Communication Vol. 255, No. 22, Issue of November 25, pp. 10547-10550. 1980 Printed in U.S.A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Effects of 17a-Ethinyl Estradiol on the Serum Lipoproteins of Cholesterol-fed Diabetic Rats*

(Received for publication, August 4, 1980, and in revised form, September 12, 1980)

Cynthia M. Arbeeny and Howard A. Eder With the technical assistance of Diane Edelstein

From the Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461

Large doses of 17a-ethinyl estradiol were adminis- tered to rats made diabetic by administration of strep- tozotocin and fed a diet containing 2% cholesterol and 1% cholic acid. Before estrogen treatment, these rats were severely hypercholesterolemic and had high con- centrations of lipoproteins of d < 1.006 g/ml (very low density lipoproteins, VLDL), d = 1.006 to 1.03 g/ml (intermediate density lipoproteins, IDL), and d = 1.03 to 1.063 g/ml (low density lipoproteins, LDL), and low concentrations of lipoproteins of d = 1.063 to 1.21 g/ml (high density lipoproteins, HDL). The VLDL contained two populations of particles with a mean diameter of 1150 and 350 A, respectively. The IDL contains only the latter particles. These particles are unusual in that they contain apolipoprotein A-I in addition to their usual complement of apolipoproteins. After estrogen treat- ment, the concentrations of both large and smaller particles in VLDL were greatly decreased as were the concentrations of IDL and LDL; apo A-I was no longer present in the residual VLDL and IDL. HDL phospho- lipid, protein, and cholesterol increased 7-fold, 4-fold, and 2-fold, respectively. Studies by others have shown that large doses of estrogen enhance uptake of LDL by the liver and this is associated with an increase in the saturable binding sites for LDL on liver membranes. The present studies suggest that these binding sites recognize lipoproteins other than LDL. Furthermore, the enhanced uptake of these lipoproteins appears to result in transfer of their surface components to HDL.

Profound hypolipidemia can be produced in rats by admin- istration of 17a-ethinyl estradiol in high doses (1,2). This has been shown by Chao et al. (2) to be due to enhanced uptake of LDL’ containing apo B and HDL containing apo E by the liver. Kovanen et al. (3) found that membranes prepared from livers of such estrogen-treated rats had a 3- to 10-fold increase in saturable binding sites for human LDL. It was speculated that this binding site may also function in the uptake of chylomicron and VLDL remnants which are known to be

* This study was supported by Research Grant HL 02965 and Training Grant HL 07383 from the National Institutes of Health and by a grant from The Barbra Streisand Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: LDL, low density lipoproteins; VLDL, very low density lipoproteins; IDL, intermediate density lipoproteins; HDL, high density lipoproteins; SDS, sodium dodecyl sulfate.

cleared rapidly by the liver and contain apo B and apo E (2, 3). We have recently shown that the feeding of a diet contain- ing cholesterol and cholic acid to rats made diabetic by administration of streptozotocin produces marked hypercho- lesterolemia characterized by high concentrations of VLDL and IDL with a reduction in the concentration of high density lipoproteins (4). The VLDL and IDL were unique in that they contained apolipoproteins A-I and A-IV in addition to apoli- poproteins B, E, and C, which are the usual constituents of rat VLDL. The high density lipoproteins from these rats were deficient in apolipoprotein E. Since previous studies of estro- gen administration were performed using normolipidemic chow-fed rats, we have investigated the effects of administra- tion of 17a-ethinyl estradiol to diabetic and nondiabetic rats with hyperlipoproteinemia produced by feeding cholesterol and cholic acid.

EXPERIMENTAL PROCEDURES

Materials-Streptozotocin and 17a-ethinyl estradiol were obtained from Sigma Chemical Co. (St. Louis, MO).

Animals-Male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 175 to 250 g were made diabetic by administration of streptozotocin, at a dose of 45 mg/kg of body weight (5). They were then fed a semipurified diet containing 21% casein, 50% corn starch, 10% cellulose, 10% lard, 2% cholesterol, and 1% cholic acid, plus vitamins and minerals (Teklad Mills, Madison, WI) (4). A second group was fed this diet but did not receive streptozotocin. An addi- tional group of nondiabetic rats was fed standard Purina Chow. After 3 weeks on the cholesterol or chow diets, the rats were injected subcutaneously with 17a-ethinyl estradiol dissolved in propylene gly- col at a dose of 5 mg/kg of body weight, as described by Chao et al. (2). Equal numbers of rats from each group were sham-injected with propylene glycol. After 7 days of treatment, the rats were exsanguin- ated from the abdominal aorta under light diethyl ether anesthesia.

Lipoprotein Separation and Apoprotein Characterization-Se- rum was fractionated into VLDL (d < 1.006 g/ml), IDL (d = 1.006 to 1.03 g/ml), LDL (d = 1.03 to 1.063 g/ml), and HDL (d = 1.063 to 1.21 g / d ) by sequential ultracentrifugation (6) and washed by recentrif- ugation at the higher density limit. The lipoprotein fractions were dialyzed, and aliquots containing 25 to 100 pg of protein were dried in uucuo and extracted with ethanol/ether (l:l, v/v). The apoproteins were dissolved in buffer and separated by SDS-slab gel electropho- resis according to the method of Swaney and Kuehl (7) utilizing gels with a gradient of 3 to 27% acrylamide.

Apoprotein Quantitation-Quantitation of apo B, apo A-IV, apo E, and apo A-I in rat serum was performed by the quantitative immunoelectrophoresis method of Laurell (8) as modified by Roheim and Vega (9). For the determination of apo A-I and apo E, the agarose contained 1% Nonidet P-40 (Sigma).

Chemical Analyses-Serum glucose was determined by an enzy- matic method (Statzyme glucose, Worthington Biochemicals, Free- hold, NJ). Cholesterol was determined by the method of Abell et al. (10); phospholipids were determined by the method of Zilversmit and Davis (11). Protein content of lipoprotein fractions was assayed by the method of Lowry et al. (12) as modified by Sata et al. (13).

RESULTS

Chow-fed, cholesterol-fed, and cholesterol-fed diabetic rats treated with l7a-ethinyl estradiol for 7 days exhibited marked decreases in serum cholesterol and phospholipid concentra- tions (Table I) with no change in serum glucose. Daily meas- urement of serum cholesterol showed that the maximal de- crease in serum cholesterol concentrations was attained after 3 days of estrogen administration.

The amount of protein recovered in VLDL, IDL, and LDL was markedly reduced in each of the three groups of rats

10547

10548 Effects of Estrogen on Lipoproteins of Cholesterol-fed Rats

treated with estradiol (Table 11). VLDL protein was decreased by 95% in each group. IDL protein was decreased by 954 in the cholesterol-fed diabetic and nondiabetic rats. LDL could not be detected in the chow-fed rats after treatment and was reduced to levels below that of untreated chow-fed rats after treatment of cholesterol-fed diabetic and nondiabetic rats. HDL protein decreased by 83% in the chow-fed rats, decreased to a lesser extent in the cholesterol-fed nondiabetic rats, and increased 4-fold in the cholesterol-fed diabetic rats.

The cholesterol in VLDL and IDL decreased to the same extent as the protein in the cholesterol-fed rats. However, in the cholesterol-fed diabetic rats, the decrease in cholesterol in VLDL was considerably greater than the decrease in protein, suggesting that the particles present after treatment are smaller, and this was confirmed by electron microscopy. The ratio of cholesterol to protein in IDL was not affected by treatment. The cholesterol in HDL decreased in the chow-fed and cholesterol-fed group but increased 2-fold in the choles- terol-fed diabetic rats. Phospholipids decreased in the chow- fed rats, increased slightly in the cholesterol-fed rats, but increased 7.5-fold in the cholesterol-fed diabetic rats.

Quantitation of apoproteins by rocket immunoelectropho- resis indicated that apo B and apo E were greatly reduced in all estradiol-treated groups (Table 111). However, apo E de- creased less in the cholesterol-fed diabetic rats. Apo A-IV was also reduced in the treated groups but to a lesser extent in the cholesterol-fed diabetic rats. Serum apo A-I levels were de- creased in the chow-fed and cholesterol-fed nondiabetic rats but remained essentially unchanged in the cholesterol-fed diabetic rats.

TABLE I Serum cholesterol and phospholipid concentrations in untreated

and estradiol-treated rats Cholesterol" Phospholipidh

mg/dI Chow

Untreated 37.0 & 5.4 80.0 Estradiol-treated 8.7 f 4.6 27.8

Cholesterol-fed Untreated 520 f 157 196 Estradiol-treated 31.7 f 15.6 58.8

Cholesterol-fed diabetic Untreated 1555 f 159 480 Estradiol-treated 202 f 112 216

" Mean f S.D., n = 4. Pooled aliquots from four rats.

SDS-gel electrophoresis indicated that, following estradiol administration, the VLDL and IDL of cholesterol-fed diabetic and nondiabetic rats no longer contained apo A-I; however, the A-I in the HDL of the diabetic rats increased markedly

TABLE 111 Effect of estradiol on serum apoprotein concentrations

Apoproteins were measured by quantitative immunoelectrophore- sis with each value representing the mean of two to four determina- tions. Concentration of each apoprotein in untreated rats (not shown in table) is expressed in arbitrary units with values in the chow-fed rats assigned a value of 100. For untreated cholesterol-fed rats, these values were: apo B, 748; A-IV, 145; E, 115; and A-I, 200. For untreated cholesterol-fed diabetic rats, these values were: apo B, 1635; A-IV. 183; E, 285; and A-I, 320. The values shown in the table represent the percentage of change in apoprotein concentration following treat- ment.

B A-IV E A-1 % change

Chow -87.0 -65.6 -96.9 -&.O Cholesterol-fed -87.4 -54.8 -93.5 -92.0 Cholesterol-fed diabetic -89.9 -27.4 -81.6 +10.0

4-IV

E

9- I

1 2 3 4 5 6 7 8 9 1 0 FIG. 1. SDS-gradient gel electrophoresis of delipidated li-

poproteins. An aliquot containing 75 pg of lipoprotein protein was applied to each position, except positions 5 and 6, where 50 pg was applied. Positions 1, 3, 5, and 7 are VLDL, IDL, LDL, and HDL, respectively, from cholesterol-fed diabetic rats treated with 17a-eth- inyl estradiol. Positions 2,4,6. and 8 are VLDL, IDL, LDL, and HDL from untreated cholesterol-fed diabetic rats. Position 9 contains HDL from chow-fed rats. Position 10 contains bovine serum albumin.

TABLE I1 Effect of I7a-ethinyl estradiol administration on serum lipoproteins

Each value was obtained from one pool of serum from four rats. VLDL IDL LDL HDL

Pr" Ch Pr Ch Pr Ch Pr Ch PI

mg/dl Chow

Untreated 6.6 2.5 - - 5.0 2.9 58.3 26.2 25.6 Estradiol-treated 1 .o - - - - 0.2 9.7 1.5 4.6

h

Cholesterol-fed Untreated 42.3 215 17.8 43.1 7.4 12.5 25.6 11.7 7.6 Estradiol-treated 2.6 14.9 0.8 2.5 3.4 3.3 7.3 3.6 10.7

Cholesterol-fed diabetic Untreated 61.3 706 101 38 1 63.8 118 21.0 19.6 2.4 Estradiol-treated 7.9 46.1 5.7 24.3 5.0 10.2 82.1 48.6 18.0

The abbreviations used are: Pr, protein; Ch, cholesterol; PI, phospholipid. -. not detectable.

Effects of Estrogen on Lipoproteins of Cholesterol-fed Rats 10549

(Fig. 1). Apo B in VLDL and IDL was present as two bands both in the treated and untreated rats. In the LDL of un- treated rats, apo B was present largely as the band of lower molecular weight, and this band decreased on treatment. The HDL also contained the lower molecular weight apo B. Apo E was present in VLDL and IDL both before and after estrogen administration. The LDL fraction was not pure since it contained, in addition to apo B, apo E and apo A-I. Follow- ing treatment, the A-I decreased but was not absent, suggest- ing that HDL was present in the LDL. The presence of apo E especially after treatment suggests that this fraction contains IDL, and the profound drop in lipid content in this fraction after treatment is consistent with this. We had previously noted that the HDL of cholesterol-fed rats, both diabetic and nondiabetic, had a reduced content of apo E, and this was further diminished by estrogen administration. C- apopro- teins, which were barely detectable in VLDL and IDL after treatment, were present in HDL after estrogen treatment.

DISCUSSION

These studies demonstrate that, in addition to causing reduction in LDL and HDL concentrations in chow-fed rats, administration of 17a-ethinyl estradiol also enhances the re- moval of the abnormal VLDL and IDL present in cholesterol- fed rats. In the cholesterol-fed diabetic rat, VLDL is heterog- enous and contains large particles with diameters ranging from 950 to 1600 A and smaller particles with diameters ranging from 300 to 500 A.* Both the large and the small particles contain apo B, apo E, apo A-I and C- apoproteins. These particles differ in their lipid composition, with the larger particles containing more than 25% triglyceride, whereas in the smaller particles the triglyceride is replaced by cholesteryl esters (4). Wisse (14) and Fraser et al. (15) have found the pore diameter of the fenestration in the endothelial lining of the liver sinusoids to average loo0 A. Fraser et al. (15) have shown that particles exceeding this size are excluded from the space of Disse. Many of the large particles in the VLDL of cholesterol-fed rats would thus be excluded from the space of Disse. Unless estrogen administration alters these pore sizes, it would be necessary for the large particles to be metabolized to smaller particles before they are removed by the liver. This could occur following lipolysis of the triglycer- ide in these particles by lipoprotein lipase. However, admin- istration of estrogen has been reported to decrease lipoprotein lipase activity (16, 17). Furthermore, there is considerable evidence that diabetes reduces lipoprotein lipase activity in man and also in rats (18, 19) so that it is unlikely that the conversion of the large particles to smaller particles could result from increased lipolytic activity due to estrogen admin- istration. The accelerated removal of the smaller particles induced by estrogen treatment may promote the conversion of the large particles to the smaller particles. The particles remaining in the VLDL fraction after estrogen administration differ from those present in the untreated animals both in the absence of A-I and in size. Most of the residual particles are similar in size to IDL, and this change is consistent with the decrease in the cholesterol/protein ratio.

The IDL (d = 1.006 to 1.03 g / d ) fraction has been found by negative stain electron microscopy and by agarose column chromatography to be homogenous, consisting of particles 300 to 500 A in diameter. These particles are similar in size and co-chromatograph with the smaller particles found in VLDL. They are similar in size to remnants produced by injection of chylomicrons and VLDL into functionally hepatectomized rats (20). The particles differ from remnants in their low

* C. M. Arbeeny and D. H. Handley, unpublished observations.

triglyceride and high cholesteryl ester content and also in that they contain apo A-I and C- apoproteins. Remnants are rap- idly removed from the liver by a saturable process (21, 22). Studies by Kris-Etherton and Cooper (23) have suggested that, in hypothyroid rats fed high cholesterol diets, remnants accumulate in plasma because of impaired removal due to partial saturation of the removal system (22, 23). Estrogen administration resulting in an increase in the number of binding sites apparently enhances the uptake of IDL from cholesterol-fed rats by the liver. In contrast to the VLDL, the residual particles in the IDL fraction are unchanged in their cholesterol/protein ratio, but they also lose apo A-I and the C- apoproteins. The reason for this alteration in composition is not readily explained, since it is unlikely that lipolysis of the small amount of triglyceride (2.1%) present in this fraction could alter the particle sufficiently to cause removal of their surface components.

The loss of A-I from VLDL and IDL and the increase in phospholipid, cholesterol, and protein in the HDL of the cholesterol-fed diabetic rats after estrogen administration sug- gest that certain surface components of the VLDL and IDL are transferred to HDL, while apo B and apo E are removed by the liver. This is supported by the immunochemical assays showing reduction in concentration of apo B and apo E, moderate reduction in apo A-IV, and no reduction in the concentration of apo A-I. The changes in HDL are similar to those described by Redgrave and Small (24) and by Tall et al. (25) who showed that, immediately after the injection of rat intestinal chylomicrons, transfer of phospholipid and apo A-I to HDL occurred. Similar studies were performed in man by Schaefer et al. (26), who demonstrated net transfer of apo A- I from injected chylomicrons to HDL. Our findings suggest that estradiol administration to cholesterol-fed diabetic rats increases the catabolism of particles resembling chylomicrons and their remnants and results in transfer of their surface components to HDL. Thus, the accelerated catabolism of lipoproteins induced by estrogen administration reveals proc- esses not apparent in the steady state.

Acknowledgments-We wish to thank Dr. Dean A. Handley for performing electron microscopy, Dr. Paul S. Roheim for anti-apo E serum, and Mrs. Joan Clarke for secretarial assistance.

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