Proc. Natl Acad. Sci. USA Vol. 79, pp. 1282-1285, February 1982 Medical Sciences Regulation of serum apolipoprotein E metabolism: Role of chylomicron metabolism (chylomicron remnant removal/high density lipoprotein apolipoprotein E/hepatic apolipoprotein E receptor/apolipoprotein E removal/ high-fat, high-cholesterol 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 lymph 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 lymphatic system, and the duodenum was cannulated Apolipoprotein E (apoE) plays a role in lipoprotein recognition for the infusion of lipid 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 triglyceride 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. Triglycerides 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). Protein 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 lipoproteins (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.