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The Clearance of Lipids from the Plasma of a Teleost Fish, the Black Bream (Acanthopagrus butcheri)
Comp. Biochem. Physiol. Vol. 116A, No. 2, pp. 167–172, 1997 Copyright 1996 Elsevier Science Inc.
ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00205-8
The Clearance of Lipids from the Plasma of a Teleost Fish, the Black Bream (Acanthopagrus butcheri) Diane E. Arnold-Reed, Peter J. Bentley, Cam T. Phan, and Trevor G. Redgrave Department of Physiology, The University of Western Australia, Nedlands, Perth, WA 6907, Australia ABSTRACT. 1. Lipid emulsions prepared to mimic the structure and lipid composition of plasma lipoproteins were injected into the vascular compartment of the black bream. The pattern of clearance of lipid emulsions from the plasma in the fish was similar to that in mammals though the time course was slower. In this fish clearance of triglyceride and cholesteryl ester reached equilibrium by 6.5 hr. 2. The greatest rate of clearance was between 15 min and 2.5 hr. 3. Triglycerides were cleared faster than cholesteryl ester from the plasma. 4. The percentage of triglyceride and cholesteryl ester remaining in the plasma at equilibrium in the fish was significantly higher than in mammals. Uptake of cholesteryl ester by the liver was much lower. 5. Gel electrophoresis of serum showed that these fish do not have apolipoprotein E. 6. Only 30% of the triglyceride and 50% of the cholesteryl ester injected could be accounted for by tissue (muscle, liver and fat) uptake or their presence in the circulation at equilibrium. Copyright 1996 Elsevier Science Inc. comp biochem physiol 116A;2:167– 172, 1997. KEY WORDS. Lipids, triglyceride, cholesteryl ester, lipoprotein, chylomicron-like emulsion, plasma clearance, teleost fish
INTRODUCTION Despite the importance of lipids in nutrition and intermediary metabolism, the details of lipid metabolism in fish are still not clear having received only limited study. The present study attempts to provide a basis for understanding lipoprotein metabolism in fish by studying the clearance of chylomicron-like lipid emulsions from the plasma of the black bream, Acanthopagrus butcheri (A. butcheri). While there is extensive literature concerning the metabolism of lipids in mammals, the way in which non-mammalian species regulate lipid metabolism has received less attention. It is thought that in fish (15), dietary lipids are absorbed across the gut primarily as free fatty acids (8) and also possibly as chylomicrons (17), i.e. triglyceride emulsion droplets, enveloped in a specific lipoprotein coating. From this study it was concluded that chylomicrons pass eventually into the systemic circulation though it is not clear whether the route is direct or via the lymphatic system as in mammals (15). In fish, subsequent metabolism of chylomicrons entering the plasma has not been investigated. Address reprint requests to: D. E. Arnold-Reed, Department of Physiology, The University of Western Australia, Nedlands, Perth, WA 6907, Australia. Fax 161 9 380 1025; E-mail: [email protected]. Abbreviations–apo B: apolipoprotein B; apo C: apolipoprotein C; apo E: apolipoprotein E; FCR: fractional clearance rate; HDL: high-density lipoprotein; LDL: low-density lipoprotein; LDLR: low-density lipoprotein receptor; LPL: lipoprotein lipase. Received 26 January 1996; accepted 14 May 1996.
Studies are hampered by the difficulty of obtaining fish chylomicrons. In the present study we have attempted to measure chylomicron clearance in fish by using a lipid emulsion to model intestinal chylomicrons. Studies in mammals have established that radioactively labelled chylomicron-like emulsions are cleared in a manner similar to lymph chylomicrons (14). Within a short time of release into the circulation, chylomicrons are depleted of most of their triglyceride content by endothelial sources of lipoprotein lipase (LPL) to be converted to cholesterol-rich remnant particles containing small amounts of triglyceride, which are taken up in the liver by as yet unidentified mechanisms and metabolised. Triglyceride clearance from the plasma therefore, is the result of lipolysis and remnant clearance while cholesterol clearance is reliant upon remnant particle uptake by the liver. Thus, by incorporating radioactively labelled triglyceride and cholesteryl ester in a chylomicron-like lipid emulsion, the path of chylomicron clearance from the plasma can be traced. While the chylomicron emulsions mimic in size and lipid composition the endogenously produced chylomicrons in fish (15), they lack the apolipoproteins necessary for clearance. In mammals it has been shown that the lipoproteins necessary for chylomicron clearance are apolipoprotein E (apo E), which acts as the ligand for the apolipoprotein B100-E (apo B-100-E) or low-density lipoprotein (LDL) receptors (22) in the liver, and apolipoprotein CII (apo CII), which acts as the cofactor of lipoprotein lipase in muscle
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and adipose tissue. The apolipoproteins important for the clearance of chylomicrons in fish are unknown. However, it is assumed that like the mammal the apolipoproteins necessary for chylomicron clearance are transferred to the emulsion particles when the emulsion is injected into blood. MATERIALS AND METHODS Animals Black bream (body weight 200–250 g) were obtained from the Aquaculture Centre, Fremantle, Western Australia. Fish stocks were held in large tanks with continuously recirculating, filtered and aerated seawater (SW) under conditions of natural day length and temperature. Fish were fed on a diet of commercial 6 mm barramundi pellets (Aquafeed Products, Australia) containing 10–11% fat. Fish used for the clearance study were fasted 24 hours prior to experimentation. Preparation of Lipid Emulsion Radiolabelled emulsions paralleling mammalian chylomicrons in size and lipid composition were prepared as described previously (12). Briefly, 70 mg triolein (TO), 3 mg cholesteryl oleate (CO), 25 mg egg yolk phosphatidylcholine (EYPC) and 2 mg free cholesterol (FC) were mixed with glycerol tri [1-14 C] oleate (Amersham) and cholesteryl [3H] oleate, prepared from [9,1(n)23H] oleic acid (Amersham), solvents were evaporated under a stream of nitrogen at 37°C and the lipids were freeze dried overnight. A solution of 8.5 ml 0.154 mM NaCl in 10 mM HEPES buffer (pH 7.4) was then added to the dried lipids and the mixture was emulsified under nitrogen by sonication (20 min, 55– 56°C) in a Vibra-Cell high-intensity ultraprocessor (Sonics and Materials, Inc.). The density of the mixture was then increased by the addition of solid potassium bromide (0.14 g/ml) and the crude emulsion was separated and refined by density gradient ultracentrifugation (Beckmann L-80 Ultracentrifuge fitted with an SW41 rotor) using sodium chloride solutions of densities 1.065, 1.020 and 1.006 g/ml. After an initial spin (35,000 g, 22 min at 20°C) the large particles were removed and discarded from the top of the salt gradient. The volume was re-adjusted with the 1.006 g/ml density salt solution and the emulsion was re-spun for 20 min at 100,000 g and 20°C. The chylomicron-like emulsion particles were then removed and diluted to 3 ml with the 1.006 g/ml density salt solution. Ten µl/ml reduced glutathione (5 mg/ml) was added to the emulsion to prevent oxidation. The emulsion was then purged with argon and stored at room temperature in a sealed tube. On the day of experiment, the size of the chylomicron-like particles was determined by laser light scattering using a BI-90 Particle Sizer (Brookhaven Instruments) and transmission electron microscopy. Most particles were in the size range 142 6 3.5 nm (n 5 11; see Fig. 1).
FIG. 1. Transmission electron micrograph of a sample of the
injected chylomicron-like lipid emulsion.
Emulsion Clearance Studies Twenty four hours before injection studies, fish were transferred to individual 10-litre tanks containing aerated artificial SW (Aquasonic, Australia). The caudal veins were cannulated under ethyl-aminobenzoate (MS222, Sigma) anaesthesia (0.05–0.1% w/v in SW buffered with an equal amount (w/w) of sodium hydrogen carbonate) as previously described (4). The fish were allowed to recover post-operatively for 24 hr before lipid emulsion injection. A solution of 2.06% (w/v) sodium citrate was used to keep the cannulae patent throughout the course of the experiment. For the clearance studies, 0.5 ml of the prepared lipid emulsion was injected via the venous cannula and 0.15 ml serial blood samples were taken at intervals of 15 min, 45 min, 1.5 hr, 2.5 hr, 3.5 hr, 4.5 hr, 6.5 hr, 8.5 hr and 24 hr. The volume of blood removed was immediately replaced with an equal volume of 0.15 M sodium chloride solution. The blood samples were then centrifuged (1300 rpm, 5 min) to separate the plasma and the radioactivity in 50 µl of plasma was counted on a liquid scintillation counter (Beckman, 6500). Counts were corrected to give an estimate of the amount of radioactivity present initially in the extracellular body fluids (mainly plasma). This was based on the amount of radioactivity in the plasma at 15 min and the effective volume of distribution was taken as 8% of body weight. Groups of fish were then sacrificed at either 6.5 hr (n 5 6) or 24 hr (n 5 9) and the body flushed intravenously with
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10 ml of saline. Liver (1 g), white muscle (1 g) and mesenteric fat (1 g) were excised and the uptake of triglyceride and cholesteryl ester into the tissues assessed by extracting the radioactively labelled material (14 C and 3H, respectively) in 30 ml of chloroform: methanol (2 : 1 v/v) and counting an aliquot in a liquid scintillation counter. Counts were corrected to give an estimate of uptake by the total mass of liver, body muscle and fat. Gel Electrophoresis for Apolipoprotein Determination A group (n 5 10) of fish were bled by direct caudal vessel puncture. Blood serum was separated by centrifugation and serum triglyceride and cholesterol were measured using commercially available colourimetric kits (Randox Laboratories, U.K.). The lipoprotein fractions were then separated from 1.8 ml serum samples by density gradient ultracentrifugation using a modification of the method previously described (7). The density of the serum was increased to 1.27 g/ml by the addition of solid potassium bromide (0.685 g) and sucrose (0.045 g). Salt gradients within the density range 1.005–1.214 g/ml were loaded successively over the serum. The samples were centrifuged at 275,000 g at 20°C for 22.5 hr in a Beckman L-80 Ultracentrifuge using an SW41 rotor. Arbitrary 1-cm fractions were then aspirated into 10 separate tubes (care was taken to maintain constancy between successive tubes) and the weight and volume of each were measured to determine density. The lipoprotein concentration in each lipoprotein fraction was then measured using a modification of the Lowry method (10). After delipidation (11) the samples were run on SDS-polyacrylamide gradient (5–25%) gels to determine the apolipoproteins present within each fraction. Molecular weight markers in the range 8.2–291 kD (Bio-Rad) were also included to provide an estimate of the molecular size of the protein bands. For comparison two Wistar rats were also bled by cardiac puncture and the apolipoprotein present in the plasma were separated and characterised using the same protocol as that described above.
14 C triglyceride and 3H cholesteryl ester from the plasma of black bream over 24 hr. Radioactivity in plasma is expressed as a percentage of the amount of radioactivity present after dilution through extracellular body fluids, i.e. the effective dose. The initial effective dose was estimated from the amount of radioactivity present in plasma 15 min after injection of the emulsion. Values are mean 6 standard error of mean, n 5 9 for 15 min–6.5 hr and n 5 7 for 8.5–24 hr.
FIG. 2. Line plot of the clearance of
clearance rates (FCR), calculated as the log-estimate for the change in plasma clearance over a defined time period. Between 15 min and 2.5 hr FCR for triglyceride was calculated as 0.53 6 0.06 hr21 (n 5 8) compared with the FCR of 0.34 6 0.03 hr21 (n 5 8) for cholesteryl ester. Clearance of triglyceride and cholesteryl ester was slightly slower between 2.5 and 4.5 hr (FCR for triglyceride was 0.36 6 0.06 hr21, n 5 7 and FCR for cholesteryl ester was 0.15 6 0.09 hr21, n 5 7) and reached equilibrium by 6.5 hr. There appeared to be no significant change in either clearance between 6.5 and 24 hr. To determine the differences in the uptake of triglyceride and cholesteryl ester by the liver, muscle and fat with time, a group of fish was sacrificed at 6.5 hr. The radioactivity present in total liver, fat and muscle after radioactive emulsion injection is shown in Table 1. There were no differences in the levels of total liver, fat and muscle and triglyceride or cholesteryl ester between 6.5 and 24 hr. A comparison of apolipoproteins present in the chylomicron/very low-density lipoprotein (VLDL) and high-den-
RESULTS Triglyceride (242.6 6 70.12 mg/100 ml, n 5 5) and cholesterol (197.09 6 33.46 mg/100 ml, n 5 5) measures of serum showed that in comparison with typical mammalian values, fish are naturally hyperlipidaemic and hypercholesterolaemic (see also (2) for other teleost species). The prepared lipid emulsions were shown by electron microscopy (Fig. 1) to be regular spherical emulsion droplets similar in size to chylomicrons and comparable in size to those previously used in mammalian studies. The clearance of injected chylomicron-like lipid emulsions from the plasma of the black bream is shown in Fig. 2. Clearance of triglyceride was faster than cholesteryl ester over the entire 24 hr of blood sampling. This was reflected in the fractional
TABLE 1. Radioactivity present in total liver, fat and muscle
6.5 and 24 hours after lipid emulsion injection Radioactivity present (% injected dose) 3
Liver
6.5 hours 24 hours Fat 6.5 hours 24 hours Muscle 6.5 hours 24 hours
H cholesteryl ester 8.76 10.24 1.10 0.67 6.89 7.50
6 6 6 6 6 6
0.79 0.90 1.00 0.66 0.92 1.57
(6) (8) (6) (8) (6) (8)
14
C triglyceride
12.48 12.25 1.37 0.42 5.56 5.63
6 6 6 6 6 6
0.95 1.37 1.39 0.44 0.76 0.43
(6)* (8) (6) (8) (6) (8)
Values are mean 6 SEM. Numbers of fish used are shown in parentheses. *P , 0.05 cholesteryl ester vs triglyceride.
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FIG. 3. Comparison of SDS-gel showing the apolipoproteins present in the very-low-density lipoprotein (VLDL) and highdensity lipoprotein (HDL) fractions of plasma from black bream (a) and the HDL fraction of rat serum (b).
sity lipoprotein (HDL) fractions of serum from the fish and the HDL fraction from a rat is shown in Fig. 3. The gel running the VLDL fraction from the fish shows a band of approximately 8.5–9 kD equivalent to apolipoprotein C (apo C) and a band of greater than 219 kD, possibly equivalent to apolipoprotein B (apo B), though this may have also degraded to a series of bands in the 67–124 kD molecular weight range (Fig. 3a). The HDL fraction of serum from both the fish and rat (Fig. 3b) shows bands equivalent to apolipoprotein AI (apo AI) and apolipoprotein AII (apo AII). The gels running the HDL fractions from rat serum (Fig. 3b) show an extra band equivalent to apolipoprotein E (apo E). It is clear that the band corresponding to apo E is absent in the VLDL and HDL fractions of the gel running serum from the black bream. DISCUSSION Our findings show that the pattern of the plasma clearance of chylomicron-like lipid emulsions after injection in black bream were similar to that found when endogenous VLDL particles were re-injected into the catfish (19). Our data showed that the plasma clearance of chylomicron-like lipid emulsions for the fish could be divided into three phases— a fast phase up to 2.5 hr, a slower phase up to 6.5 hr and a phase of no change between 6.5 hr and 24 hr. It was thus concluded that equilibrium was reached at about 6.5 hr. The overall pattern of lipid clearance from the plasma was also similar to that described for mammals (14). However, the time course was slower in the fish. There are a
number of possible explanations for the slower rate of clearance in the fish compared with the mammals. It is possible that the slower rate of clearance is no more than a reflection of the generally slower rate of metabolism of fish compared with mammals. Postprandial peaks in lipid absorption have been reported to be slower in fish compared with mammals (2,20). It has also been suggested that fish livers are capable of de novo lipid synthesis by utilising free fatty acids as well as lipid deposited from the plasma lipoproteins (9). Thus, the high plasma concentrations and the low liver concentrations of lipid 24 hr after injection of the emulsion could reflect the slower rate of metabolism of cold-blooded vertebrates, which results in a reduced rate of assimilation by the liver. The lipid emulsions used in the present study mimicked in size and composition the chylomicrons produced by teleost fish, as exemplified by the trout, where they are composed of predominantly (80%) triglyceride with small amounts of cholesterol and phospholipids (17). Under normal conditions fish dietary lipid is present in the plasma as fatty acids and chylomicron-like particles (15). Thus, fish appear to have the necessary mechanisms to utilize complex emulsions containing cholesteryl ester. However, our data show that clearance from the plasma was slow and liver uptake of cholesteryl ester small compared with mammals (12). A possible reason for the slow clearance is that the emulsion injection combined with the high dietary lipid entry into the plasma could have resulted in lipid overload with which the normal dietary lipid clearance processes were unable to cope. Though the fish were fasted for 24 hr
Lipid Clearance in Fish
prior to emulsion injection, this time was insufficient to allow complete gastric emptying of dietary food (ArnoldReed, personal observation). In fact, it has been previously shown that when trout are held at 14°C, postprandial lipid absorption occurs around 22 hr (21). In the present study black bream were fasted for 24 hr after a normal dietary regimen (containing 10–11% fat) before injection of lipid emulsion. The timing of the emulsion injection thus may have been coincidental with peak dietary lipid absorption. The amount of injected cholesteryl ester remaining in the blood of black bream at equilibrium was 25–30% greater than in mammals (compare with 14). By contrast the liver content of radiolabelled cholesteryl ester at equilibrium (6.5 hr) was significantly less than that in the rat (12) and the mouse (Mortimer, personal communication) at equilibrium (20 min). However, triglyceride uptake in the liver at equilibrium was similar to that of the rat and mouse. The lesser uptake of cholesteryl ester could be due, as shown by the gel electrophoretic data in this and other studies (6,18,19), to the absence of apo E in fish. In fish the pathway for chylomicron remnant uptake by the liver may be different from mammals where apo E is the essential ligand for the apo E/ LDL receptor (LDLR), which enables uptake of remnant lipoproteins by the liver and other tissues (1,22). The absence of apo E has also been reported in birds (5). In birds it has been suggested that apo B acts as the ligand for uptake of VLDL particles by the maturing oocyte during development of the egg (13). From the gel electrophoretic data in the present and previous studies (6,18,19), it seems that endogenously produced chylomicron/VLDL lipoproteins in fish have an apo B-like component. In salmon (3) apo B has an LDLR binding domain; it is thus likely apo B acts as the ligand for a receptor mediated (possibly the LDLR) remnant particle uptake by the liver in fish. However, the lack of apo E in the plasma of the black bream and the possibility that apo B does not transfer from plasma to the injected chylomicron-like lipid emulsions, would suggest that chylomicron remnant uptake by the liver in the black bream also involves unidentified ligand-receptor interaction. This suggestion would be consistent with the possible pathway for lipid transport in fish put forward previously (15). The mesenteric fat in the black bream took up only a small amount of the injected triglyceride and cholesteryl ester. In agreement with earlier (16) observations, the muscle uptake of the injected lipid was greater than mesenteric fat. Though triglyceride uptake for mesenteric fat was three times greater at 6.5 hr than at 24 hr, the difference was not statistically significantly different due to inter-animal variation. Accumulated lipids in muscle did not vary between 6.5 hr and 24 hr. Thus, it would seem that muscle acts as a longer-term storage depot than the mesenteric fat, contrary to Sheridan’s (16) suggestion. Only 30% of the triglyceride and 50% of the cholesteryl ester injected could be accounted for by tissue (muscle, liver and fat) uptake
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and presence in the plasma at equilibrium. The remainder that is unaccounted for was presumably lost due to metabolism and/or excretion. In summary therefore, it would appear that the rate of clearance of triglyceride and cholesteryl ester in fish was slower than that of the mammal. The mechanism of chylomicron remnant clearance from the plasma was not clearly defined from the current study though there appear to be differences from mammals, possibly involving a pathway that does not require apo E. This study was supported in part by grants from the National Health and Medical Research Council (Australia) and The Raine Medical Research Foundation of The University of Western Australia. The authors thank Mr Alan Light for the electron microscopy of emulsions and Mr Laszlo Bubrik for photography.
References 1. Arbeeny, C.M.; Rifici, V.A. The uptake of chylomicron remnants and very low density lipoprotein remnants by the perfused rat liver. J. Biol. Chem. 259:9662 –9666;1984. 2. Babin, P.J.; Vernier, J.-M. Plasma lipoproteins in fish. J. Lipid Res. 30:467–489;1989. 3. Babin, P.J.; Deryckere, F.; Gannon, F. Presence of an extended duplication in the putative low-density lipoprotein receptor binding domain of apolipoprotein B. Cloning and characterization of the domain in salmon. Eur. J. Biochem. 230:45–51; 1995. 4. Balment, R.J.; Carrick, S. The endogenous renin angiotensin system and drinking behaviour in the flounder. Am. J. Physiol. 248:R157–R160;1985. 5. Barakat, H.A.; St. Clair, R.W. Characterization of plasma lipoproteins of grain- and cholesterol-fed White Carneau and Show Racer pigeons. J. Lipid Res. 26:1252–1268;1985. 6. Chapman, M.J.; Goldstein, S.; Mills, G.L.; Leger, C. Distribution and characterization of the serum lipoproteins and their apoproteins in the rainbow trout (Salmo gairdneri). Biochemistry 17:4455 –4464;1978. 7. Demacker, P.N.; van Sommeren-Zondag, D.F.; Stalenhoef, A.F.; Styt, P.M.; van’t Laar, A. Ultracentrifugation in swinging bucket and fixed angle rotors evaluated for isolation and determination of high density lipoprotein fractions HDL2 and HDL3. Clin. Chem. 29:656–663;1983. 8. Kayama, M.; Iijima, N. Studies on the lipid transport mechanisms in the fish. Bull. Jap. Soc. Sci. Fish 42:987–996;1976. 9. Lin, H.; Romsos, D.R.; Tack, P.I.; Leveille, G.A. Influence of diet on in vitro and in vivo rates of fatty acid synthesis in coho salmon [Onchorhynchus kisutch (Walbaum)]. J.Nutri. 107: 1677–1682;1977. 10. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with a folin phenol reagent. J. Biol. Chem. 193:265–275;1951. 11. Mindham, M.A.; Mayers, P.A. A simple and rapid method for the preparation of apolipoproteins for electrophoresis. J. Lipid Res. 33:1084–1088;1992. 12. Mortimer, B-C.; Simmonds, W.J.; Joll, C.A.; Stick, R.V.; Redgrave, T.G. The effect of added monacylglycerols on the removal from plasma of chylomicron-like emulsions injected intravenously in rats. Biochim. Biophys. Acta 1002:359 –364; 1989. 13. Perry, M.M.; Griffin, H.D.; Gilbert, A.B. The binding of very low density and low density lipoproteins to the plasma mem-
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brane of the hen’s oocyte: a morphological study. Exp. Cell. Res. 151:433–446;1984. Redgrave, T.G.; Ly, H.L.; Quintao, E.C.R.; Ramberg, C.F.; Boston, R.C. Clearance from plasma of triglycerol and cholesterol ester after intravenous injection of chylomicron-like lipid emulsions in rats and man. Biochem. J. 290:843–847; 1993. Sheridan, M.A. Lipid dynamics in fish: aspects of absorption, transportation, deposition and mobilization. Comp. Biochem. Physiol. 90B:679–690;1988. Sheridan, M.A. Regulation of lipid metabolism in poikilothermic vertebrates. Comp. Biochem. Physiol. 107B:495–508; 1994. Sheridan, M.A.; Friedlander, J.K.L.; Allen, W.V. Chylomicra in the serum of postprandial steelhead trout (Salmo gairdneri). Comp. Biochem. Physiol. 81B:281–284;1985.
18. Skinner, E.R.; Rogie, A. The isolation and partial characterisation of the serum lipoproteins and apolipoproteins of the rainbow trout. Biochem. J. 173:507–520;1978. 19. Smith, M.A.K.; McKay, M.C.; Lee, R.F. Catfish plasma lipoproteins: In vivo studies of apoprotein synthesis and catabolism. J. Exp. Zool. 246:223–235;1988. 20. Wallaert, C.; Babin, P.J. Effects of 17β-estradiol and starvation on trout plasma lipoproteins. Lipids 27:1032–1041;1992. 21. Wallaert, C.; Babin, P.J. Effects of temperature variations on dietary lipid absorption and plasma lipoprotein concentrations in trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 109B:473–487;1994. 22. Windler, E.; Chao, Y.; Havel, R.J. Regulation of the hepatic uptake of triglyceride-rich lipoproteins in the rat. Opposing effects of homologous apolipoprotein E and individual C apoproteins. J. Biol. Chem. 225:8303–8307;1980.
ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00205-8
The Clearance of Lipids from the Plasma of a Teleost Fish, the Black Bream (Acanthopagrus butcheri) Diane E. Arnold-Reed, Peter J. Bentley, Cam T. Phan, and Trevor G. Redgrave Department of Physiology, The University of Western Australia, Nedlands, Perth, WA 6907, Australia ABSTRACT. 1. Lipid emulsions prepared to mimic the structure and lipid composition of plasma lipoproteins were injected into the vascular compartment of the black bream. The pattern of clearance of lipid emulsions from the plasma in the fish was similar to that in mammals though the time course was slower. In this fish clearance of triglyceride and cholesteryl ester reached equilibrium by 6.5 hr. 2. The greatest rate of clearance was between 15 min and 2.5 hr. 3. Triglycerides were cleared faster than cholesteryl ester from the plasma. 4. The percentage of triglyceride and cholesteryl ester remaining in the plasma at equilibrium in the fish was significantly higher than in mammals. Uptake of cholesteryl ester by the liver was much lower. 5. Gel electrophoresis of serum showed that these fish do not have apolipoprotein E. 6. Only 30% of the triglyceride and 50% of the cholesteryl ester injected could be accounted for by tissue (muscle, liver and fat) uptake or their presence in the circulation at equilibrium. Copyright 1996 Elsevier Science Inc. comp biochem physiol 116A;2:167– 172, 1997. KEY WORDS. Lipids, triglyceride, cholesteryl ester, lipoprotein, chylomicron-like emulsion, plasma clearance, teleost fish
INTRODUCTION Despite the importance of lipids in nutrition and intermediary metabolism, the details of lipid metabolism in fish are still not clear having received only limited study. The present study attempts to provide a basis for understanding lipoprotein metabolism in fish by studying the clearance of chylomicron-like lipid emulsions from the plasma of the black bream, Acanthopagrus butcheri (A. butcheri). While there is extensive literature concerning the metabolism of lipids in mammals, the way in which non-mammalian species regulate lipid metabolism has received less attention. It is thought that in fish (15), dietary lipids are absorbed across the gut primarily as free fatty acids (8) and also possibly as chylomicrons (17), i.e. triglyceride emulsion droplets, enveloped in a specific lipoprotein coating. From this study it was concluded that chylomicrons pass eventually into the systemic circulation though it is not clear whether the route is direct or via the lymphatic system as in mammals (15). In fish, subsequent metabolism of chylomicrons entering the plasma has not been investigated. Address reprint requests to: D. E. Arnold-Reed, Department of Physiology, The University of Western Australia, Nedlands, Perth, WA 6907, Australia. Fax 161 9 380 1025; E-mail: [email protected]. Abbreviations–apo B: apolipoprotein B; apo C: apolipoprotein C; apo E: apolipoprotein E; FCR: fractional clearance rate; HDL: high-density lipoprotein; LDL: low-density lipoprotein; LDLR: low-density lipoprotein receptor; LPL: lipoprotein lipase. Received 26 January 1996; accepted 14 May 1996.
Studies are hampered by the difficulty of obtaining fish chylomicrons. In the present study we have attempted to measure chylomicron clearance in fish by using a lipid emulsion to model intestinal chylomicrons. Studies in mammals have established that radioactively labelled chylomicron-like emulsions are cleared in a manner similar to lymph chylomicrons (14). Within a short time of release into the circulation, chylomicrons are depleted of most of their triglyceride content by endothelial sources of lipoprotein lipase (LPL) to be converted to cholesterol-rich remnant particles containing small amounts of triglyceride, which are taken up in the liver by as yet unidentified mechanisms and metabolised. Triglyceride clearance from the plasma therefore, is the result of lipolysis and remnant clearance while cholesterol clearance is reliant upon remnant particle uptake by the liver. Thus, by incorporating radioactively labelled triglyceride and cholesteryl ester in a chylomicron-like lipid emulsion, the path of chylomicron clearance from the plasma can be traced. While the chylomicron emulsions mimic in size and lipid composition the endogenously produced chylomicrons in fish (15), they lack the apolipoproteins necessary for clearance. In mammals it has been shown that the lipoproteins necessary for chylomicron clearance are apolipoprotein E (apo E), which acts as the ligand for the apolipoprotein B100-E (apo B-100-E) or low-density lipoprotein (LDL) receptors (22) in the liver, and apolipoprotein CII (apo CII), which acts as the cofactor of lipoprotein lipase in muscle
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and adipose tissue. The apolipoproteins important for the clearance of chylomicrons in fish are unknown. However, it is assumed that like the mammal the apolipoproteins necessary for chylomicron clearance are transferred to the emulsion particles when the emulsion is injected into blood. MATERIALS AND METHODS Animals Black bream (body weight 200–250 g) were obtained from the Aquaculture Centre, Fremantle, Western Australia. Fish stocks were held in large tanks with continuously recirculating, filtered and aerated seawater (SW) under conditions of natural day length and temperature. Fish were fed on a diet of commercial 6 mm barramundi pellets (Aquafeed Products, Australia) containing 10–11% fat. Fish used for the clearance study were fasted 24 hours prior to experimentation. Preparation of Lipid Emulsion Radiolabelled emulsions paralleling mammalian chylomicrons in size and lipid composition were prepared as described previously (12). Briefly, 70 mg triolein (TO), 3 mg cholesteryl oleate (CO), 25 mg egg yolk phosphatidylcholine (EYPC) and 2 mg free cholesterol (FC) were mixed with glycerol tri [1-14 C] oleate (Amersham) and cholesteryl [3H] oleate, prepared from [9,1(n)23H] oleic acid (Amersham), solvents were evaporated under a stream of nitrogen at 37°C and the lipids were freeze dried overnight. A solution of 8.5 ml 0.154 mM NaCl in 10 mM HEPES buffer (pH 7.4) was then added to the dried lipids and the mixture was emulsified under nitrogen by sonication (20 min, 55– 56°C) in a Vibra-Cell high-intensity ultraprocessor (Sonics and Materials, Inc.). The density of the mixture was then increased by the addition of solid potassium bromide (0.14 g/ml) and the crude emulsion was separated and refined by density gradient ultracentrifugation (Beckmann L-80 Ultracentrifuge fitted with an SW41 rotor) using sodium chloride solutions of densities 1.065, 1.020 and 1.006 g/ml. After an initial spin (35,000 g, 22 min at 20°C) the large particles were removed and discarded from the top of the salt gradient. The volume was re-adjusted with the 1.006 g/ml density salt solution and the emulsion was re-spun for 20 min at 100,000 g and 20°C. The chylomicron-like emulsion particles were then removed and diluted to 3 ml with the 1.006 g/ml density salt solution. Ten µl/ml reduced glutathione (5 mg/ml) was added to the emulsion to prevent oxidation. The emulsion was then purged with argon and stored at room temperature in a sealed tube. On the day of experiment, the size of the chylomicron-like particles was determined by laser light scattering using a BI-90 Particle Sizer (Brookhaven Instruments) and transmission electron microscopy. Most particles were in the size range 142 6 3.5 nm (n 5 11; see Fig. 1).
FIG. 1. Transmission electron micrograph of a sample of the
injected chylomicron-like lipid emulsion.
Emulsion Clearance Studies Twenty four hours before injection studies, fish were transferred to individual 10-litre tanks containing aerated artificial SW (Aquasonic, Australia). The caudal veins were cannulated under ethyl-aminobenzoate (MS222, Sigma) anaesthesia (0.05–0.1% w/v in SW buffered with an equal amount (w/w) of sodium hydrogen carbonate) as previously described (4). The fish were allowed to recover post-operatively for 24 hr before lipid emulsion injection. A solution of 2.06% (w/v) sodium citrate was used to keep the cannulae patent throughout the course of the experiment. For the clearance studies, 0.5 ml of the prepared lipid emulsion was injected via the venous cannula and 0.15 ml serial blood samples were taken at intervals of 15 min, 45 min, 1.5 hr, 2.5 hr, 3.5 hr, 4.5 hr, 6.5 hr, 8.5 hr and 24 hr. The volume of blood removed was immediately replaced with an equal volume of 0.15 M sodium chloride solution. The blood samples were then centrifuged (1300 rpm, 5 min) to separate the plasma and the radioactivity in 50 µl of plasma was counted on a liquid scintillation counter (Beckman, 6500). Counts were corrected to give an estimate of the amount of radioactivity present initially in the extracellular body fluids (mainly plasma). This was based on the amount of radioactivity in the plasma at 15 min and the effective volume of distribution was taken as 8% of body weight. Groups of fish were then sacrificed at either 6.5 hr (n 5 6) or 24 hr (n 5 9) and the body flushed intravenously with
Lipid Clearance in Fish
169
10 ml of saline. Liver (1 g), white muscle (1 g) and mesenteric fat (1 g) were excised and the uptake of triglyceride and cholesteryl ester into the tissues assessed by extracting the radioactively labelled material (14 C and 3H, respectively) in 30 ml of chloroform: methanol (2 : 1 v/v) and counting an aliquot in a liquid scintillation counter. Counts were corrected to give an estimate of uptake by the total mass of liver, body muscle and fat. Gel Electrophoresis for Apolipoprotein Determination A group (n 5 10) of fish were bled by direct caudal vessel puncture. Blood serum was separated by centrifugation and serum triglyceride and cholesterol were measured using commercially available colourimetric kits (Randox Laboratories, U.K.). The lipoprotein fractions were then separated from 1.8 ml serum samples by density gradient ultracentrifugation using a modification of the method previously described (7). The density of the serum was increased to 1.27 g/ml by the addition of solid potassium bromide (0.685 g) and sucrose (0.045 g). Salt gradients within the density range 1.005–1.214 g/ml were loaded successively over the serum. The samples were centrifuged at 275,000 g at 20°C for 22.5 hr in a Beckman L-80 Ultracentrifuge using an SW41 rotor. Arbitrary 1-cm fractions were then aspirated into 10 separate tubes (care was taken to maintain constancy between successive tubes) and the weight and volume of each were measured to determine density. The lipoprotein concentration in each lipoprotein fraction was then measured using a modification of the Lowry method (10). After delipidation (11) the samples were run on SDS-polyacrylamide gradient (5–25%) gels to determine the apolipoproteins present within each fraction. Molecular weight markers in the range 8.2–291 kD (Bio-Rad) were also included to provide an estimate of the molecular size of the protein bands. For comparison two Wistar rats were also bled by cardiac puncture and the apolipoprotein present in the plasma were separated and characterised using the same protocol as that described above.
14 C triglyceride and 3H cholesteryl ester from the plasma of black bream over 24 hr. Radioactivity in plasma is expressed as a percentage of the amount of radioactivity present after dilution through extracellular body fluids, i.e. the effective dose. The initial effective dose was estimated from the amount of radioactivity present in plasma 15 min after injection of the emulsion. Values are mean 6 standard error of mean, n 5 9 for 15 min–6.5 hr and n 5 7 for 8.5–24 hr.
FIG. 2. Line plot of the clearance of
clearance rates (FCR), calculated as the log-estimate for the change in plasma clearance over a defined time period. Between 15 min and 2.5 hr FCR for triglyceride was calculated as 0.53 6 0.06 hr21 (n 5 8) compared with the FCR of 0.34 6 0.03 hr21 (n 5 8) for cholesteryl ester. Clearance of triglyceride and cholesteryl ester was slightly slower between 2.5 and 4.5 hr (FCR for triglyceride was 0.36 6 0.06 hr21, n 5 7 and FCR for cholesteryl ester was 0.15 6 0.09 hr21, n 5 7) and reached equilibrium by 6.5 hr. There appeared to be no significant change in either clearance between 6.5 and 24 hr. To determine the differences in the uptake of triglyceride and cholesteryl ester by the liver, muscle and fat with time, a group of fish was sacrificed at 6.5 hr. The radioactivity present in total liver, fat and muscle after radioactive emulsion injection is shown in Table 1. There were no differences in the levels of total liver, fat and muscle and triglyceride or cholesteryl ester between 6.5 and 24 hr. A comparison of apolipoproteins present in the chylomicron/very low-density lipoprotein (VLDL) and high-den-
RESULTS Triglyceride (242.6 6 70.12 mg/100 ml, n 5 5) and cholesterol (197.09 6 33.46 mg/100 ml, n 5 5) measures of serum showed that in comparison with typical mammalian values, fish are naturally hyperlipidaemic and hypercholesterolaemic (see also (2) for other teleost species). The prepared lipid emulsions were shown by electron microscopy (Fig. 1) to be regular spherical emulsion droplets similar in size to chylomicrons and comparable in size to those previously used in mammalian studies. The clearance of injected chylomicron-like lipid emulsions from the plasma of the black bream is shown in Fig. 2. Clearance of triglyceride was faster than cholesteryl ester over the entire 24 hr of blood sampling. This was reflected in the fractional
TABLE 1. Radioactivity present in total liver, fat and muscle
6.5 and 24 hours after lipid emulsion injection Radioactivity present (% injected dose) 3
Liver
6.5 hours 24 hours Fat 6.5 hours 24 hours Muscle 6.5 hours 24 hours
H cholesteryl ester 8.76 10.24 1.10 0.67 6.89 7.50
6 6 6 6 6 6
0.79 0.90 1.00 0.66 0.92 1.57
(6) (8) (6) (8) (6) (8)
14
C triglyceride
12.48 12.25 1.37 0.42 5.56 5.63
6 6 6 6 6 6
0.95 1.37 1.39 0.44 0.76 0.43
(6)* (8) (6) (8) (6) (8)
Values are mean 6 SEM. Numbers of fish used are shown in parentheses. *P , 0.05 cholesteryl ester vs triglyceride.
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D. E. Arnold-Reed et al.
FIG. 3. Comparison of SDS-gel showing the apolipoproteins present in the very-low-density lipoprotein (VLDL) and highdensity lipoprotein (HDL) fractions of plasma from black bream (a) and the HDL fraction of rat serum (b).
sity lipoprotein (HDL) fractions of serum from the fish and the HDL fraction from a rat is shown in Fig. 3. The gel running the VLDL fraction from the fish shows a band of approximately 8.5–9 kD equivalent to apolipoprotein C (apo C) and a band of greater than 219 kD, possibly equivalent to apolipoprotein B (apo B), though this may have also degraded to a series of bands in the 67–124 kD molecular weight range (Fig. 3a). The HDL fraction of serum from both the fish and rat (Fig. 3b) shows bands equivalent to apolipoprotein AI (apo AI) and apolipoprotein AII (apo AII). The gels running the HDL fractions from rat serum (Fig. 3b) show an extra band equivalent to apolipoprotein E (apo E). It is clear that the band corresponding to apo E is absent in the VLDL and HDL fractions of the gel running serum from the black bream. DISCUSSION Our findings show that the pattern of the plasma clearance of chylomicron-like lipid emulsions after injection in black bream were similar to that found when endogenous VLDL particles were re-injected into the catfish (19). Our data showed that the plasma clearance of chylomicron-like lipid emulsions for the fish could be divided into three phases— a fast phase up to 2.5 hr, a slower phase up to 6.5 hr and a phase of no change between 6.5 hr and 24 hr. It was thus concluded that equilibrium was reached at about 6.5 hr. The overall pattern of lipid clearance from the plasma was also similar to that described for mammals (14). However, the time course was slower in the fish. There are a
number of possible explanations for the slower rate of clearance in the fish compared with the mammals. It is possible that the slower rate of clearance is no more than a reflection of the generally slower rate of metabolism of fish compared with mammals. Postprandial peaks in lipid absorption have been reported to be slower in fish compared with mammals (2,20). It has also been suggested that fish livers are capable of de novo lipid synthesis by utilising free fatty acids as well as lipid deposited from the plasma lipoproteins (9). Thus, the high plasma concentrations and the low liver concentrations of lipid 24 hr after injection of the emulsion could reflect the slower rate of metabolism of cold-blooded vertebrates, which results in a reduced rate of assimilation by the liver. The lipid emulsions used in the present study mimicked in size and composition the chylomicrons produced by teleost fish, as exemplified by the trout, where they are composed of predominantly (80%) triglyceride with small amounts of cholesterol and phospholipids (17). Under normal conditions fish dietary lipid is present in the plasma as fatty acids and chylomicron-like particles (15). Thus, fish appear to have the necessary mechanisms to utilize complex emulsions containing cholesteryl ester. However, our data show that clearance from the plasma was slow and liver uptake of cholesteryl ester small compared with mammals (12). A possible reason for the slow clearance is that the emulsion injection combined with the high dietary lipid entry into the plasma could have resulted in lipid overload with which the normal dietary lipid clearance processes were unable to cope. Though the fish were fasted for 24 hr
Lipid Clearance in Fish
prior to emulsion injection, this time was insufficient to allow complete gastric emptying of dietary food (ArnoldReed, personal observation). In fact, it has been previously shown that when trout are held at 14°C, postprandial lipid absorption occurs around 22 hr (21). In the present study black bream were fasted for 24 hr after a normal dietary regimen (containing 10–11% fat) before injection of lipid emulsion. The timing of the emulsion injection thus may have been coincidental with peak dietary lipid absorption. The amount of injected cholesteryl ester remaining in the blood of black bream at equilibrium was 25–30% greater than in mammals (compare with 14). By contrast the liver content of radiolabelled cholesteryl ester at equilibrium (6.5 hr) was significantly less than that in the rat (12) and the mouse (Mortimer, personal communication) at equilibrium (20 min). However, triglyceride uptake in the liver at equilibrium was similar to that of the rat and mouse. The lesser uptake of cholesteryl ester could be due, as shown by the gel electrophoretic data in this and other studies (6,18,19), to the absence of apo E in fish. In fish the pathway for chylomicron remnant uptake by the liver may be different from mammals where apo E is the essential ligand for the apo E/ LDL receptor (LDLR), which enables uptake of remnant lipoproteins by the liver and other tissues (1,22). The absence of apo E has also been reported in birds (5). In birds it has been suggested that apo B acts as the ligand for uptake of VLDL particles by the maturing oocyte during development of the egg (13). From the gel electrophoretic data in the present and previous studies (6,18,19), it seems that endogenously produced chylomicron/VLDL lipoproteins in fish have an apo B-like component. In salmon (3) apo B has an LDLR binding domain; it is thus likely apo B acts as the ligand for a receptor mediated (possibly the LDLR) remnant particle uptake by the liver in fish. However, the lack of apo E in the plasma of the black bream and the possibility that apo B does not transfer from plasma to the injected chylomicron-like lipid emulsions, would suggest that chylomicron remnant uptake by the liver in the black bream also involves unidentified ligand-receptor interaction. This suggestion would be consistent with the possible pathway for lipid transport in fish put forward previously (15). The mesenteric fat in the black bream took up only a small amount of the injected triglyceride and cholesteryl ester. In agreement with earlier (16) observations, the muscle uptake of the injected lipid was greater than mesenteric fat. Though triglyceride uptake for mesenteric fat was three times greater at 6.5 hr than at 24 hr, the difference was not statistically significantly different due to inter-animal variation. Accumulated lipids in muscle did not vary between 6.5 hr and 24 hr. Thus, it would seem that muscle acts as a longer-term storage depot than the mesenteric fat, contrary to Sheridan’s (16) suggestion. Only 30% of the triglyceride and 50% of the cholesteryl ester injected could be accounted for by tissue (muscle, liver and fat) uptake
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and presence in the plasma at equilibrium. The remainder that is unaccounted for was presumably lost due to metabolism and/or excretion. In summary therefore, it would appear that the rate of clearance of triglyceride and cholesteryl ester in fish was slower than that of the mammal. The mechanism of chylomicron remnant clearance from the plasma was not clearly defined from the current study though there appear to be differences from mammals, possibly involving a pathway that does not require apo E. This study was supported in part by grants from the National Health and Medical Research Council (Australia) and The Raine Medical Research Foundation of The University of Western Australia. The authors thank Mr Alan Light for the electron microscopy of emulsions and Mr Laszlo Bubrik for photography.
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