saline, and the radiolabeling was terminated by the addition of KI to a final concentration of 0.01 M. The I-labeled proteins were separated from the free I by Sephadex G10 (Pharmacia Biotech, Inc.) column chromatography. The specific activity of the radiolabeled monoclonal antibodies ranged from 20 to 731 cpm/ng of protein.

Plasma Clearance and Tissue Uptake of Radiolabeled Chylomicrons and I-Labeled 9D9 in Normal, Cholesterol-fed, and WHHL Rabbits

These studies were performed using one of two experimental protocols that serve as controls for the cross-circulation and lipoprotein infusion studies described below. The anesthetized rabbits were either infused with saline over a period of 5-8 min or subjected to an arterial-venous (A-V) shunt for 20 min. Chylomicrons and/or 9D9 were injected 20 min after the start of either of these procedures. Blood samples were obtained at designated intervals for an additional 40 min, at which time the animals were euthanized and tissue samples obtained. There was no significant difference between the results obtained by either protocol; therefore, the results were combined (Figs. 2, 3, and 5).

For chylomicron clearance studies, the [H]retinol- and [C]cholesterol-labeled chylomicrons were injected into the animals at a dose of 75 mg of triglyceride/kg of body weight. Blood samples and liver biopsies were obtained at designated intervals. The rabbits were euthanized at 40 min by intravenous injection of euthanasia solution (Anthony Products Co., Arcadia, CA, 200 mg of sodium pentobarbital/kg of body weight) followed by bilateral thoracotomy or perfusion fixation (22, 23) . Tissues (liver, bone marrow, spleen, kidney, adrenals, heart, lung, and perinephric fat) were obtained. To determine receptor levels, monoclonal antibodies were injected into the three groups of rabbits at a dose of 25 µg of protein/kg of body weight, and experiments were performed as described above for chylomicrons.

Plasma Clearance and Tissue Uptake in Cross-circulated Rabbits

To perform cross-circulation between two rabbits, the femoral artery and vein were exposed and catheterized (16 gauge Medicut, Sherwood Medical, Tullamore, Ireland). As shown in Fig. 1, the femoral artery from one rabbit was connected to the femoral vein of a second rabbit using silastic tubing (⅛" internal diameter, Dow Corning Corp., Midland, MI). Reciprocally, the femoral artery of the second rabbit was connected to the femoral vein of the first rabbit. The silastic tubing was pretreated with heparin (5% tridodecylmethylammonium chloride heparin complex, Polysciences, Inc., Warrington, PA). To determine plasma lipid levels, blood samples (1.0 ml) were collected at 5-min intervals from stopcocks in the arterial port. At 20 min after initiation of the cross-circulation, each rabbit was simultaneously injected with radiolabeled lipoproteins through the stopcocks into the femoral vein, and metabolic studies were performed as described above. As a control for the cross-circulation studies, normal, cholesterol-fed, and WHHL rabbits were subjected to A-V shunts. The catheterized artery and vein of the rabbit were connected by silastic tubing.

Plasma Clearance and Tissue Uptake of Lipoproteins in Infusion Studies

Lipoproteins (1-2 ml/kg of body weight) were infused into rabbits through the ear vein over a period of 5-8 min. At 20 min after the start of the infusion, radiolabeled chylomicrons were injected into these animals. Blood samples were obtained from the vernicular artery of the contralateral ear. The experiments were terminated 40 min after the injection of chylomicrons as described above. As a control for lipoprotein infusion, normal rabbits were infused with saline.

The amount of isolated lipoproteins to be injected was calculated to elevate the plasma cholesterol levels of the normal rabbits from a base line of 30-80 mg/dl to 200 mg/dl. This value was chosen because levels in this range were typically obtained in the cross-circulated rabbits. The plasma cholesterol levels were determined just before the injection of the chylomicrons and 20 min after the beginning of the lipoprotein infusion. It was possible in most cases to obtain these levels using the d <1.21 and d <1.02 g/ml fractions and LDL. However, much lower levels were obtained with HDL. Plasma Clearance of Chylomicrons from the d < 1.006 g/mlFraction-To determine the plasma clearance of chylomicrons from the d <1.006 g/ml fraction, plasma samples (0.2-0.5 ml) were overlaid with 0.5-0.8 ml of saline and centrifuged (Beckman tabletop ultracentrifuge, TLA100.2 rotor, 100,000 rpm, 2.5 h, 4 °C), and the top 200 µl and bottom fractions were removed and counted. The density distribution of the radiolabeled chylomicrons mixed with plasma (0.2 ml) was determined by centrifugation in parallel with samples obtained from the rabbits.

Effect of ApoE on Chylomicron Catabolism in Cholesterol-fed Rabbits and in Cross-circulated Pairs of Rabbits

To study the effect of apoE on chylomicrons, chylomicrons (75 mg of triglyceride/kg) were incubated with apoE (10-12 mg of protein/kg) for 1 h at 37 °C and injected into the rabbits for metabolic studies. The apoE used in these studies was either human recombinant apoE or rabbit apoE purified from cholesterol-fed rabbits.

Analysis of Plasma and Tissues

To determine the amount of radioactivity present in plasma, blood samples (3.0 ml) were collected as described (22, 23) . In those cases where I-labeled 9D9 was injected with the radiolabeled chylomicrons, 0.5 ml of plasma was mixed with 0.5 ml of 100% ethanol, vortexed, and counted in a counter for 5-10 min. After counting, [H]retinol and [C]cholesterol were extracted twice into hexane, which was evaporated, and the sample was counted in the presence of 0.5 ml of 100% ethanol and 10 ml of Beckman scintillation mixture. In those cases where only radiolabeled chylomicrons were injected, the samples were subjected to direct liquid scintillation counting (22, 23) .

To determine the tissue uptake of the monoclonal antibody I-9D9 and radiolabeled chylomicrons, samples of different tissues were obtained in duplicate, weighed, and counted for I in a counter. The tissues were then digested with 0.5 ml of 6 N KOH, and lipids were extracted into ethanol and hexane as described earlier (22, 23) . In those cases where chylomicrons were injected alone, the tissues were digested and extracted as described (22, 23) .

Determination of Plasma Volume and Tissue Weights

To determine plasma volume (51) , rabbit albumin (fraction V, Sigma) was iodinated using Iodobeads as described earlier for monoclonal antibodies. The I-labeled rabbit albumin was injected into the ear vein of unanesthetized normal, cholesterol-fed, and WHHL rabbits. Blood samples (2.0 ml each) were collected at designated time points for 1 h. Plasma volume was deduced by extrapolating the albumin die-away curve to 0 min. Determination of plasma volume in normal and cholesterol-fed rabbits revealed no differences in plasma volume (3.5 ± 0.4% ( n = 11) and 3.6 ± 0.3% ( n = 13) of body weight, respectively). In contrast, WHHL rabbits had significantly lower ( p < 0.001) plasma volumes, i.e. 2.7 ± 0.1% of body weight ( n = 7). To determine tissue weights, tissues were rinsed with ice-cold saline, blot dried, and weighed. Results were expressed as percent of body weight. The weight of the liver of cholesterol-fed rabbits (3.3 ± 0.5%, n = 8) was significantly greater ( p < 0.05) than the weight of the liver of normal (2.9 ± 0.3%, n = 18) and WHHL (2.5 ± 0.3%, n = 3) rabbits. Other tissues had similar weights when expressed as a percent of body weight. No significant differences were observed between the weights of different tissues in normal and WHHL rabbits.

Other Analyses

Lipid (total cholesterol, triglyceride, free cholesterol, phospholipid) analyses were performed using a Spectrum lipid analyzer (Abbott Laboratories, North Chicago, IL). Protein concentrations were determined by the method of Lowry et al. (52) . All the data are presented as mean ± S.D. Data were evaluated by unpaired Student's t test using an IBM PC program called ``Primer'' (written by Dr. Stanton Glantz, University of California, San Francisco) (McGraw-Hill, Inc., New York, NY).


RESULTS

Chylomicron Metabolism in Normal, Cholesterol-fed, and WHHL Rabbits

Previously, we demonstrated that when [H]retinol- and [C]cholesterol-labeled chylomicrons were injected intravenously into normal rabbits, the chylomicrons were cleared from the plasma very rapidly, mainly by the liver (22, 23) . In the present study, at 40 min, 14% of [H]retinol- and 17% of [C]cholesterol-labeled chylomicrons remained in the plasma of normal rabbits, i.e. 85% of the chylomicrons were removed from the plasma (Fig. 2, A and C). In contrast, chylomicron clearance in cholesterol-fed and WHHL rabbits was significantly slower. At 40 min, 40-50% of the radiolabeled chylomicrons remained in the plasma of the WHHL and cholesterol-fed rabbits. Analysis of chylomicron uptake by the liver, during the course of in vivo studies, showed that the livers of normal rabbits accumulated greater amounts of chylomicrons compared with the livers of cholesterol-fed and WHHL rabbits (Figs. 2, B and D, and 3). For example, the liver of the normal rabbit took up 44% of the injected dose of [C]cholesterol-labeled chylomicrons, whereas the livers of cholesterol-fed and WHHL rabbits took up 21 and 22% of the [C]cholesterol, respectively (Fig. 2 D).


Figure 2: Chylomicron metabolism in normal, cholesterol-fed, and WHHL rabbits. The metabolism studies were performed as described under ``Materials and Methods.'' The [H]retinol- and [C]cholesterol-labeled chylomicrons (75 mg of triglyceride/kg) were injected into the ear or femoral vein. Blood samples and liver biopsies were obtained at designated times after the injection of chylomicrons. At 40 min after the injection of chylomicrons, rabbits were euthanized, and plasma and tissue samples were analyzed (see ``Materials and Methods''). Panels A and C show the plasma clearance of [H]retinol- and [C]cholesterol-labeled chylomicrons in normal, cholesterol-fed, and WHHL rabbits. Plasma clearance of chylomicrons in cholesterol-fed and WHHL rabbits was compared with plasma clearance in normal rabbits and evaluated using the Student's t test. The plasma clearance of chylomicrons was significantly slower ( p < 0.001) in the cholesterol-fed and WHHL rabbits at 30 and 40 min compared with normal rabbits. Panels B and D show uptake by the liver of [H]retinol- and [C]cholesterol-labeled chylomicrons in normal, cholesterol-fed, and WHHL rabbits.



Uptake of the [C]cholesterol-labeled chylomicrons by various tissues at 40 min is summarized in Fig. 3. A significant decrease in chylomicron uptake is seen in both the liver and bone marrow of cholesterol-fed and WHHL rabbits. Bone marrow uptake was decreased from 24% of the injected dose in normal rabbits to 10-11% in cholesterol-fed and WHHL rabbits. Other tissues studied, i.e. spleen, kidney, lung, adrenal, and heart, each contained less than 3% of the injected chylomicrons. These results indicate that chylomicron clearance from the plasma of cholesterol-fed and WHHL rabbits was significantly slower, as both the liver and bone marrow uptake was decreased by 50%.


Figure 3: Chylomicron uptake by tissues of normal, cholesterol-fed, and WHHL rabbits. Chylomicron metabolism studies were performed in normal, cholesterol-fed, and WHHL rabbits as described under ``Materials and Methods'' and Fig. 2. Rabbits were euthanized at 40 min, and tissues were collected. Small portions (0.1-0.2 g) of each tissue were digested, extracted, and counted as described under ``Materials and Methods.'' Differences in the uptake of [C]cholesterol-labeled chylomicrons were evaluated by Student's t test. The uptake by cholesterol-fed ( p < 0.001) and WHHL ( p < 0.05) rabbit liver and by cholesterol-fed ( p < 0.001) and WHHL ( p < 0.05) rabbit bone marrow were significantly lower, as compared with uptake in the normal rabbit.



The clearance of the [C]cholesterol and [H]retinol from the plasma (Fig. 2) was compared with the clearance of radiolabeled chylomicrons specifically from the d <1.006 g/ml ultracentrifugal fraction in normal, cholesterol-fed, and WHHL rabbits (Fig. 4). As contrasted with the normal rabbits, clearance of the radiolabeled chylomicrons from the d <1.006 g/ml fraction was retarded in the cholesterol-fed and WHHL rabbits. A comparison of Figs. 2 and 4 suggests that at each time point some of the [C]cholesterol and [H]retinol occurred in the d > 1.006 g/ml fraction. Nevertheless, the clearance of radiolabeled chylomicrons from whole plasma was followed in subsequent studies since it is impossible to ascertain whether the label in the d > 1.006 g/ml fraction represents metabolic conversion or exchange or an artifact of ultracentrifugation.

Effect of ApoE on Chylomicron Clearance in Cholesterol-fed Rabbits

The possibility existed that the amount of apoE associated with the chylomicrons could be rate limiting for clearance in the cholesterol-fed rabbits. Chylomicrons were preincubated with saline or apoE for 1 h at 37 °C as described under ``Materials and Methods'' and injected into cholesterol-fed rabbits. Previously, it was demonstrated that apoE enrichment resulted in enhanced initial rates of clearance in normal rabbits (16, 22) . In three cholesterol-fed rabbits injected with saline-incubated chylomicrons, 46-47% of the injected dose remained in the plasma, whereas in two cholesterol-fed rabbits injected with apoE-enriched chylomicrons, 40 and 60% of the injected dose remained in the plasma at 40 min. The liver and bone marrow of the three rabbits injected with chylomicrons alone took up 15-19% (17 ± 2) and 15-18% (17 ± 2) of the injected dose, respectively. The percentages of injected apoE-enriched chylomicrons recovered from the liver (17 and 20%) and bone marrow (15 and 18%) of two rabbits were similar to the values obtained in rabbits injected with chylomicrons without added apoE. These studies demonstrated that added apoE did not increase chylomicron clearance in cholesterol-fed rabbits, and thus the amount of apoE on the chylomicrons does not appear to be rate limiting.

LDL Receptor and LRP Expression in Normal, Cholesterol-fed, and WHHL Rabbits

The levels of LDL receptor and LRP expressed in these rabbits were investigated using monoclonal antibodies against these receptors. The expression of LDL receptors was studied using the monoclonal antibody 9D9, as described by Huettinger et al. (50) . During in vivo studies, I-labeled 9D9 immunoglobulin was cleared more rapidly from the plasma of normal rabbits compared with cholesterol-fed and WHHL rabbits (Fig. 5 A). At 40 min, about 49% of injected I-labeled 9D9 remained in the plasma of normal rabbits, i.e. 51% had been cleared. In contrast, about 62 and 74% of the injected dose of the radiolabeled 9D9 remained in the plasma of cholesterol-fed and WHHL rabbits, respectively. Analysis of 9D9 uptake by the liver during the in vivo studies demonstrated that, compared with the livers of WHHL rabbits, the livers of normal rabbits accumulated greater amounts of the antibodies (Fig. 5 B). Cholesterol-fed rabbits also demonstrated significantly lower amounts of I-labeled 9D9 in the liver than did the normal rabbits, but their livers still expressed higher levels of functional LDL receptors than those of the WHHL rabbits. At 40 min, 37% of the injected dose was present in the livers of normal rabbits, whereas the livers of cholesterol-fed rabbits contained 27% of the injected dose ( p < 0.02). In contrast, the livers of WHHL rabbits contained 10% of the injected dose, and this value was significantly ( p < 0.005) lower than the values seen in the livers of normal and cholesterol-fed rabbits. Other tissues (bone marrow, spleen, kidney, lung, adrenals, and heart) took up less than 5% of the injected antibodies (data not shown).


Figure 5:I-Labeled 9D9 and LRP-515 monoclonal antibody metabolism in normal, cholesterol-fed, and WHHL rabbits. I-Labeled monoclonal antibodies (25 µg/kg) were injected into animals with or without chylomicrons (as described in Fig. 2 and under ``Materials and Methods''). Blood samples and liver biopsies were obtained at designated time points. At 40 min after the injection of chylomicrons, rabbits were euthanized, and plasma and tissue samples were analyzed (see ``Materials and Methods''). Panel A shows the plasma clearance of I-labeled 9D9 in normal, cholesterol-fed, and WHHL rabbits. Plasma clearance of 9D9 in these rabbits was evaluated using the Student's t test. The plasma clearance of 9D9 was significantly slower in the cholesterol-fed and WHHL rabbits at 2 min ( p < 0.05) and at 10-40 min ( p < 0.001) as compared with normal rabbits. Moreover, the plasma clearance of 9D9 was significantly slower in WHHL rabbits at 30 min ( p < 0.05) and 40 min ( p < 0.005) as compared with cholesterol-fed rabbits. Panel B shows the uptake of 9D9 by the liver in normal, cholesterol-fed, and WHHL rabbits. The uptake of 9D9 by cholesterol-fed ( p < 0.01) and WHHL ( p < 0.001) rabbit liver was significantly lower than the uptake by the normal rabbit liver. Moreover, the uptake of 9D9 by the liver in WHHL rabbits was significantly lower ( p < 0.02) than the uptake by the liver in cholesterol-fed rabbits. Panels C and D show the plasma clearance and liver uptake of I-labeled LRP-515 in normal, cholesterol-fed, and WHHL rabbits.



The expression of the LRP was studied using monoclonal antibody LRP-515 as described by Herz et al. (18) . The I-LRP-515 was cleared rapidly from the plasma of normal, cholesterol-fed, and WHHL rabbits (Fig. 5 C). No differences in the rate of clearance of this antibody were observed. The clearance from the plasma was due primarily to uptake by the liver (Fig. 5 D). The uptake of LRP-515 by the livers of normal, cholesterol-fed, and WHHL rabbits was similar. Other tissues took up less than 5% of the injected antibodies. In all animals, the rate of uptake increased for the first 20 min, and thereafter the amount of radioactivity decreased in the liver. These studies indicate that the amount of LRP expressed in these animals is similar.

These studies demonstrated that cholesterol-fed rabbits expressed lower amounts of LDL receptor activity than did normal rabbits and that WHHL rabbits expressed significantly lower amounts of LDL receptor activity compared with normal and cholesterol-fed rabbits. Even though cholesterol-fed and WHHL rabbits expressed different amounts of LDL receptor activity, they catabolized the radiolabeled chylomicrons to a similar extent, i.e. 50% of the injected dose. This finding suggests that the reduced chylomicron catabolism in these rabbits could only be explained in part by a decreased expression of LDL receptors.

Inhibition of Chylomicron Uptake by Accumulated Plasma Lipoproteins

Consideration was given to the possibility that the decreased chylomicron clearance in cholesterol-fed and WHHL rabbits might be due to the accumulation of lipoproteins that compete with chylomicrons for the uptake process. Two protocols were developed to test for competing lipoproteins in these rabbits that might impair or saturate the removal process. In the first protocol, normal and cholesterol-fed or normal and WHHL rabbits were cross-circulated as a means of rapidly introducing a large amount of accumulated lipoproteins into the circulation of the normal rabbit and to reduce the lipoprotein concentrations in the circulation of the cholesterol-fed and WHHL rabbits. In the second protocol, lipoproteins were isolated from cholesterol-fed and WHHL rabbits and infused into normal rabbits to study the effect of these lipoproteins on chylomicron catabolism.

Cross-circulation of the Blood of Normal and Cholesterol-fed or Normal and WHHL Rabbits

A normal and a cholesterol-fed rabbit or a normal and a WHHL rabbit were cross-circulated by connecting the pair via an arterial-venous anastomosis (Fig. 1). This A-V shunt allowed the rapid infusion of the cholesterol-induced or WHHL plasma lipoproteins into the circulation of the normal rabbit and created a new circulating level of plasma lipoproteins in common between the two rabbits. As shown in Fig. 6, the plasma cholesterol and triglyceride levels equilibrated in the cross-circulated rabbits in approximately 10 min and remained similar in both rabbits. At 20 min after initiating cross-circulation, radiolabeled chylomicrons were injected simultaneously into each rabbit, and their plasma clearance and tissue uptake determined over a period of 40 min. The blood in these rabbits was cross-circulated throughout the experiment.


Figure 1: Schematic diagram of cross-circulation of blood between two rabbits. Two rabbits were anesthetized; then, the femoral vein and artery of each were exposed, and an arterial-venous shunt between these rabbits was established as described under ``Materials and Methods.'' The blood in both rabbits was allowed to circulate for 20 min. Blood samples were obtained 5, 10, 15, and 20 min after the start of cross-circulation. At 20 min, radiolabeled chylomicrons with or without I-labeled 9D9 were injected into the venous circulation of both rabbits simultaneously. Blood samples and liver biopsies were obtained at designated times to study the metabolism of chylomicrons and 9D9.



Chylomicron Metabolism in Cross-circulated Normal and Cholesterol-fed Rabbits

Data from a single pair of rabbits are shown in Fig. 7, and all the data for all rabbit pairs are tabulated in Table I. At 20 min after the start of cross-circulation, chylomicrons were injected into the rabbits. At 40 min, 41% of [C]cholesterol-labeled chylomicrons remained in the plasma of the cross-circulated pair, i.e. only 59% of the injected dose was cleared (Fig. 7 A). The clearance was significantly slower than that observed in normal rabbits (compare with Fig. 2C) but was similar to that observed in cholesterol-fed rabbits (compare with Fig. 2 C). As shown in , the uptake by the normal liver of all cross-circulated pairs was less than that observed for the liver of the A-V shunt control rabbits and was similar to that observed in cholesterol-fed rabbits subjected to A-V shunt. This finding suggested that the cholesterol-induced lipoproteins infused into the normal rabbit (by cross-circulation) competed for the uptake of chylomicrons by the normal liver and that the system was readily saturated by competing diet-induced lipoproteins. Similarly, the cholesterol-induced lipoproteins competed with the uptake of chylomicrons by the bone marrow in the normal cross-circulated rabbit. Thus, the competing lipoproteins reduced chylomicron catabolism by 34-66% in normal rabbits (). In contrast, the decrease of the plasma cholesterol and competing lipoproteins in the cholesterol-fed rabbit after cross-circulation was not sufficient to allow for significantly increased chylomicron catabolism by the liver of the cholesterol-fed rabbit.

Chylomicron Metabolism in Cross-circulated Normal and WHHL Rabbits

Data from a single pair of rabbits are shown in Fig. 8, and all the data for all rabbit pairs are tabulated in . The plasma clearance of [C]cholesterol-labeled chylomicrons was slow in the cross-circulating pair (Fig. 8 A); At 40 min, 58% of the chylomicrons remained in the plasma, i.e. only 42% of the chylomicrons were cleared. The slow clearance of chylomicrons was due to less uptake of chylomicrons by the liver and bone marrow of the normal cross-circulated rabbit, i.e. the normal animal took up approximately 27% of the injected dose () as compared with approximately 50% in the normal A-V shunt control (). The uptake by the liver of the WHHL rabbit (cross-circulated) was unchanged compared to that of the WHHL control. As summarized in , the introduction of the WHHL lipoproteins into the normal rabbit by cross-circulation decreased the liver clearance of the radiolabeled chylomicrons. Uptake by the bone marrow may or may not be affected in the cross-circulated rabbits. Thus, these studies suggest that lipoproteins in the plasma of WHHL rabbits compete with the uptake of chylomicrons by the liver of normal rabbits. These lipoproteins reduce the uptake of chylomicrons by the liver of normal rabbits 30-46% as compared with the rabbits receiving saline infusion. As a result of cross-circulation, there was a 50% reduction in the levels of circulating, competing lipoproteins. This did not increase chylomicron uptake by the livers of WHHL rabbits.


Figure 8: Chylomicron metabolism in cross-circulated normal and WHHL rabbits. Normal and WHHL rabbits were cross-circulated for 20 min as described in Fig. 1 and under ``Materials and Methods.'' The plasma cholesterol values equilibrated at 230 mg/dl. At 20 min, chylomicrons (75 mg of triglyceride/kg of body weight) were injected into both rabbits. Plasma samples were obtained at the designated times. After euthanasia, both rabbits were perfusion-fixed, and tissues were obtained for analysis. Panel A, plasma clearance of radiolabeled chylomicrons; panel B, tissue uptake of [C]cholesterol-labeled chylomicrons.



Identification and Characterization of Lipoproteins That Compete with Chylomicron Clearance

The cross-circulation studies demonstrated that there were, in fact, competing lipoproteins in the plasma of cholesterol-fed and WHHL rabbits. To identify the fraction of lipoproteins that competed with the liver uptake of chylomicrons and to study the extent of their inhibition, lipoproteins isolated from the plasma of cholesterol-fed and WHHL rabbits, cholesterol-fed dogs, and humans were used. Table II presents data comparing the uptake of chylomicrons by the liver and bone marrow following the infusion of the various classes of lipoproteins. Infusions of d <l.21 g/ml lipoproteins from cholesterol-fed and WHHL rabbits significantly retarded (33-45% inhibition) the uptake of chylomicrons by the liver (). For example, the liver of the normal rabbit infused with saline took up 45% of the injected dose of [C]cholesterol-labeled chylomicrons as compared with an uptake of 25 and 30% by the livers of normal rabbits infused with d <1.21 g/ml lipoproteins from cholesterol-fed or WHHL rabbits, respectively. This inhibition of chylomicron uptake by the livers of normal rabbits could be attributed to the lipoproteins of d <1.02 g/ml, which are primarily VLDL and chylomicron remnants (). The d <1.006 g/ml lipoproteins from cholesterol-fed rabbits and dogs and d <1.02 g/ml lipoproteins from cholesterol-fed rabbits decreased the liver uptake of chylomicrons to approximately 20-30% of the injected dose compared with 45% in the control animals. In addition, the HDL, which are apoE-containing lipoproteins from cholesterol-fed dogs (49) , competed with the uptake of chylomicrons by the liver (). Previously, it was shown that canine HDLwas a competitive inhibitor of chylomicron remnants for uptake by the perfused rat liver (53) , and in addition to their ability to bind with high affinity to the LDL receptor (1, 49) , HDLhave now been demonstrated to bind to cell-surface HSPG in cell-culture studies (8, 9) . These data suggest that lipoprotein fractions that are enriched in VLDL and chylomicron remnants (and apoE-containing HDL) compete for chylomicron catabolism. However, the uptake of chylomicrons by the bone marrow was not significantly inhibited by the infused lipoproteins. A protein necessary for the recognition of lipoproteins by the bone marrow may have been lost during the isolation of individual lipoproteins.

In contrast, LDL from cholesterol-fed rabbits ( d = 1.02-1.063 g/ml) and from humans ( d = 1.02-1.05 g/ml) did not significantly inhibit the uptake of chylomicrons by the liver. Similarly, human and cholesterol-fed rabbit HDL do not appear to compete with chylomicrons for plasma clearance (), suggesting that the competition was specific for the apoE-enriched HDLand d <1.02 g/ml lipoproteins present in the plasma of the hyperlipidemic rabbits. Therefore, the results obtained both for cross-circulated pairs or lipoprotein-infused rabbits suggest that there are competing lipoproteins in the plasma of hyperlipidemic rabbits and that these lipoproteins rapidly saturate and inhibit remnant clearance from the plasma.


DISCUSSION

The ease of induction of hypercholesterolemia in rabbits by dietary fat and cholesterol suggests that the remnant lipoprotein clearance pathway in this species may be very sensitive to elevated lipoprotein levels. Previously, we demonstrated that the level of apoE may be rate limiting under certain conditions in the rabbit (24) , and clearly apoE is a critical determinant of normal remnant clearance (1, 2, 3) . In addition, hepatic lipase has been shown to be involved in remnant lipoprotein binding to cells in culture (11) and in plasma clearance (6, 7) , and it has been established that the rabbit is deficient in this enzyme (54, 55) . Recently, Fan et al. (56, 57) overexpressed human hepatic lipase in transgenic rabbits and demonstrated a reduced plasma cholesterol level in these rabbits on a normal diet and a reduced tendency to develop diet-induced hypercholesterolemia. Thus, impaired clearance of remnants may reflect abnormalities at one of several points in the pathway, including sequestration in the space of Disse, further lipolytic processing, or receptor-mediated uptake. Other species, such as rats (32) , dogs (40, 41) , and humans (58) , may have remnant pathways that are less sensitive to dietary perturbations, and care must be exercised in trying to extend results obtained in rabbits to other species, including humans.

The studies reported in this paper demonstrate that chylomicron clearance is retarded in both cholesterol-fed and WHHL rabbits. Experiments were performed to define the mechanism(s) responsible for the impaired clearance of remnant lipoproteins. Several possibilities to explain the impaired clearance of chylomicrons were considered.

It was demonstrated that the retarded clearance did not appear to be due primarily to the exchange or transfer of the [C]cholesterol or [H]retinol from the chylomicrons to higher density lipoproteins that are cleared more slowly. Although there was a difference in the die away of the radiolabeled chylomicrons when the whole plasma clearance was compared with that of the d < 1.006 g/ml fraction, the interpretations were similar after the initial time points. It is appreciated that exchange or transfer of these radiolabeled moieties occurs (42, 43) ; however, whole plasma clearance of chylomicrons is considered an appropriate measure of the metabolic activity of these lipoproteins since ultracentrifugal manipulation and time delays necessitated by lipoprotein fractionation can introduce artifacts. Clearly, the whole plasma die away reflected the trends seen with the d < 1.006 g/ml fraction.

The level of apoE on the chylomicrons did not appear to be rate limiting. The addition of excess apoE to the chylomicrons did not result in an accelerated clearance of these lipoproteins from the plasma of the hyperlipidemic animals. As will be discussed later, it appears that the apoE-mediated clearance process is saturated in the hyperlipidemic animals.

The retarded clearance in hyperlipidemic rabbits was not correlated with the expression of hepatic receptors. No significant difference was observed among the rabbits in the level of LRP activity. However, the level of LDL receptor expression differed among the rabbits. The cholesterol-fed rabbits had levels of LDL receptor activity that were lower than those in normal rabbits, but they expressed considerably more LDL receptor activity than was found in the WHHL rabbits. It is reasonable to conclude that the reduced expression of LDL receptors in hyperlipidemic rabbits could result in a reduced capacity of the hepatocytes to take up the remnant lipoproteins. However, since the cholesterol-fed and WHHL rabbits had similar impaired chylomicron clearance but very different levels of LDL receptor activity, the reduced expression of hepatic LDL receptors did not appear to be the primary rate-limiting factor in the reduced clearance of chylomicron remnants in these rabbits.

Consistent with data from the present study is the conclusion that lipoproteins that accumulate in the plasma of cholesterol-fed and WHHL rabbits retard remnant clearance by interfering with an initial rapid phase of clearance. Cross-circulation of normal and cholesterol-fed or normal and WHHL rabbits allowed the rapid infusion of cholesterol-induced or WHHL plasma lipoproteins into the circulation of normal rabbits and allowed the determination of the acute effects of plasma lipoproteins on liver uptake. This obviated criticism of the effects of isolation on lipoprotein composition and function. Furthermore, these studies were completed within 1 h, a short time interval during which major changes in LDL receptor activity would not be expected to play a major role. The rapid influx of the hyperlipidemic plasma during cross-circulation from cholesterol-fed and WHHL rabbits retarded the liver uptake of chylomicrons in normal rabbits. We interpret these results as indicating that the competing lipoproteins saturate the early steps involved in the clearance of chylomicrons ( i.e. sequestration in the space of Disse). It is reasonable to speculate that the apoE-containing remnants rapidly interact with the HSPG in the space of Disse and saturate the initial step in the remnant clearance pathway. We have established that HSPG-remnant interaction is essential for the binding of remnant lipoproteins and facilitates the actual uptake of the lipoproteins, presumably in association with the LRP (8, 9) . This process may be facilitated by the enrichment of the particles with apoE (8, 9) or by the interaction of the remnants with hepatic lipase (6, 7, 11) in the space of Disse.

Canine HDLand remnant lipoproteins competed for chylomicron clearance. However, when comparing the effects of different lipoproteins, it is difficult to know how to calculate the amount to be infused. The ideal approach would be to infuse similar numbers of particles, but this is impossible when comparing very heterogeneous lipoprotein classes such as VLDL or remnants. Therefore, we decided to inject sufficient d <1.21 g/ml, d <1.02 g/ml, and LDL to elevate the plasma cholesterol from a normal level of 30-50 mg/dl to 200 mg/dl. The protocol was designed to mimic the cross-circulation studies ( i.e. infusion of the isolated lipoproteins, a 12-15-min period of equilibration, and completion of the study in 1 h). As shown, the d <1.02 g/ml lipoproteins (remnants and intermediate density lipoproteins) and cholesterol-induced canine lipoproteins containing primarily apoE (HDL) inhibited chylomicron clearance (). These in vivo data confirm previous results obtained in the perfused rat liver, demonstrating that HDLinhibit chylomicron remnant clearance (53) .

In contrast to the inhibition seen with remnant lipoproteins and HDL, LDL did not appear to inhibit liver uptake. Likewise, HDL did not appear to inhibit chylomicron uptake. However, since HDL are less cholesterol-rich lipoproteins, it was difficult to obtain sufficient HDL to raise the plasma cholesterol to 200 mg/dl. Nevertheless, tremendous amounts of HDL were infused and did not appear to affect chylomicron clearance. These results indicate that specific lipoproteins, e.g. apoE-containing and remnant lipoproteins, compete for lipoprotein clearance.

In summary, chylomicron catabolism was markedly retarded in cholesterol-fed and WHHL rabbits. The primary factor for the retarded clearance appears to be competition of the specific plasma lipoproteins that interfere with the clearance step involved in remnant uptake, i.e. saturation of sequestration in the space of Disse. Enrichment of the remnants with apoE may not have enhanced remnant clearance because of impaired access of these lipoproteins to the already saturated binding sites involved in the sequestration phase of clearance.

  
Table: Liver and bone marrow uptake of [C]cholesterol-labeled chylomicrons in the cross-circulated pairs of rabbits


  
Table: Effect of isolated lipoproteins on [C]cholesterol-labeled chylomicron uptake by the liver and bone marrow of normal rabbits

Isolated lipoproteins were infused intravenously into normal rabbits for a period of 5-8 min. Control animals received a saline infusion for 5-8 min. At 20 min after the start of infusion, chylomicrons were injected into these rabbits, and blood samples were collected for an additional 40 min, at which time the animals were euthanized, and tissue samples were obtained (see ``Materials and Methods'').



FOOTNOTES

*
This work was supported in part by American Heart Association Research Grant 90-1327 and by National Institutes of Health Program Project Grant HL 41633. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Depts. of Pathology and Biochemistry, Medical College of Pennsylvania, Philadelphia, PA 19129.

To whom correspondence should be addressed: Gladstone Institute of Cardiovascular Disease, P. O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-826-7500; Fax: 415-285-5632.

The abbreviations used are: apoE, apolipoprotein E; HDL, high density lipoproteins; HDL, cholesterol-induced canine lipoproteins containing primarily apoE; HSPG, heparan sulfate proteoglycans; LDL, low density lipoproteins; LRP, LDL receptor-related protein; VLDL, very low density lipoproteins; WHHL, Watanabe heritable hyperlipidemic.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Robert C. Kowal of Brigham and Women's Hospital in Boston for the monoclonal antibody LRP-515; Dr. Karl H. Weisgraber for rabbit apoE; BioTechnology General in Israel for human recombinant apoE; R. Dennis Miranda, Peter A. Lindquist, and Marilyn Hathaway for technical support; Sylvia Richmond for manuscript preparation; Charles Benedict and Tom Rolain for graphics; and Dawn Levy and Lewis DeSimone for editorial assistance. We appreciate the skilled surgical expertise of Peter A. Lindquist, who performed the surgeries for the cross-circulation studies.


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