Proc. Natl Acad. Sci. USA Vol. 79, pp. 1282-1285, February 1982 Medical Sciences

Regulation of serum E metabolism: Role of chylomicron metabolism (chylomicron remnant removal/high density /hepatic apolipoprotein E receptor/apolipoprotein E removal/ high-fat, high- meals) JOHN G. DELAMATRE, BRIAN R. KRAUSE, AND PAUL S. ROHEIM Louisiana State University Medical Center, Department of Physiology, New Orleans, Louisiana 70112-1393 Communicated by Alex B. Novikoff, November 6, 1981

ABSTRACT The mechanism by which high-fat, high-choles- MATERIALS AND METHODS terol diets lower serum apolipoprotein E (apoE) concentration was studied in rats by feeding a single high-fat, high-cholesterol meal Animals. Male, Sprague-Dawley rats (275-300 g) were and by intravenously infusing chylomicrons containing low and maintained on laboratory rat chow on a 12-hr-light/12-hr-dark high amounts of cholesterol. Serum apoE concentrations were cycle (6 a.m.-6 p.m.). At 3 p.m. on the day before an experi- unchanged 9 hours after an olive oil meal but were decreased by ment, the food was removed from the cages. At midnight the 35% after an olive oil/cholesterol-enriched meal. The decrease in rats were given their respective diets and allowed free access serum apoE concentrations with the olive oil/cholesterol meal was to the food until the time ofsacrifice. The following meals were accompanied by a decrease in apoE concentration in the high den- given: ground laboratory rat chow (control), 20% olive oil in lab- sity lipoprotein fraction. Three hours after the intravenous infu- oratory rat chow (olive oil meal), and 20% olive oil/2% choles- sion of cholesterol-enriched chylomicrons, serum apoE concen- terol in laboratory rat chow (olive oil/cholesterol meal). The rats trations decreased 40%, whereas serum apoE concentrations were then bled through the abdominal aorta at midnight, 3 decreased by only 10% when chylomicrons with low cholesterol a.m., or 9 a.m. concentrations or saline were infused. It is concluded that the To obtain chylomicrons, rats (300- to 350-g males) metabolism of cholesterol-enriched chylomicrons results in an in- were given a creased removal of serum apoE and that the cholesterol content subcutaneous injection of EE at a dose of 5 mg/ of is a determinant of kg of body weight per day for 5 days before and during the chylomicrons serum apoE removal. lymph collection (11). Lymph was collected from the main mes- enteric , and the duodenum was cannulated Apolipoprotein E (apoE) plays a role in lipoprotein recognition for the infusion of as described (10). Intralipid (Cutter and removal by the liver and is also thought to be the hepatic Laboratories, Berkeley, CA) alone was infused to produce chy- recognition site on chylomicron remnants (1-5). This concept lomicrons containing low cholesterol concentrations (LoChol- is supported by the fact that high density lipoprotein (HDL) chylos). To produce chylomicrons containing high cholesterol particles (density, 1.063-1.21) that contain only apoE (apoE, concentrations (HiChol-chylos), cholesterol (1%) was added to HDLc) compete effectively for hepatic binding with rat chy- the Intralipid. Chylomicrons were isolated by ultracentrifuga- lomicron remnants in a rat liver perfusion system (1). We have tion for 2.0 x 106 g-min in a Beckman L5-50 ultracentrifuge with demonstrated that plasma apoE concentrations decreased in an SW-41 rotor. The recipient rats received a chylomicron tri- euthyroid rats fed a high-fat, high-cholesterol diet for several glyceride mass equal to 1.25 mg of per g of body weeks. We postulated that the decrease of serum apoE con- weight. This amount of chylomicron triglyceride is approxi- centrations was associated with the removal of chylomicron mately equal to that normally produced during a fatty meal (10, remnants (6). Because chylomicrons are rapidly removed from 12). the plasma (7, 8), we tested whether the decrease in serum apoE Recipient rats (175- to 250-g males) were prepared as de- concentrations would occur acutely. Therefore, we studied the scribed (13) so that the chylomicrons could be infused into and effects of a single high-fat, high-cholesterol meal and of the in- blood samples withdrawn from the jugular vein without the use travenous infusion of chylomicrons with different cholesterol ofanesthesia or restraint. Such preparation is important because contents on serum apoE concentrations. Chylomicrons col- anesthesia (14) or restraint (15) alter triglyceride metabolism. lected from the mesenteric lymph ofa normal rat contain apoE, Chylomicrons (4.5 ml) were infused into the jugular vein at a whereas nascent chylomicrons do not (9, 10). To accurately as- constant rate for 3 hr. Blood samples were taken immediately sess the effect that chylomicrons have on serum apoE concen- before the infusion and at 1, 3, and 6 hr after the infusion period. trations, it is necessary to infuse chylomicrons that do not con- Serum was separated and stored in the presence of0. 1% EDTA/ tain apoE. It is possible to obtain chylomicrons with no apoE 0. 1% azide. from a rat treated with 17a-ethynylestradiol (EE) because EE Analytical Methods. and cholesterol were de- treatment lowers serum apoE concentrations dramatically (10, termined enzymatically by the methods of Bucolo and David 11). Therefore, chylomicrons collected from the mesenteric (16) and of Allain et aL (17), respectively. Free and esterified lymph of EE-treated rats were used so that apoE would not be cholesterol ofthe chylomicrons were separated by column chro- introduced into the serum during the chylomicron infusion and matography (18, 19). was determined by the method interfere with the results. The present study demonstrates that of Lowry et al. (20) as modified by Sata, Havel, and Jones (21) the metabolism of cholesterol-enriched chylomicrons is asso- ciated with serum apoE removal. Abbreviations: HiChol-chylos, chylomicrons containing high choles- terol concentrations; LoChol-chylos, chylomicrons containing low cho- The publication costs ofthis article were defrayed in part by page charge lesterol concentrations; HDL, high density (density, payment. This article must therefore be hereby marked "advertise- 1.063-1.21 g/ml); EE, 17a-ethynylestradiol; VLDL, very low density ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. lipoproteins (density, <1.006 g/ml). 1282 Downloaded by guest on October 1, 2021 Medical Sciences: DeLamatre et aL Proc. Natl. Acad. Sci. USA 79 (1982) 1283

Table 1. Serum concentrations of cholesterol, triglyceride, and Table 3. Serum cholesterol, triglyceride, and apoE concentrations apoE of rats 3 hr after feeding immediately before feeding and 9 hr after feeding control, olive-oil, Assay* and olive-oil/cholesterol meals Cholesterol, Triglyceride, ApoE, Assay* Meal mg/dl mg/dl arbitrary units Cholesterol, Triglyceride, ApoE, Control 56 ± 2 93 ± 10 96 ± 8 Meal mg/dl mg/dl arbitrary units Olive oil 67 ± 4t* .146 ± 20 108 ± 8 Control,Ohr 53 ± 3 23 ± 3 124 ± 7 Olive oil/cholesterol 76 ± 2tt 179 ± 24t 91 ± 6 Control, 9 hr 60 ± 4 46 ± 6t 117 ± 7t Olive 9 hr 67 ± 4 89 ± 6tt 129 ± 13tt * ± oil, Values SEM; number of samples: 11 rats. Olive oil/cholesterol, 9 hr 93 ± 88 ± 11tt 86 ± 7tf§ t Significantly different from control (P, <0.05). 5t*§ t Significantly different from other experimental group (P, <0.05). * Values ± SEM; number of samples: six rats. t Significantly different from 0-hr control group (P, <0.05). * Significantly different from 9-hr control group (P, <0.05). with bovine serum albumin as a standard. Lipoproteins were § Significantly different from olive oil-fed group (P, <0.05). separated and apoE was quantified by electroimmunoassay in the presence of Nonidet P.40 as described (6). For statistical analysis, one-way analysis ofvariance was used followed by the or olive oil-fed groups; serum apoE concentrations decreased Student-Neuman-Keuls multiple-range test (22). A P value of only in the rats given the olive oil/cholesterol meal, confirming <0.05 was considered significant. NaDodSO4polyacrylamide the data presented in Table 2. gel electrophoresis was performed with 10% acrylamide gels by Previously we found that while total serum apoE concentra- the method of Shapiro, Vinuela, and Maizel (23) as described tions decrease with chronic cholesterol feeding, apoE concen- (24). trations increase in the lower density lipoproteins and decrease in HDL (6). We also have shown that the quantitation ofapoE RESULTS by electroimmunoassay agrees closely with values obtained by densitometric scanning of Coomassie blue-stained NaDod- To determine whether there is a specific effect of acute cho- S04/polyacrylamide gels. Fig. 1 depicts the apoE concentra- lesterol feeding on the serum concentrations ofapoE, rats were tions in the various lipoprotein fractions at 9 hr after feed- fed the control, olive oil, and olive oil/cholesterol meals. Three ing in the control and experimental groups. There were no dif- hours after feeding (Table 1), there was a significant increase ferences in apoE distribution between control and olive oil- in the serum cholesterol and triglyceride concentrations in the fed groups. In the olive oil/cholesterol-fed group, where serum olive oil- and olive oil/cholesterol-fed groups but serum apoE apoE concentrations were decreased by 35% (Table 2), the apoE concentrations did not change. At 9 hr after feeding (Table 2), concentration in HDL decreased by 94% and decreased by 50% a significant increase in serum cholesterol and triglyceride con- in the fraction with adensity of>1.21 g/ml. The increased apoE centrations also was seen in both the olive oil- and the olive oil/ in the fraction with a density of <1.063 g/ml is consistent with cholesterol-fed groups; in the latter group, the increase in our chronic experiment (6) and with another acute feeding ex- serum cholesterol was of greater magnitude and was accom- periment (data not shown); this fraction represented 63% ofthe panied by a significant decrease in serum apoE concentrations. total apoE in the olive oil/cholesterol rats and only 30% in the At this 9-hr time point, serum apoE concentration was not other groups. Not only did the apoE concentration decrease in changed in the olive oil-fed group. This response suggests that dietary cholesterol specifically influences the serum concen- trations of apoE. We also ascertained whether serum apoE concentrations change temporally from the basal level before feeding until 9 hr after feeding. The two experiments represented by Tables 1 and 2 were done at different times and the data may not be directly comparable. Therefore, in aseparate experiment (Table

3), we determined serum cholesterol, triglyceride, and apoE Go -4--l .q concentrations in the same group of rats immediately before 0 0 feeding (0 hr) and 9 hr after feeding. This procedure allowed c Cd us to determine iffeeding per se results in a lowering of serum 1.4 :t. apoE concentrations (Table 3). The data clearly show that feed- -2 ing does not lower serum apoE concentrations in the control 14

Table 2. Serum concentrations of cholesterol, triglyceride, and apoE of rats 9 hr after feeding Assay* Cholesterol, Triglyceride, ApoE, Meal mg/dl mg/dl arbitrary units <1.063 1.063- >1.21 Control 61 2 102 ± 8 77 ± 5 1.21 Olive oil 69 ± 1tt 210 ± 16t 74 ± 2t Density, g/ml Olive oil/cholesterol 102 ± 3t1 212 ± 30t 51 ± 3t4 FIG. 1. The effect of control (El, olive oil (A), and olive oil/cho- * Values ± SEM; number of samples: four pools of four rats per pool. lesterol (3) meals on the distribution of apoE 9 hr afterfeeding. Values t Significantly different from control (P, <0.05). + SEM are based on four pools of three rats per pool. t Significantly different from other experimental group (P, <0.05). *Significantly different from other two groups (P, <0.05). Downloaded by guest on October 1, 2021 1284 Medical Sciences: DeLamatre et alPProc. Natl. Acad. Sci. USA 79 (1982)

Table 4. Concentration of cholesterol and protein in HDL of rats 100 T 9 hr after feeding Assay* N1%N. b: 80 + IS Cholesterol, Protein, a Meal mg/dl mg/dl Control 23 ± 2 39 ± 3 -4 60 + Olive oil 25 ± It 43 ± it 0as Olive oil/cholesterol 19 ± 1t 31 ± 1t:

a) * Values ± SEM; number of animals: four pools of four rats per pool. CI) 40 + t Significantly different from control group (P, <0.05). t Signficantly different from other experimental group (P, <0.05). Chylomicron 20 infusion HDL, but total HDL protein and cholesterol also decreased, as shown in Table 4. These data show that the decrease ofapoE I I in the HDL and in fractions with a density >1.21 g/ml occurred I I I I I I I I only when cholesterol was added to the high-fat diet, indicating 0 1 2 3 4 5 6 7 8 9 that this was a specific effect of cholesterol. Time, hr The feeding data implied that the decrease in serum apoE concentration was associated with the metabolism of choles- FIG. 2. The effect of infusing saline, LoChol-chylos, and HiChol- terol-enriched chylomicrons. To more directly test the effect chylos on the serum concentration of apoE (expressed as the percent- ofchylomicrons on serum apoE concentrations, HiChol-chylos age of apoE concentration at time 0). o----o, Saline infusion; and LoChol-chylos were infused into *-*, LoChol-chylo infusion; *----e, HiChol-chylo infusion. Values unanesthetized/unre- SEM are based on: saline, n = 3; LoChol-chylos, n = 5; HiChol-chy- strained rats. The lipid concentrations in the two chylomicron los, n = 4, where n = the number of rats. preparations are shown in Table 5. The total cholesterol-to-tri- glyceride ratio was 3.5-fold higher in the HiChol-chylos than diet is probably associated with the metabolism of cholesterol- in the LoChol-chylos. The apoprotein composition in both types enriched chylomicrons. of chylomicrons was determined by NaDodSOpolyacryla- To determine whether the decrease in serum apoE concen- mide gel electrophoresis and, as previously reported, no apoE trations was directly associated with chylomicron metabolism, was found (10). Fig. 2 shows the effect ofa 3-hr infusion ofthese chylomicrons or saline were infused into a recipient rat, and the chylomicrons on the plasma concentrations ofapoE. In this ex- serum apoE concentrations were measured at various times periment the LoChol-chylo infusion and the saline infusion after the infusion. The feeding study indicated that the choles- served as controls and resulted in a 10% decrease in plasma terol content of the chylomicron was an important determinant apoE concentrations 3 hr after cessation ofthe infusion. Serum of the response. Therefore, two types of chylomicrons were apoE concentrations were decreased by 40% 3 hr after the in- produced-HiChol-chylos and LoChol-chylos. By infusing fusion of HiChol-chylos. These data show that the infusion of these two types ofchylomicrons, it was possible to observe the cholesterol-enriched chylomicrons also results in a marked de- effect that the cholesterol content of the chylomicron particle crease in serum apoE concentrations. had on serum apoE concentrations. Serum apoE concentrations decreased to a limited extent when LoChol-chylos or saline DISCUSSION were infused. The serum apoE concentration decreased dra- matically when HiChol-chylos were infused. These results are Previous studies from our laboratory have shown that serum consistent with the feeding experiments and support the con- apoE concentrations are significantly lower than control in eu- cept that the cholesterol content ofthe chylomicrons influences thyroid rats chronically fed olive oil/cholesterol diets (6). The serum apoE concentrations. present study extends this observation by demonstrating that It is likely that plasma apoE removal is greatly increased in the decrease in serum apoE concentrations occurs after a single the olive oil/cholesterol-fed rats. These rats have a decreased olive oil/cholesterol meal but not after the olive oil meal with serum apoE concentration (6) despite a greatly increased syn- no added cholesterol. The data presented here also show that thesis ofapoE shown by studies using cultured from feeding per se does not change the serum apoE concentrations. euthyroid rats fed high-fat/high-cholesterol diets (R. A. Davis, When the animals ate the control or olive oil meal, the serum personal communication). To understand the possible meta- apoE concentrations were not significantly lower than prefeed- bolic implications of these data, it is necessary to consider what ing values. This shows that the high cholesterol content of the is known about chylomicron metabolism. When the triglycer- high-fat meal specifically influenced the serum apoE concen- ides of the chylomicrons are hydrolyzed, two types of particles trations. The feeding study also suggests that the lowering of are produced-the core remnants and the surface remnants (25, serum apoE concentrations seen with the olive oil/cholesterol 26). The core remnants consist ofthe esterified cholesterol and Table 5. Lipid concentrations and ratios for a typical chylomicron preparation* Lipid concentrations, jig/ml Ratio x lo-3 Cholesterol Triglyceride FC EC TC Total (TC) Free (FC) Esterified (EC) (TG) TG TG TG LoChol-chylos 318 193 124 69 x 103 2.8 1.8 4.6 HiChol-chylos 1098 593 505 69 x 103 8.6 7.3 15.9 * Chylomicrons were collected from the mesenteric lymph of a rat that had been previously treated with EE. Downloaded by guest on October 1, 2021 Medical Sciences: DeLamatre et al. Proc. NatL Acad. Sci. USA 79 (1982) 1285

the remaining triglyceride along with the , free apoE from the plasma. Because the serum apoE concentration cholesterol, and apolipoprotein that remains with the core ma- is related to the cholesterol content of the chylomicrons, cho- terial to form a particle. The surface remnants consist of the lesterol seems to be intimately associated with the process and phospholipid, free cholesterol, and apolipoprotein that are re- is likely to be a controlling factor that increases serum apoE moved from the chylomicron surface in the course of chylo- removal. It is proposed that chylomicron metabolism could be micron metabolism. The core remnants are removed from the considered a part ofa physiological pathway for apoE-catabolism. plasma by the liver, whereas the surface remnants become com- ponents of HDL rat (25-27). Hepatic uptake of chylomicron The authors gratefully acknowledge the expert technical assistance core remnants is effectively inhibited by competition with an of Mr. Craig Hoffineier. This research was supported by U.S. Public HDL particle that contains only apoE (apoE,. HDLC) (1). This Health Service Grant HL 25596 and Cardiovascular Training Grant HL indicates that apoE may be a recognition site for chylomicron 07098B. B.R.K. is a recipient of National Heart, Lung, and Blood In- core remnants. ApoE is known to transfer to nascent chylo- stitute Research Fellowship Award 1-F2-HL06002. microns from HDL; subsequently, when triglyceride hydrolysis occurs, a portion ofthe apoE is removed from the chylomicron 1. Sherrill, B. C., Innerarity, T. L. & Mahley, R. W. (1980)J. Biol as a surface remnant (25, 28, 29). One explanation for our ob- Chem. 255, 1804-1807. servations is that the chylomicron core remnant from a choles- 2. Hui, D. Y., Innerarity, T. L. & Mahley, R. W. (1981) J. Biol terol-enriched chylomicron may have a greater affinity for apoE Chem. 256, 5646-5655. and, thus, retain more apoE than a chylomicron core remnant 3. Shelburne, F., Hanks, J., Meyers, W. & Quarfordt, S. (1980)J. from a relatively-cholesterol-poor chylomicron. Our observa- Clin. Invest. 65, 652-658. 4. Windler, E., Chao, Y. & Havel, R. J. (1980)J. Biol Chem. 255, tions are similar to the finding in humans that when cholesterol 5475-5480. ester concentrations in very low density lipoproteins (density, 5. Windler, E., Chao, Y. & Havel, R. J. (1980)J. Biol Chem. 255, <1.006 g/ml; VLDL) increase due to transfer from HDL, 8303-8307. VLDL apoE concentrations increase (30). Therefore, when the 6. DeLamatre, J. G. & Roheim, P. S. (1981) J. Lipid Res. 22, 297-306. core remnant from a relatively cholesterol-enriched chylomi- 7. Cooper, A. D. & Yu, P. Y. S. (1978)J. Lipid Res. 19, 635-643. cron is taken up by the liver, more apoE per particle is removed 8. Sherrill, B. C. & Dietschy, J. M. (1978) J. Biol Chem. 253, from the serum. This mechanism may at least partially explain 1859-1867. the lowering of serum apoE concentrations in the olive oil/ 9. Wu, A.-L. & Windmueller, H. G. (1978) J. Biol Chem. 253, cholesterol group. 2525-2528. 10. Krause, B. R., Sloop, C. H., Castle, C. K. & Roheim, P. S. Although part of the decrease in serum apoE concentration (1981) J. Lipid Res. 22, 610-619. can be accounted for by chylomicron core-remnant removal, 11. Davis, R. A. & Roheim, P. S. (1978) Atherosclerosis 30, 293-299. this probably does not fully explain the decrease. It is probable 12. Imaizumi, K., Havel, R. J., Fainaru, M. & Vigne, J. L. (1978)J. that a significant portion of the decrease in serum apoE con- Lipid Res. 19, 1038-1046. centration is due to a mechanism other than core-remnant re- 13. Krause, B. R., Sloop, C. H. & Roheim, P. S. (1981) Biochim. moval, because the serum apoE concentration continues to Biophys. Acta 665, 165-169. decrease up to 3 hr after the HiChol-chylo infusion 14. Harris, K. L. & Felts, J. M. (1970) J. Lipid Res. 11, 75-81. (Fig. 2), 15. Risser, T. A., Reaven, G. M. & Reaven, E. P. (1978) Am.J. Phys- whereas the remnant-removal process is extremely fast (7, 8). iol 234, E277-E281. The Vm. for hepatic chylomicron remnant removal in a 250-g 16. Bucolo, G. & David, H. (1973) Clin. Chem. 19, 476-482. rat is calculated to be 345 ,ug ofchylomicron cholesterol per min 17. Allain, C. C., Poon, L. S., Chan, C. S. G., Richmond, W. & Fu, (7, 8), whereas the maximum amount ofchylomicron cholesterol P. C. (1974) Clin. Chem. 20, 470-474. infused in this study-was 29 ,ug of cholesterol per min. Thus, 18. Krause, B. R. & Hartman, A. D. (1978)J. Lipid Res. 19, 774-777. remnant removal is not 19. Cham, B. F., Harwood, J. J., Knowles, B. R. & Powell, L. W. rate-limiting, and the decrease in apoE (1973) Clin. Chim. Acta 49, 109-113. concentrations seen after the infusion is due to a mechanism 20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. other than chylomicron core-remnant removal. (1951)1. Biol Chem. 193, 256-275. The metabolism of cholesterol-enriched chylomicrons may 21. Sata, T., Havel, R. J. & Jones, A. L. (1972) J. Lipid Res. 13, result in the formation of surface remnants that increase the 755-768. removal of HDL apoE. Chylomicrons with high cholesterol 22. Zar, J. H. (1974) in Biostatistical Analysis (Prentice-Hall, Engle- an wood Cliffs, NJ), pp. 133-139, 151-154. concentrations have increased quantity of free cholesterol 23. Shapiro, A. L., Vinuela, E. & Maizel, J. V., Jr. (1967) Biochem. (Table 5). Therefore, the surface remnants produced from the Biophys. Res. Commun. 28, 815-820. cholesterol-enriched chylomicrons have a high cholesterol con- 24. Bar-On, H., Roheim, P. S. & Eder, H. A. (1976)J. Clin. Invest. tent and bring a large quantity ofcholesterol to HDL (25, 26). 57, 714-721. This loading of HDL with cholesterol may result in the for- 25. Tall, A. R. & Small, D. M. (1978) N. Engl J. Med. 299, mation ofa specific apoE-enriched HDL particle. This particle 1232-1236. 26. Redgrave, T. G. & Small, D. M. (1979) J. Clin. Invest. 64, may be rapidly removed without accumulating in the plasma, 162-171. resulting in decreased HDL apoE and cholesterol concentra- 27. Redgrave, T. G. (1970)J. Clin. Invest. 49, 465-471. tions (Fig. 1 and Table 4). This implies that apoE also may have 28. Robinson, S. F. & Quarfordt, S. H. (1978) Biochim. Biophys. a role in returning HDL cholesterol to the liver. The mechanism Acta 541, 492-503. proposed above is consistent with the observation that HDL 29. Imaizumi, K., Fainaru, M. & Havel, R. J. (1978)J. Lipid Res. 19, apoE concentrations increase 1 hr after removing the liver from 712-722. a rat 30. Glomset, J. A., Mitchell, C. D., King, W. C., Applegate, K. A., (31). However, decreased apoE synthesis cannot be ruled Forte, T., Norum, K. R. & Gjone, E.. (1980) Ann. N.Y. Acad. Sci. out by the present study. 348, 224-243. From these results it is concluded that the metabolism of 31. Davis, R. A., Hartman, A. D., Dory, L., Van Lenten, B. J. & cholesterol-enriched chylomicrons increases the removal of Roheim, P. S. (1981) Biochim. Biophys. Acta 665, 154-164. Downloaded by guest on October 1, 2021