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Metabolic regulation of plasma apolipoprotein E by estrogen and progesterone in the baboon (Papio sp)
Metabolic
Regulation
Rampratap
S. Kushwaha,
of Plasma Apolipoprotein E by Estrogen and Progesterone in the Baboon (Papio sp) David M. Foster, P. Hugh R. Barrett, K. Dee Carey, and Michael
G. Bernard
Apolipoprotein (apo) E plays an important role in the metabolism of lipoproteins. To determine the effects of estrogen and progesterone on plasma levels and metabolism of apo E, we used 12 ovariectomized baboons fed a cholesterol- and fat-enriched diet. These baboons were divided into four groups and treated with estrogen, progesterone, estrogen + progesterone, and a placebo control. After 10 months, although the lipid levels were not different among the treatment groups, low-density lipoprotein (LDL)/high-density lipoprotein (HDL) ratios in the estrogen + progesterone group were significantly lower than those in the control and progesterone groups. Estrogen alone or in combination with progesterone decreased plasma apo E levels significantly compared with those in the control group. Plasma apo E levels in the progesterone group were similar to those in the control group. In all groups, most ( >60%) of the apo E was present in HDL. HDL apo E concentrations in the estrogen and estrogen + progesterone groups were significantly lower than those in the control and progesterone groups. To determine the metabolic mechanisms of these changes in apo E levels, turnover studies were conducted by injection of iodinated apo E-labeled very-low-density lipoprotein (VLDL) and HDL. Residence times were calculated using multicompartment modeling. Progesterone alone and in combination with estrogen decreased residence times of apo E injected in both HDL and VLDL compared with estrogen alone and control groups. Progesterone alone also increased the apo E production rate compared with other groups. On the other hand, estrogen decreased the production rate of apo E in HDL and VLDL and, in addition, decreased the residence time of VLDL apo E compared with the control group. When estrogen was administered with progesterone, the production rate of apo E was not decreased and was similar to that in the control group. This observation suggests that the effect of estrogen on apo E production is counterbalanced by progesterone. Copyright 0 1991 by W.B. Saunders Company
A
POLIPOPROTEIN (apo) E was first identified by Shore and Shore’ as a constituent of very-low-density lipoproteins (VLDL) and since it was rich in arginine, they termed it arginine-rich protein. Utermann’ also isolated a similar apolipoprotein from VLDL that was rich in arginine and called it apo E. Utermann et al’ later described genetic poiymorphism of apo E and reported that one of the isoforms of apo E (apo E-II) was associated with type III hyperlipoproteinemia, which leads to premature atheroaclerosis.‘~’ Another apo E isoform (apo E-IV) has been reported to be associated with an increased concentration of low-density lipoproteins (LDL) in plasma.6 According to this study, up to 30% of the variability in LDL cholesterol was attributable to apo E polymorphism.6 It is well established that apo E-III and E-IV are recognized by two high-affinity hepatic receptors, which bind and internalize apo E-containing lipoproteins.‘.* Homozygous familial apo E deficiency has been suggested to be associated with type III hyperlipoproteinemia, premature atherosclerosis, and tuberoeruptive xanthomas.’ Metabolic studies in individuals with apo E deficiency have suggested that apo E is essential for the normal catabolism of triglyceride-rich lipoproteins.” Apo E thus plays an important role in the metabolism of lipoproteins. A number of factors have been shown to affect apo E level in the plasma. For example, dietary cholesterol causes pronounced elevation of apo E in the plasma due to the overproduction.‘” Similarly, sexrelated differences in the concentration of apo E in human plasma have been reported.” According to this report, serum apo E levels were significantly lower among women taking contraceptive drugs than among nonusers of similar age and VLDL-triglyceride level. Women taking estrogens without progestins also had lower serum apo-E than nonusers, but the mean difference was not statistically significant. Thus, both estrogen and progesterone affect apo E levels in the plasma. However, the mechanism by which apo E levels
Metabolism,
Vol40,No
1 (Januaryj.1991:
pp 93-100
are affected by these female sex hormones is not known. These studies were undertaken to determine the mechanism of changes induced by estrogen and progesterone in apo E levels in the plasma. We used ovariectomized and hysterectomized baboons, which have been employed successfully as models of lipoprotein metabolism and atherosclerosis.“~” MATERIALS
AND METHODS
Animals and Diet These studies were conducted using 12 adult female baboons ranging from 7 to 9 years of age. The animals selected for this experiment had similar lipoprotein profiles on both a low cholesterol. low fat diet (monkey diet, Purina, St Louis, MO) and a high cholesterol, high fat diet. The lipoprotein levels and body weights of these animals are described in Table 1. The composition of the high cholesterol. high fat diet was similar to that described earlier.‘3~‘4 Lard was used as a source of fat and provided 40% of the total calories in the diet. Cholesterol content of the diet was 1.7 mg/kcal. All the animals used for this study were ovariectomized and hysterectomized and maintained on a high cholesterol. high fat diet.
Experimental Design and Treatment Two blocks of 12 baboons estrogen and progesterone
each were used to study the effects of on lipoprotein metabolism. The first
From the Department of Physiology and Medicine, Southwest Foundation for Biomedical Research, San Antonio, TX 78228; and the Center for Bioengineering, University of Washington, Seattle, WA. Supported by National Institutes of Health Grants No. HL34982, HL28972, HL41256, and RR021 76, and contract No. HV-53030. Address reprint requests to Rampratap S. Kushwaha. PhD, Department of Physiology and Medicine. Southwest Foundation for Biomedical Research. PO Box 28147San Antonio, TX 78228-0147. Copyright 8 1991 by W.B. Saunders Cornpan! 0026-0495/9114OOl-0017$03.0010 93
94
KUSHWAHA
Table 1. Characterization
ET AL
of Animals and Their Plasma Lipids and Lipoproteins During Apo E Turnover Studies (10 months of hormone treatment) Cholesterol(mg/dL)
Treatment Group/ BaboonNo.
Body Weight (Kg)
Plasma
VLDL + IDL
LDL
HDL
Triglycerides(mg/dL) LDUHDL Cholesterol VLDL + Ratio Plasma IDL LDL HDL
APOE (mg/dL) VLDL + PiZ3lla
HDL
IDL
Control x7054
13.0
202
5
106
91
1.16
66
16
24
26
10.0
8.3
1.2
x7057
10.4
171
6
66
99
0.67
67
11
15
41
9.5
6.8
2.1
X7061
14.6
205
6
88
111
0.79
52
11
14
27
9.8
7.3
1.8
Mean
12.7
193
6
87
100
0.87
62
13
18
31
9.8
7.5
+19
20.6
+0.26*
28
k2.9
k5.5
28.4
*0.3t
20.8s
*SD
22.1
k20
*lo
1.7 20.5
Estrogen x7055
13.4
173
5
63
105
0.60
44
8
11
25
6.7
5.0
1.2
X7056
14.5
176
9
66
101
0.65
47
10
12
26
6.7
4.8
1.6
X7060
14.5
138
11
44
83
0.53
40
8
11
21
7.2
5.6
1.3
Mean
14.1
162
8
58
96
0.59
44
9
11
24
6.9
5.1
*SD
20.6
r21
kO.06
44
21.2
~0.6
22.7
23.1
212
212
kO.3
20.4
1.4 kO.2
Estrogen + progesterone X7058
13.3
136
5
28
103
0.27
37
9
9
19
5.0
3.4
1.3
X7062
13.1
143
2
34
107
0.32
60
15
18
26
9.5
5.0
2.4
X7064
14.5
197
2
62
133
0.47
56
18
16
22
6.7
5.1
1.2
Mean
13.6
159
3
41
114
0.35
51
14
14
22
7.1
4.8
1.6
*SD
20.8
218
516
k4.6
k-4.7
23.5
k2.3
k1.3
kO.7
233
k1.7
20.1
+12
Progesterone x7059
14.0
193
7
73
113
0.65
59
11
17
31
10.6
8.0
1.9
X7063
11.4
174
22
61
91
0.67
31
12
9
11
7.8
6.7
1.0 1.6
X7065
11.6
178
14
72
92
0.78
49
20
9
19
8.9
6.8
Mean
12.3
182
14
69
99
0.70
46
14
12
20
9.1
7.2
27.5
+7
kO.07
214
24.9
t4.6
-cSD
lLDi_/HDL cholesterol
21.4
210
212
210
21.4
20.7
1.5 20.5
ratios are significantly different among treatment groups (P < .Ol). LDUHDL ratios in the estrogen + progesterone group
are significantly lower (P < .Ol) than those in the control and progesterone groups. tPlasma apo E levels in the estrogen and estrogen + progesterone groups are significantly lower (P < ,051 than those in the control group. SHDL apo E concentrations in the estrogen and estrogen + progesterone groups are significantly lower (P < ,025) than those in the control and progesterone groups.
block was used to study apo B, apo A-I, and apo A-II metabolism as described earlier.‘3.‘4 The second block was used to study apo E metabolism. Animals in each group were randomly divided into four treatment groups: untreated control, estrogen group, progesterone group, and estrogen + progesterone group (three baboons per group). The untreated control group was injected weekly with cottonseed oil (estradiol vehicle) and fed a fig bar daily. The estrogen group was treated with estradiol (UpJohn, Kalamazoo, Ml) injected intramuscularly at 100 ug/‘kg/wk and was also fed a fig bar daily. The progesterone group was treated with progesterone at 3 mgkg/d in a fig bar and injected with the estrogen vehicle weekly. The estrogen + progesterone group was treated with both estradiol and progesterone as in the estrogen and the progesterone groups. Treatments were given for 3 weeks and then withdrawn for the fourth week. This schedule was maintained for approximately 18 months. The apo E metabolic studies were conducted 10 months after starting the treatment. The protocol for this experiment was approved by the institutional Animal Research Committee. The Southwest Foundation for Biomedical Research is accredited by the American Association for Accreditation of Laboratory Animal Care and is registered with the US Department of Agriculture. Isolation and Labeling of apo E Apo E for turnover studies was isolated from VLDL + intermediate-density lipoprotein (IDL) obtained from plasma of cholesterol-fed donor baboons. Four baboons were anesthetized with
ketamine HCl (10 mgkg) and bled via the femoral vein (50 mL each). Blood was received in tubes containing EDTA (1 mg/mL) and was centrifuged at a low speed to obtain plasma. Plasma was adjusted to a density of 1.019 g/mL by adding solid potassium bromide and ultracentrifuged at 45,000 rpm in a 60 Ti rotor at 6°C for 22 hours using Beckman ultracentrifuge model LS-70 (Beckman, Palo Alto, CA). After ultracentrifugation, the VLDL + IDL fraction was obtained by tube slicing.” The VLDL + IDL fraction was ultracentrifuged again after dilution with saline (d = 1.019 g/mL) under similar conditions. The purified fraction of VLDL + IDL was delipidated first with ethanol:ether 3:1, and then with increasing concentrations of ether, and finally with ether alone. The delipidated apoproteins were separated by a DEAE cellulose column as described by Shore and Shore.’ The purity of apo E was checked by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis.‘5 Iodination of apo E was performed by the iodine monochloride procedure’6 using 0.1 to 0.2 mg of protein dissolved in 0.20 mL buffer containing 0.1 mol/L glycine and 0.5 mol/L sodium chloride (pH 10). Most of the free iodine was removed by chromatography on a Sephadex G-25 column equilibrated with 0.15 mol/L saline containing 0.01 mol/L Tris and 0.001 mol/L EDTA (pH 7.4) and by dialyzing the pooled apo E fractions against the same buffer with five to six changes of buffer. Most (> 98%) of the iodinated apo E was TCA precipitable. The specific activity of apo E was approximately 800 cpming.
HORMONAL
REGULATION
95
OF APO E METABOLISM
Incorporation of Labeled Apo E into VLDL and HDL To isolate VLDL and high-density lipoprotein (HDL) for incorporation of iodinated apo E, blood was obtained from each animal during surgery to implant venous catheters. Blood from the three animals in each treatment group was pooled and centrifuged to obtain plasma. The plasma was ultracentrifuged sequentially using a 50 Ti rotor to obtain VLDL and HDL.1”‘4VLDL and HDL were dialyzed before incubation with iodinated apo E. The iodinated apo E was incorporated into lipoproteins by a technique described previously.” In each study, a fraction of apo E was labeled with “‘1 and another with 13’I.VLDL was incubated with “‘I-labeled apo E and HDL was incubated with ‘Wabeled apo E for 4 hours at 37°C. Following incubation, VLDL and HDL were reisolated by ultracentrifugation and the lipoproteins obtained by tube slicing. VLDL and HDL were dialyzed against saline with several changes of dialysate and used for injections. FL DL and HDL apo E Turnover Procedure Six baboons were studied at one time. Two weeks before turnover studies, baboons were put on a dummy tether to accustom them to the tether system. The next week, venous catheters were implanted surgically and the animals were maintained on the tether. Catheters were implanted on the day animals were started on hormone treatment. Animals were injected with iodinated lipoproteins at the beginning of the second week of the hormone treatment and the turnover studies were completed during the third week of the hormone treatment. All animals followed the same cycle of treatment and were studied at the same point of the treatment cycle. The blood for measurements of lipids and apo E was obtained prior to the injection of iodinated lipoproteins (O-hour sample). The tether allowed us to inject and bleed animals without the use of anesthesia. Each animal was injected with 3 to 5 FCi of “‘I-labeled VLDL (0.2 to 0.4 mg of VLDL protein) and 30 to 40 $Zi of “‘I-labeled HDL (1.5 to 2.0 mg of HDL protein). After injection of iodinated lipoproteins, each animal was bled to obtain h mL of blood at 5 minutes, 0.5, 0.75, 1, 2, 3, 8, 12, 24, 48, 72, 96, 120. 144, 192. and 240 hours. Blood samples at each time point were collected in tubes containing EDTA (1 mg/mL) and were centrifuged to obtain plasma. A small aliquot of plasma (0.1 mL) was counted to determine the decay of radioactivity in plasma. Another aliquot of plasma (2 mL) was used to isolate VLDL (d < 1.006 g/mL), IDL + LDL (d = 1.006 to 1.063 gimL), and HDL (d = 1.063 to 1.21 gimL) by sequential ultracentrifugation as described earlier.‘3,‘4 The radioactivity in each lipoprotein fraction and the fraction with density > 1.21 gimLwas determined by a gamma counter (Nuclear Chicago. Des Plaines, IL). To avoid quenching due to high salt concentration, the lipoproteins were diluted lo-fold with water. The radioactivity spilling over from the “‘I channel into the ‘“‘I chsnnel was subtracted using standards and the counts were corrected for isotopic decay.
(Atlantic Antibodies, Scarborough, ME). Since the apo E concentration of lipoproteins, especially in VLDL, was very low, the apo E in lipoproteins was measured by immunoblotting following separation of plasma by gradient (2% to 16%) polyacrylamide gel electrophoresis. The gradient gels were run as described by Nichols et al” and the proteins were blotted onto nitrocellulose sheets by the method of Towbin et a1.22The sheets were incubated with antibody against human apo E after blocking the nonspecific binding sites and again with iodinated secondary antibody as described.” The sheets were cut into 5-mm pieces and counted for radioactivity. The radioactivity in each lipoprotein region was calculated and was used to estimate the amount of apo E in each lipoprotein from total apo E concentration of plasma. Each analysis was performed in duplicate and the results were reproducible. Kinetic Analysis The kinetic data were analyzed using the CONSAM” computer program on a MicroVax II computer (Digital Equipment Corporation, Maynard, MA). Apo E radioactivity disappearance curves were analyzed using two- or three-compartment models to account for the multiexponential nature of the disappearance curves. Residence time (inverse of fractional catabolic rate [FCR]) describes the period of time apo E is expected to remain in the plasma, on average, prior to removal. These residence times represent the weighted mean residence time for apo E reinjected on either VLDL or HDL particles. These values, therefore, reflect the initial distribution of apo E on either HDL or VLDL. and the metabolism and conversion of apo E from and between different lipoprotein fractions. The residence times presented for HDL apo E, like those for plasma apo E, represent a weighted mean residence time for apo E on the HDL particle. The effect of labeled apo E returning to the HDL particle from other lipoprotein particles on the residence time of HDL apo E could not be determined. Due to the complex nature of the VLDL apo E radioactivity curves following the reinjection of labeled VLDL apo E, residence times for the VLDL fraction could not be determined using the simple approach used for the analysis of the plasma HDL and VLDL apo E data. The producton rates of apo E in plasma and HDL were calculated by multiplying the fractional catabolic rate by the apo E pool in plasma and HDL, respectively. The production rates were expressed as mg/kg/d. StatisticalAnalysis Lipid and apo E levels in plasma and lipoproteins and the kinetic parameters in different groups of animals were compared by ANOVA. If significant differences among these values were detected, the individual means were compared using Tukey’s test. Data in tables are presented as mean + SD. along with individual values. RESULTS
Analytical Procedures Plasma lipoproteins for the measurement of lipids and apo E were isolated by density gradient ultracentrifugation using 2.0 mL of plasma with a modified procedure of Redgrave et al’* as described earlier.” Cholesterol and triglycerides in plasma and lipoproteins were measured by the enzymatic method using assay kits (Sigma Chemical). The purity of apo E for iodination and standard was tested by polyacrylamide slab gel electrophoresis with SDS.“ The apo E in plasma was measured by electroimmunoassay as described by Laurell.‘” using antibody against human apo E
Effect of Hormone Treatment on the Concentration and Distribution of Apo E Table 1 describes the body weight, lipid and apo E levels in the plasma and lipoproteins of these animals during apo E turnover studies (10 months of hormone treatment). Animals did not differ in body weight after undergoing 10 months of treatment with pharmacological levels of sex steroid hormones. As reported earlier’3.‘” for the first block of animals treated similarly, there was no significant differ-
KUSHWAHA
96
ence in lipid levels among these treatments, but there were significant differences in LDL/HDL cholesterol ratio.13.14 The LDL/HDL cholesterol ratio in the estrogen + progesterone group was significantly lower than that in the control and progesterone groups. Plasma apo E levels in the estrogen (6.9 f 0.3 mg/dL) and estrogen + progesterone (7.1 f 2.3 mg/dL) groups were lower than in the control (9.8 k 0.25 mg/dL) and progesterone (9.1 2 1.4 mg/dL) groups. Most (> 60%) of the plasma apo E was present in HDL in all animals (Table 1). HDL apo E concentrations were significantly different among treatment groups. HDL apo E content in the estrogen and estrogen + progesterone groups was significantly less (P < .05) than HDL apo E content in the control and progesterone groups. Apo E content in apo B-containing lipoproteins (VLDL + IDL and LDL) was less than 40% of the total plasma apo E, was variable among the treatment groups, and did not differ significantly. Since the plasma samples for analysis of lipids and apo E were obtained at the same time point of the treatment cycle, these differences will not be affected by the treatment cycle. Effect of Estrogen and Progesterone on the Metabolism of apo E in HDL
The decay of radioactivity from apo E in HDL into the plasma in representative animals from the control, estrogen, estrogen + progesterone, and progesterone groups is given in Figs 1A and B, and 2A and B, respectively. The data were consistent from animals in ail groups. The residence times of plasma apo E, when injected in HDL particles, are given in Table 2. Plasma apo E residence times differed significantly (P < .05) among treatment groups. The residence times of plasma apo E (injected in HDL) in the control and estrogen groups were significantly longer than those in the progesterone or estrogen + progesterone groups. Plasma apo E decayed twice as rapidly in the progesterone and estrogen + progesterone did in the control or estrogen group.
II
0
I....I
50
.I....!.
100
150
Hours
-.
200
ET AL
The decay of radioactivity from apo E in the HDL fraction (d = 1.063 to 1.21 g/mL) in representative animals from the control, estrogen, estrogen + progesterone and progesterone groups is given in Figs 3A and B, and 4A and B, respectively. Most of the radioactivity remained in HDL at all times. A small amount of radioactivity (<20%) was transferred to VLDL, and IDL + LDL. The decay of apo E radioactivity in HDL was very similar to that of apo E in whole plasma. The residence times of HDL apo E are given in Table 2. HDL apo E residence times differed significantly (P < .OS) among treatment groups as did plasma apo E. HDL apo E residence times in the control and estrogen groups were significantly longer than those in the estrogen + progesterone and progesterone groups. Apo E residence times in HDL in the control and estrogen groups were similar to or slightly shorter than residence time for apo E in plasma. These observations suggest that most of the apo E in HDL remains as part of HDL and is removed from there. A small amount of apo E is transferred to other particles where it exchanges slowly causing plasma apo E removal to be slightly slower than that for HDL apo E. On the other hand, HDL apo E residence time in the estrogen + progesterone and progesterone groups was similar to or longer than plasma apo E residence time. These observations suggest that apo E in other lipoproteins is lost more rapidly than that in HDL, and HDL constantly loses apo E to these lipoproteins. Production rates for apo E in plasma and HDL, when iodinated apo E was injected in HDL, are given in Table 2. A greater proportion ( > 50%) of plasma apo E production appeared to be in HDL in all treatment groups. However, the proportion of total plasma apo E production in HDL was far greater (> 80%) in baboons from the control and estrogen groups than that (<60%) in baboons from the estrogen + progesterone and progesterone groups. Plasma and HDL apo E production rates in the estrogen group were significantly lower than those in other groups. On the other hand, plasma and HDL apo E production rates in the
1
2.50 0
so
100 I so Hours
’ 200
250
Fig 1. Disappearance of plasma apo E radioactivity in representative animals from the (A) control and (B) estrogen groups when injected in VLDL (0-O) and HDL (A-A).The apo E injected in VLDL was labeled with 13’1and in HDL with ‘=I.
97
HORMONAL REGULATION OF APO E METABOLISM
Fig 2. Disappearance of plasma apo E radioactivity in representative animals from the (A) estrogen + progesterone groups when injected in VLDL (fl--Cl) and HDL (A-A). The apo E in VLDL was labeled with “‘I and in HDL with ‘251.
and (6) progesterone
Table 2. Plasma Residence Times for Apo E Injected on Either HDL or VLDL Particles in Baboons VLDLApo E* Treatment
PlasmaApo E*
PlasmaApo E*
Group/
Residence
FSDt
Production
Baboon No.
Time(h)
1%)
(w/kg/d)
x7054
85.1
3.7
x7057
81.9
3.3
X7061
81.7
5.2
Mean
82.9
ZSD
+ 1.95ll
Rate
HDLApo E*
HDL Apo E
VLDL Apo Et
Production
Residence
FSDt
Production
Residence
FSDt
Rate in Plasma
Time (h)
W)
Rate (mglkgid)
Time(h)
I%1
(w/kg/d1
1.30
84.5
4.1
1.08
69.5
4.1
1.59
1.28
77.2
2.2
0.97
75.2
5.4
1.39
1.32
78.0
1.5
1.03
60.1
5.0
1.80
1.30
79.9
1.03
68.2
+-4.05
zO.O6/1
Control
20.22
1.59
+7.6§#
kO.21
Estrogen x7055
76.5
3.6
0.97
69.4
2.2
0.80
56.2
4.2
1.32
X7056
84.9
7.9
0.87
75.4
3.0
0.70
61.2
5.6
1.21
X7060
78.9
2.6
1.Ol
73.3
2.2
0.84
60.4
2.9
1.32
Mean
80.1
0.95
72.7
0.78
59.3
1.28
*SD
?4.3§8#
22.75
i-0.06tt
LO.07
*3.0§**
kO.07
Estrogen + progesterone X7058
34.0
4.9
1.62
39.8
6.9
0.94
36.5
5.0
1.51
X7062
44.0
9.2
2.38
62.4
3.3
1.06
59.3
13.4
1.77
X7064
45.2
6.7
1.64
44.9
2.5
1.25
42.1
5.7
1.76
Mean
41.1
1.88
55.5
1.08
45.9
*SD
k6.1
-0.43/l
+9.2
+0.1611
1.68
k11.9
to.15
Progesterone x7059
45.7
4.5
2.56
65.1
3.5
1.36
50.9
6.8
2.30
X7063
38.3
7.3
2.25
54.7
3.9
1.35
39.7
4.4
2.17
X7065
34.9
5.5
2.89
46.8
5.5
1.60
48.3
6.5
2.03
Mean
39.6
2.57
55.5
1.44
45.3
2.17
?SD
25.5
+0.32**
+9.2#
+0.14/j
~5.8
20.14tt
*Labeled apo E was incubated with HDL prior to reinjection. tFractional standard deviation, expressed as %. *Labeled apo E was incubated with VLDL prior to reinjection. These values describe the residence time and production rate of apo E within plasma. SValues are significantly different from progesterone and estrogen + progesterone groups (P < .05). Walue for plasma apo E residence time when injected in HDL was significantly (P < ,051 higher than that when injected in VLDL in control and estrogen groups. I/Production rates for plasma and HDL apo E (when injected as HDL) in progesterone groups ware significantly (P < .05) higher than that in other groups. Production rate in plasma in the progesterone group was also significantly higher (P < .05) than that in estrogen + progesterone group. Production rate in plasma and HDL in the control group was significantly (P < .05) higher than that in estrogen group. #Values are significantly different from estrogen VLDL residence time (P < .Ol). *Walues ttApo
are significantly different from plasma (HDL) residence time (P < .05).
E production rate in plasma (when injected as VLDL) in the estrogen group was significantly (P < .05) lower than that in other groups. Apo E
production rate in the progesterone group was significantly higher (P < .Ol) than that in control or estrogen + progesterone groups.
98
KUSHWAHA
ll....I....1....1....1.... 0 50 loo
Il....I....l....l....I....,
I
150
200
250
0
50
progesterone group were significantly higher than those in other groups. Plasma apo E production rate in the estrogen + progesterone group was also significantly (P < .05) higher than that in the control and estrogen groups. Effect of Estrogen and Progesterone on the Metabolism of Apo E in I/LDL
The decay of radioactivity from apo E in VLDL in plasma from representative animals from the control, estrogen, estrogen + progesterone, and progesterone groups is given in Figs 1A and B, and 2A and B, respectively. The data were consistent for VLDL apo E in all animals in each group. Immediately after injection, most of the radioactivity from VLDL apo E was transferred to HDL. The disappearance of radioactivity in the VLDL fraction from representative animals from the controi, estrogen, estrogen + progesterone, and progesterone groups is given in Figs 3A and B, and 4A and B, respectively. Due to a continuous exchange of apo E radioactivity between VLDL and HDL, the VLDL
100
IS0
Z(K)
250
Hours
Fig 3. Disappearance of apo E radioactivity in VLDL (O---O) and HDL (a---a) groups when injected in VLDL (‘3’1) and HDL (“I).
SO
I 100
Hours
0
ET AL
150
200
in representative
animals from the (A) control and (8) estrogen
apo E decay curves were complex, and therefore, residence times for VLDL apo E were measured for plasma only and not for the VLDL fraction. The residence times for VLDL apo E in plasma (Table 2) were considerably different among treatment groups: they were longest in the control group (68.2 5 7.6 hours), followed by the estrogen (59.3 2 2.7 hours), estrogen + progesterone (45.9 k 11.9 hours), and progesterone (45.3 + 5.8 hours) groups. VLDL apo E residence times in the control group were significantly (P < .05) longer than those in other groups. Similarly, VLDL apo E residence times in the estrogen group were significantly (P < .05) longer than those in the estrogen + progesterone and progesterone groups. VLDL apo E residence times in plasma in the control and estrogen groups were significantly (P < .05) shorter than those of plasma and HDL apo E (when apo E was injected in HDL). However, VLDL and HDL apo E residence times in plasma were similar in the estrogen + progesterone and progesterone groups.
250
0
so
Hours Fig 4. Disappearance of apo E radioactivity in VLDL (O---O) and HDL (A-A) and (B) progesterone groups when injected in VLDL (“‘I) and HDL (‘?).
in representative
I(WI Hours
150
animals from the (A) estrogen +
2.50
progesterone
HORMONAL
REGULATION
99
OF APO E METABOLISM
As with plasma HDL apo E, VLDL apo E production rates in plasma were significantly (P < .OS)different among the groups. Progesterone administered alone or with estrogen significantly (P < .05) increased VLDL apo E production rate compared with that in the control or estrogen group. On the other hand, estrogen significantly decreased (P < .05) VLDL apo E production rate in plasma compared with that of the control group. Production rates of VL,DL apo E and HDL apo E in plasma were not significantly different. However, there was a significant (P < .05) interaction with treatments. VLDL apo E production rates in plasma were slightly but consistently higher than HDL apo E production rates in plasma from baboons in 1he control and estrogen groups. In contrast, VLDL apo E production rates in plasma were slightly but consistently lower than HDL apo E production rates in plasma from baboons in the estrogen + progesterone and progesterone groups. DISCUSSION
Summary of Results
These studies present evidence that, like apo B,” apo A-I, and apo A-II metabolism,‘4 the metabolism of apo E is also influenced by estrogen and progesterone administered in pharmacological doses. Estrogen treatment alone or combined with progesterone decreased apo E content in plasma and HDL. Progesterone treatment had a profound effect on the catabolic rate of apo E. Progesterone alone or in combination with estrogen decreased residence times of apo E injected in HDL or in VLDL. On the other hand, progesterone treatment increased the apo E production rate. Since both the production and catabolism of apo E were increased by progesterone treatment, baboons in the progesterone group had levels of apo E in plasma and HDL similar to those in the control group. Estrogen treatment decreased the residence time of VLDL apo E compared with that in the control group. Estrogen treatment also decreased the production rate of apo E. Thus, increased catabolism and decreased production of apo E were responsible for the reduced level of apo E in the estrogen-treated group. Ro/e of Apo E and Its Isoform in Lipoprotein Metabolism
Ape E plays an important role in the metabolism of apo B-containing lipoproteins in the plasma.’ Since apo E is recognized by the chylomicron remnant and apo B/E receptors, it mediates the catabolism of apo E-rich chylomicron and VLDL remnants.8 Apo E has also been suggested to play an important role in the reverse cholesterol transport process by directing cholesteryl ester-enriched large HDL particles to the liver; in some cases, these particles contain only apo E and have been shown to be rapidly removed.” Apo E is also believed to be involved in the conversion of VLDL remnants into LDL.*’ The latter role for apo E has been suggested because subjects with type III hyperlipoproteinemia who lack functional isoforms of apo E (apo E-III or E-IV) cannot convert VLDL remnants to LDL and consequently VLDL remnants accumulate in
their plasma.” Apo E exists in polymorphic forms in humans.’ Apo E polymorphism in humans has been shown to regulate lipoprotein cholesterol in the plasma.h In contrast to that in humans, baboon apo E is not polymorphic, but is like apo E-IV, which does not contain cysteine.” The present studies, therefore, may explain the role of estrogen and progesterone in the regulation of the functional form of apo E and the receptors that recognize it. Effect of Estrogen and Progesterone on Metabolic Heterogeneity of Apo E in VLDL and HDL
Most of the apo E in baboons was present in HDL, consistent with the fact that baboons have very little plasma VLDL.‘*,‘j Apo E injected in HDL or VLDL particles had different residence times in plasma and lipoproteins. Apo E radioactivity in VLDL or in HDL rapidly exchanged with other plasma lipoproteins and the difference in residence times may be due to differential catabolism of apo E in various plasma lipoproteins. Similarly, it has been reported that apo E injected in vivo rapidly exchanged among plasma lipoproteinszx Most such studies have reported the removal of apo E from whole plasma.2y.3”Our studies were conducted using labeled apo E incorporated into both HDL and VLDL to determine if apo E was metabolically heterogeneous in these lipoproteins and if this was affected by estrogen and progesterone treatment. Apo E in HDL had higher residence times than that in VLDL in plasma of all animals. This observation suggests that the metabolism of apo E in VLDL and HDL is heterogeneous. Apo E is more susceptible to catabolism when it is in the VLDL particle; it is relatively more protected when it is incorporated in HDL. Estrogen treatment decreased plasma VLDL apo E residence time compared with that in the control group. As reported earlier, estrogen treatment also decreased the residence time of LDL apo B in these animals.‘” Estrogen treatment in rats has been suggested to increase LDL receptor activity.“,” Thus, the decrease in VLDL apo E residence time in estrogen treated animals may be due to the effect of estrogen on LDL receptor activity. The estrogen + progesterone and progesterone groups had much shorter residence times for apo E in VLDL and HDL than did the estrogen and control groups. However. progesterone treatment did not affect the catabolism of apo B” and, therefore, the decrease in apo E residence time in VLDL and HDL could not be due to LDL receptor activity. The effect of progesterone on VLDL apo E catabolism may be explained by one of two mechanisms. First, progesterone may induce another receptor, possibly the chylomicron receptor, which removes the larger, apo E-enriched VLDL particles, thus causing apo E to be lost rapidly by the VLDL compartment. Second, progesterone may increase VLDL production causing more apo E to be lost as a component of the VLDL remnant. Progesterone treatment increased catabolism of apoproteins of HDL in baboons.“’ Thus, the increase in catabolism of apo E in HDL may be due to the increased catabolism of HDL caused by progesterone treatment. Production of apo E was also affected by the estrogen and progesterone treatment. In general. most of the apo E
100
KUSHWAHA
ET AL
was secreted into HDL. Estrogen decreased production rate but did not affect the route of secretion into the plasma. Like in the control, most (> 80%) of the plasma apo E was secreted through HDL. In contrast, progesterone increased the production of apo E in the plasma and affected the route of secretion. Even though a greater proportion of apo E was secreted in HDL, it was considerably lower ( < 60%) than that in the estrogen or the control group. Thus, progesterone increased the production of apo E via the apo B-containing lipoproteins. The decrease of
the estrogen + progesterone group. This observation suggests that effect of estrogen on apo E production is counterbalanced by progesterone.
apo E production
of the data.
in plasma
and HDL was not observed
in
ACKNOWLEDGMENT
We thank Maggie Garcia and William Ehler for providing technical assistance for these studies. We also thank Thomas Cooper and David Weaver for bleeding and maintaining the tethered baboons. We gratefully acknowledge Jo Fletcher for editorial assistance and Dr Douglas S. Lewis for statistical analysis
REFERENCES
1. Shore VG, Shore B: Heterogeneity of human plasma very low density lipoproteins. Separation of species differing in protein components. Biochemistry 12:502-507,1973 2. Utermann G: Isolation and partial characterization of an arginine-rich apoprotein from human plasma very-low-densitylipoprotein: Apolipoprotein E. Hoppe-Seyler’s Z Physiol Chem 356:1113-1121,197s 3. Utermann G, Hess M, Steinmetz A: Polymorphism of apolipoprotein E and occurrence of dysbetalipoproteinemia in man. Nature 269:604-607.1977 4. Hazzard WR: Primary type III hyperlipoproteinemia, in Rifkind BM, Levy RI (eds): Hyperlipidemia Diagnosis and Therapy. Philadelphia, PA, Grune & Stratton, 1977, pp 137-175 5. Davignon J, Gregg RE, Sing CF: Apolipoprotein E polymorphism and atherosclerosis. Arteriosclerosis 8:1-21, 1988 6. Bouthillier D, Sing CF, Davignon J: Apolipoprotein E phenotyping with a single gel method: Application to the study of informative matings. J Lipid Res 24:1060-1069,1983 7. Mahley RW, Innerarity TL, Rall SC Jr, et al: Plasma lipoproteins: Apolipoprotein structure and function. J Lipid Res 25:1277-1294,1984 8. Mahley RW: Apolipoprotein E: Cholesterol transport protein with an expanding role in cell biology. Science 240:622-630,1988 9. Schaefer EJ, Gregg RE, Ghiselli G, et al: Familial apolipoprotein E deficiency. J Clin Invest 78:1206-1219, 1986 10. Roth RI, Gaubatz JW, Gott AM Jr, et al: Effect of cholesterol feeding on the distribution of plasma lipoproteins and on the metabolism of apolipoprotein E in the rabbit. J Lipid Res 24:1-11, 1983 11. Phillips NR, Have1 RJ, Kane JP: Sex-related differences in the concentrations of apolipoprotein E in human blood plasma and plasma lipoproteins. J Lipid Res 24:1525-1531,1983 12. Kushwaha RS, Hazzard WR, Harker LA, et al: Lipoprotein metabolism in baboons: Effect of feeding cholesterol-rich diet. Atherosclerosis 31:65-77,1978 13. Kushwaha RS, Foster DM, Barrett PHR, et al: Effect of estrogen and progesterone on the metabolism of apoprotein B in the baboon. Am J Physiol258:E172-E183,1990 14. Kushwaha RS, Foster DM, Murthy VN, et al: Metabolic regulation of apoproteins of high density lipoproteins by estrogen and progesterone in the baboon. Metabolism 39:544-552, 1990 15. Weber K, Osborne J: The reliability of molecular weight determinations by dodecylsulfate polyacrylamide gel electrophoresis. J Biol Chem 244:4406-4412, 1969 16. McFarlane AS: Efficient trace labeling of protein with iodine. Nature 182:53-54, 1958 17. Steinmetz A, Kushwaha R, Foster D, et al: The differential catabolism of apolipoprotein E in very low and high density lipoproteins. Circulation 66:11-12(48), 1982 18. Redgrave TG, Roberts DCK, West CE: Separation of
plasma lipoproteins by density-gradient ultracentrifugation. Anal Biochem 65:42-49.1975 19. McGill HC Jr, McMahan CA, Kushwaha RS, et al: Dietary effects on serum lipoproteins of dyslipoproteinemic baboons with high HDL,. Arteriosclerosis 6:651-663, 1986 20. Laurel1 CB: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 15:45-52, 1966 21. Nichols AV, Blanche PJ, Gong EL: Gradient gel electrophoresis of human plasma high density lipoproteins, in Lewis LA (ed): CRC Handbook of Electrophoresis, vol 3. Lipoprotein Methodology and Human Studies. Boca Raton, FL, CRC, 1983, pp 29-47 22. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Nat1 Acad Sci USA 76:4350,1979 23. Berman M, Weiss MF: SAAM Manual. DHEW Publication No. (NIH)78-180, Washington, DC, US Government Printing Office 24. Sherrill BC, Innerarity TL, Mahley RW: Rapid hepatic clearance of the canine lipoproteins containing only the E apoprotein by a high affinity receptor: Identity with the chylomicron remnant transport process. J Biol Chem 255:1804-1807,198O 25. Ehnholm C, Mahley RW, Chappell DA, et al: Role of apolipoprotein E in the lipolytic conversion of B-very low density lipoproteins to low density lipoproteins in type III hyperlipoproteinemia. Proc Nat1 Acad Sci USA 81:5566-5570,1984 26. Innerarity TL, Hui DY, Bersot TP, et al: Type III hyperlipoproteinemia: A focus on lipoprotein receptor-apolipoprotein E2 interactions. Adv Exp Med Biol201:273-2881986 27. Hixson JE, Cox LA, Borenstein S: The baboon apolipoprotein E gene: Structure, expression, and linkage with the gene for apolipoprotein C-I. Genomics 2:315-323,1988 28. Weisgraber KH, Mahley RW, Assman G: The rat argininerich apoprotein and its redistribution following injection of iodinated lipoproteins into normal and hypercholesterolemic rats. Atherosclerosis 28:140-212,1977 29. Wong L, Rubinstein D: Turnover of apo E in normal and hypercholesterolemic rats. Atherosclerosis 34:249-258,1979 30. Van Hooft F, Have1 RJ: Metabolism of chromatographically separated rat serum lipoproteins specifically labeled with “‘Iapolipoprotein E. J Biol Chem 256:3963-3968,198l 31. Kovanen PT, Brown MS, Goldstein JL: Increased binding of low density lipoprotein to liver membranes from rats treated with 17a-ethinyl estradiol. J Biol Chem 254:11367-l 1373, 1979 32. Windler EE, Kovanen PT, Chao YS, et al: The estradiolstimulated lipoprotein receptor of rat liver. A binding site that mediates the uptake of rat lipoproteins containing apoprotein B and E J Biol Chem 255:10464-10471,198O
Regulation
Rampratap
S. Kushwaha,
of Plasma Apolipoprotein E by Estrogen and Progesterone in the Baboon (Papio sp) David M. Foster, P. Hugh R. Barrett, K. Dee Carey, and Michael
G. Bernard
Apolipoprotein (apo) E plays an important role in the metabolism of lipoproteins. To determine the effects of estrogen and progesterone on plasma levels and metabolism of apo E, we used 12 ovariectomized baboons fed a cholesterol- and fat-enriched diet. These baboons were divided into four groups and treated with estrogen, progesterone, estrogen + progesterone, and a placebo control. After 10 months, although the lipid levels were not different among the treatment groups, low-density lipoprotein (LDL)/high-density lipoprotein (HDL) ratios in the estrogen + progesterone group were significantly lower than those in the control and progesterone groups. Estrogen alone or in combination with progesterone decreased plasma apo E levels significantly compared with those in the control group. Plasma apo E levels in the progesterone group were similar to those in the control group. In all groups, most ( >60%) of the apo E was present in HDL. HDL apo E concentrations in the estrogen and estrogen + progesterone groups were significantly lower than those in the control and progesterone groups. To determine the metabolic mechanisms of these changes in apo E levels, turnover studies were conducted by injection of iodinated apo E-labeled very-low-density lipoprotein (VLDL) and HDL. Residence times were calculated using multicompartment modeling. Progesterone alone and in combination with estrogen decreased residence times of apo E injected in both HDL and VLDL compared with estrogen alone and control groups. Progesterone alone also increased the apo E production rate compared with other groups. On the other hand, estrogen decreased the production rate of apo E in HDL and VLDL and, in addition, decreased the residence time of VLDL apo E compared with the control group. When estrogen was administered with progesterone, the production rate of apo E was not decreased and was similar to that in the control group. This observation suggests that the effect of estrogen on apo E production is counterbalanced by progesterone. Copyright 0 1991 by W.B. Saunders Company
A
POLIPOPROTEIN (apo) E was first identified by Shore and Shore’ as a constituent of very-low-density lipoproteins (VLDL) and since it was rich in arginine, they termed it arginine-rich protein. Utermann’ also isolated a similar apolipoprotein from VLDL that was rich in arginine and called it apo E. Utermann et al’ later described genetic poiymorphism of apo E and reported that one of the isoforms of apo E (apo E-II) was associated with type III hyperlipoproteinemia, which leads to premature atheroaclerosis.‘~’ Another apo E isoform (apo E-IV) has been reported to be associated with an increased concentration of low-density lipoproteins (LDL) in plasma.6 According to this study, up to 30% of the variability in LDL cholesterol was attributable to apo E polymorphism.6 It is well established that apo E-III and E-IV are recognized by two high-affinity hepatic receptors, which bind and internalize apo E-containing lipoproteins.‘.* Homozygous familial apo E deficiency has been suggested to be associated with type III hyperlipoproteinemia, premature atherosclerosis, and tuberoeruptive xanthomas.’ Metabolic studies in individuals with apo E deficiency have suggested that apo E is essential for the normal catabolism of triglyceride-rich lipoproteins.” Apo E thus plays an important role in the metabolism of lipoproteins. A number of factors have been shown to affect apo E level in the plasma. For example, dietary cholesterol causes pronounced elevation of apo E in the plasma due to the overproduction.‘” Similarly, sexrelated differences in the concentration of apo E in human plasma have been reported.” According to this report, serum apo E levels were significantly lower among women taking contraceptive drugs than among nonusers of similar age and VLDL-triglyceride level. Women taking estrogens without progestins also had lower serum apo-E than nonusers, but the mean difference was not statistically significant. Thus, both estrogen and progesterone affect apo E levels in the plasma. However, the mechanism by which apo E levels
Metabolism,
Vol40,No
1 (Januaryj.1991:
pp 93-100
are affected by these female sex hormones is not known. These studies were undertaken to determine the mechanism of changes induced by estrogen and progesterone in apo E levels in the plasma. We used ovariectomized and hysterectomized baboons, which have been employed successfully as models of lipoprotein metabolism and atherosclerosis.“~” MATERIALS
AND METHODS
Animals and Diet These studies were conducted using 12 adult female baboons ranging from 7 to 9 years of age. The animals selected for this experiment had similar lipoprotein profiles on both a low cholesterol. low fat diet (monkey diet, Purina, St Louis, MO) and a high cholesterol, high fat diet. The lipoprotein levels and body weights of these animals are described in Table 1. The composition of the high cholesterol. high fat diet was similar to that described earlier.‘3~‘4 Lard was used as a source of fat and provided 40% of the total calories in the diet. Cholesterol content of the diet was 1.7 mg/kcal. All the animals used for this study were ovariectomized and hysterectomized and maintained on a high cholesterol. high fat diet.
Experimental Design and Treatment Two blocks of 12 baboons estrogen and progesterone
each were used to study the effects of on lipoprotein metabolism. The first
From the Department of Physiology and Medicine, Southwest Foundation for Biomedical Research, San Antonio, TX 78228; and the Center for Bioengineering, University of Washington, Seattle, WA. Supported by National Institutes of Health Grants No. HL34982, HL28972, HL41256, and RR021 76, and contract No. HV-53030. Address reprint requests to Rampratap S. Kushwaha. PhD, Department of Physiology and Medicine. Southwest Foundation for Biomedical Research. PO Box 28147San Antonio, TX 78228-0147. Copyright 8 1991 by W.B. Saunders Cornpan! 0026-0495/9114OOl-0017$03.0010 93
94
KUSHWAHA
Table 1. Characterization
ET AL
of Animals and Their Plasma Lipids and Lipoproteins During Apo E Turnover Studies (10 months of hormone treatment) Cholesterol(mg/dL)
Treatment Group/ BaboonNo.
Body Weight (Kg)
Plasma
VLDL + IDL
LDL
HDL
Triglycerides(mg/dL) LDUHDL Cholesterol VLDL + Ratio Plasma IDL LDL HDL
APOE (mg/dL) VLDL + PiZ3lla
HDL
IDL
Control x7054
13.0
202
5
106
91
1.16
66
16
24
26
10.0
8.3
1.2
x7057
10.4
171
6
66
99
0.67
67
11
15
41
9.5
6.8
2.1
X7061
14.6
205
6
88
111
0.79
52
11
14
27
9.8
7.3
1.8
Mean
12.7
193
6
87
100
0.87
62
13
18
31
9.8
7.5
+19
20.6
+0.26*
28
k2.9
k5.5
28.4
*0.3t
20.8s
*SD
22.1
k20
*lo
1.7 20.5
Estrogen x7055
13.4
173
5
63
105
0.60
44
8
11
25
6.7
5.0
1.2
X7056
14.5
176
9
66
101
0.65
47
10
12
26
6.7
4.8
1.6
X7060
14.5
138
11
44
83
0.53
40
8
11
21
7.2
5.6
1.3
Mean
14.1
162
8
58
96
0.59
44
9
11
24
6.9
5.1
*SD
20.6
r21
kO.06
44
21.2
~0.6
22.7
23.1
212
212
kO.3
20.4
1.4 kO.2
Estrogen + progesterone X7058
13.3
136
5
28
103
0.27
37
9
9
19
5.0
3.4
1.3
X7062
13.1
143
2
34
107
0.32
60
15
18
26
9.5
5.0
2.4
X7064
14.5
197
2
62
133
0.47
56
18
16
22
6.7
5.1
1.2
Mean
13.6
159
3
41
114
0.35
51
14
14
22
7.1
4.8
1.6
*SD
20.8
218
516
k4.6
k-4.7
23.5
k2.3
k1.3
kO.7
233
k1.7
20.1
+12
Progesterone x7059
14.0
193
7
73
113
0.65
59
11
17
31
10.6
8.0
1.9
X7063
11.4
174
22
61
91
0.67
31
12
9
11
7.8
6.7
1.0 1.6
X7065
11.6
178
14
72
92
0.78
49
20
9
19
8.9
6.8
Mean
12.3
182
14
69
99
0.70
46
14
12
20
9.1
7.2
27.5
+7
kO.07
214
24.9
t4.6
-cSD
lLDi_/HDL cholesterol
21.4
210
212
210
21.4
20.7
1.5 20.5
ratios are significantly different among treatment groups (P < .Ol). LDUHDL ratios in the estrogen + progesterone group
are significantly lower (P < .Ol) than those in the control and progesterone groups. tPlasma apo E levels in the estrogen and estrogen + progesterone groups are significantly lower (P < ,051 than those in the control group. SHDL apo E concentrations in the estrogen and estrogen + progesterone groups are significantly lower (P < ,025) than those in the control and progesterone groups.
block was used to study apo B, apo A-I, and apo A-II metabolism as described earlier.‘3.‘4 The second block was used to study apo E metabolism. Animals in each group were randomly divided into four treatment groups: untreated control, estrogen group, progesterone group, and estrogen + progesterone group (three baboons per group). The untreated control group was injected weekly with cottonseed oil (estradiol vehicle) and fed a fig bar daily. The estrogen group was treated with estradiol (UpJohn, Kalamazoo, Ml) injected intramuscularly at 100 ug/‘kg/wk and was also fed a fig bar daily. The progesterone group was treated with progesterone at 3 mgkg/d in a fig bar and injected with the estrogen vehicle weekly. The estrogen + progesterone group was treated with both estradiol and progesterone as in the estrogen and the progesterone groups. Treatments were given for 3 weeks and then withdrawn for the fourth week. This schedule was maintained for approximately 18 months. The apo E metabolic studies were conducted 10 months after starting the treatment. The protocol for this experiment was approved by the institutional Animal Research Committee. The Southwest Foundation for Biomedical Research is accredited by the American Association for Accreditation of Laboratory Animal Care and is registered with the US Department of Agriculture. Isolation and Labeling of apo E Apo E for turnover studies was isolated from VLDL + intermediate-density lipoprotein (IDL) obtained from plasma of cholesterol-fed donor baboons. Four baboons were anesthetized with
ketamine HCl (10 mgkg) and bled via the femoral vein (50 mL each). Blood was received in tubes containing EDTA (1 mg/mL) and was centrifuged at a low speed to obtain plasma. Plasma was adjusted to a density of 1.019 g/mL by adding solid potassium bromide and ultracentrifuged at 45,000 rpm in a 60 Ti rotor at 6°C for 22 hours using Beckman ultracentrifuge model LS-70 (Beckman, Palo Alto, CA). After ultracentrifugation, the VLDL + IDL fraction was obtained by tube slicing.” The VLDL + IDL fraction was ultracentrifuged again after dilution with saline (d = 1.019 g/mL) under similar conditions. The purified fraction of VLDL + IDL was delipidated first with ethanol:ether 3:1, and then with increasing concentrations of ether, and finally with ether alone. The delipidated apoproteins were separated by a DEAE cellulose column as described by Shore and Shore.’ The purity of apo E was checked by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis.‘5 Iodination of apo E was performed by the iodine monochloride procedure’6 using 0.1 to 0.2 mg of protein dissolved in 0.20 mL buffer containing 0.1 mol/L glycine and 0.5 mol/L sodium chloride (pH 10). Most of the free iodine was removed by chromatography on a Sephadex G-25 column equilibrated with 0.15 mol/L saline containing 0.01 mol/L Tris and 0.001 mol/L EDTA (pH 7.4) and by dialyzing the pooled apo E fractions against the same buffer with five to six changes of buffer. Most (> 98%) of the iodinated apo E was TCA precipitable. The specific activity of apo E was approximately 800 cpming.
HORMONAL
REGULATION
95
OF APO E METABOLISM
Incorporation of Labeled Apo E into VLDL and HDL To isolate VLDL and high-density lipoprotein (HDL) for incorporation of iodinated apo E, blood was obtained from each animal during surgery to implant venous catheters. Blood from the three animals in each treatment group was pooled and centrifuged to obtain plasma. The plasma was ultracentrifuged sequentially using a 50 Ti rotor to obtain VLDL and HDL.1”‘4VLDL and HDL were dialyzed before incubation with iodinated apo E. The iodinated apo E was incorporated into lipoproteins by a technique described previously.” In each study, a fraction of apo E was labeled with “‘1 and another with 13’I.VLDL was incubated with “‘I-labeled apo E and HDL was incubated with ‘Wabeled apo E for 4 hours at 37°C. Following incubation, VLDL and HDL were reisolated by ultracentrifugation and the lipoproteins obtained by tube slicing. VLDL and HDL were dialyzed against saline with several changes of dialysate and used for injections. FL DL and HDL apo E Turnover Procedure Six baboons were studied at one time. Two weeks before turnover studies, baboons were put on a dummy tether to accustom them to the tether system. The next week, venous catheters were implanted surgically and the animals were maintained on the tether. Catheters were implanted on the day animals were started on hormone treatment. Animals were injected with iodinated lipoproteins at the beginning of the second week of the hormone treatment and the turnover studies were completed during the third week of the hormone treatment. All animals followed the same cycle of treatment and were studied at the same point of the treatment cycle. The blood for measurements of lipids and apo E was obtained prior to the injection of iodinated lipoproteins (O-hour sample). The tether allowed us to inject and bleed animals without the use of anesthesia. Each animal was injected with 3 to 5 FCi of “‘I-labeled VLDL (0.2 to 0.4 mg of VLDL protein) and 30 to 40 $Zi of “‘I-labeled HDL (1.5 to 2.0 mg of HDL protein). After injection of iodinated lipoproteins, each animal was bled to obtain h mL of blood at 5 minutes, 0.5, 0.75, 1, 2, 3, 8, 12, 24, 48, 72, 96, 120. 144, 192. and 240 hours. Blood samples at each time point were collected in tubes containing EDTA (1 mg/mL) and were centrifuged to obtain plasma. A small aliquot of plasma (0.1 mL) was counted to determine the decay of radioactivity in plasma. Another aliquot of plasma (2 mL) was used to isolate VLDL (d < 1.006 g/mL), IDL + LDL (d = 1.006 to 1.063 gimL), and HDL (d = 1.063 to 1.21 gimL) by sequential ultracentrifugation as described earlier.‘3,‘4 The radioactivity in each lipoprotein fraction and the fraction with density > 1.21 gimLwas determined by a gamma counter (Nuclear Chicago. Des Plaines, IL). To avoid quenching due to high salt concentration, the lipoproteins were diluted lo-fold with water. The radioactivity spilling over from the “‘I channel into the ‘“‘I chsnnel was subtracted using standards and the counts were corrected for isotopic decay.
(Atlantic Antibodies, Scarborough, ME). Since the apo E concentration of lipoproteins, especially in VLDL, was very low, the apo E in lipoproteins was measured by immunoblotting following separation of plasma by gradient (2% to 16%) polyacrylamide gel electrophoresis. The gradient gels were run as described by Nichols et al” and the proteins were blotted onto nitrocellulose sheets by the method of Towbin et a1.22The sheets were incubated with antibody against human apo E after blocking the nonspecific binding sites and again with iodinated secondary antibody as described.” The sheets were cut into 5-mm pieces and counted for radioactivity. The radioactivity in each lipoprotein region was calculated and was used to estimate the amount of apo E in each lipoprotein from total apo E concentration of plasma. Each analysis was performed in duplicate and the results were reproducible. Kinetic Analysis The kinetic data were analyzed using the CONSAM” computer program on a MicroVax II computer (Digital Equipment Corporation, Maynard, MA). Apo E radioactivity disappearance curves were analyzed using two- or three-compartment models to account for the multiexponential nature of the disappearance curves. Residence time (inverse of fractional catabolic rate [FCR]) describes the period of time apo E is expected to remain in the plasma, on average, prior to removal. These residence times represent the weighted mean residence time for apo E reinjected on either VLDL or HDL particles. These values, therefore, reflect the initial distribution of apo E on either HDL or VLDL. and the metabolism and conversion of apo E from and between different lipoprotein fractions. The residence times presented for HDL apo E, like those for plasma apo E, represent a weighted mean residence time for apo E on the HDL particle. The effect of labeled apo E returning to the HDL particle from other lipoprotein particles on the residence time of HDL apo E could not be determined. Due to the complex nature of the VLDL apo E radioactivity curves following the reinjection of labeled VLDL apo E, residence times for the VLDL fraction could not be determined using the simple approach used for the analysis of the plasma HDL and VLDL apo E data. The producton rates of apo E in plasma and HDL were calculated by multiplying the fractional catabolic rate by the apo E pool in plasma and HDL, respectively. The production rates were expressed as mg/kg/d. StatisticalAnalysis Lipid and apo E levels in plasma and lipoproteins and the kinetic parameters in different groups of animals were compared by ANOVA. If significant differences among these values were detected, the individual means were compared using Tukey’s test. Data in tables are presented as mean + SD. along with individual values. RESULTS
Analytical Procedures Plasma lipoproteins for the measurement of lipids and apo E were isolated by density gradient ultracentrifugation using 2.0 mL of plasma with a modified procedure of Redgrave et al’* as described earlier.” Cholesterol and triglycerides in plasma and lipoproteins were measured by the enzymatic method using assay kits (Sigma Chemical). The purity of apo E for iodination and standard was tested by polyacrylamide slab gel electrophoresis with SDS.“ The apo E in plasma was measured by electroimmunoassay as described by Laurell.‘” using antibody against human apo E
Effect of Hormone Treatment on the Concentration and Distribution of Apo E Table 1 describes the body weight, lipid and apo E levels in the plasma and lipoproteins of these animals during apo E turnover studies (10 months of hormone treatment). Animals did not differ in body weight after undergoing 10 months of treatment with pharmacological levels of sex steroid hormones. As reported earlier’3.‘” for the first block of animals treated similarly, there was no significant differ-
KUSHWAHA
96
ence in lipid levels among these treatments, but there were significant differences in LDL/HDL cholesterol ratio.13.14 The LDL/HDL cholesterol ratio in the estrogen + progesterone group was significantly lower than that in the control and progesterone groups. Plasma apo E levels in the estrogen (6.9 f 0.3 mg/dL) and estrogen + progesterone (7.1 f 2.3 mg/dL) groups were lower than in the control (9.8 k 0.25 mg/dL) and progesterone (9.1 2 1.4 mg/dL) groups. Most (> 60%) of the plasma apo E was present in HDL in all animals (Table 1). HDL apo E concentrations were significantly different among treatment groups. HDL apo E content in the estrogen and estrogen + progesterone groups was significantly less (P < .05) than HDL apo E content in the control and progesterone groups. Apo E content in apo B-containing lipoproteins (VLDL + IDL and LDL) was less than 40% of the total plasma apo E, was variable among the treatment groups, and did not differ significantly. Since the plasma samples for analysis of lipids and apo E were obtained at the same time point of the treatment cycle, these differences will not be affected by the treatment cycle. Effect of Estrogen and Progesterone on the Metabolism of apo E in HDL
The decay of radioactivity from apo E in HDL into the plasma in representative animals from the control, estrogen, estrogen + progesterone, and progesterone groups is given in Figs 1A and B, and 2A and B, respectively. The data were consistent from animals in ail groups. The residence times of plasma apo E, when injected in HDL particles, are given in Table 2. Plasma apo E residence times differed significantly (P < .05) among treatment groups. The residence times of plasma apo E (injected in HDL) in the control and estrogen groups were significantly longer than those in the progesterone or estrogen + progesterone groups. Plasma apo E decayed twice as rapidly in the progesterone and estrogen + progesterone did in the control or estrogen group.
II
0
I....I
50
.I....!.
100
150
Hours
-.
200
ET AL
The decay of radioactivity from apo E in the HDL fraction (d = 1.063 to 1.21 g/mL) in representative animals from the control, estrogen, estrogen + progesterone and progesterone groups is given in Figs 3A and B, and 4A and B, respectively. Most of the radioactivity remained in HDL at all times. A small amount of radioactivity (<20%) was transferred to VLDL, and IDL + LDL. The decay of apo E radioactivity in HDL was very similar to that of apo E in whole plasma. The residence times of HDL apo E are given in Table 2. HDL apo E residence times differed significantly (P < .OS) among treatment groups as did plasma apo E. HDL apo E residence times in the control and estrogen groups were significantly longer than those in the estrogen + progesterone and progesterone groups. Apo E residence times in HDL in the control and estrogen groups were similar to or slightly shorter than residence time for apo E in plasma. These observations suggest that most of the apo E in HDL remains as part of HDL and is removed from there. A small amount of apo E is transferred to other particles where it exchanges slowly causing plasma apo E removal to be slightly slower than that for HDL apo E. On the other hand, HDL apo E residence time in the estrogen + progesterone and progesterone groups was similar to or longer than plasma apo E residence time. These observations suggest that apo E in other lipoproteins is lost more rapidly than that in HDL, and HDL constantly loses apo E to these lipoproteins. Production rates for apo E in plasma and HDL, when iodinated apo E was injected in HDL, are given in Table 2. A greater proportion ( > 50%) of plasma apo E production appeared to be in HDL in all treatment groups. However, the proportion of total plasma apo E production in HDL was far greater (> 80%) in baboons from the control and estrogen groups than that (<60%) in baboons from the estrogen + progesterone and progesterone groups. Plasma and HDL apo E production rates in the estrogen group were significantly lower than those in other groups. On the other hand, plasma and HDL apo E production rates in the
1
2.50 0
so
100 I so Hours
’ 200
250
Fig 1. Disappearance of plasma apo E radioactivity in representative animals from the (A) control and (B) estrogen groups when injected in VLDL (0-O) and HDL (A-A).The apo E injected in VLDL was labeled with 13’1and in HDL with ‘=I.
97
HORMONAL REGULATION OF APO E METABOLISM
Fig 2. Disappearance of plasma apo E radioactivity in representative animals from the (A) estrogen + progesterone groups when injected in VLDL (fl--Cl) and HDL (A-A). The apo E in VLDL was labeled with “‘I and in HDL with ‘251.
and (6) progesterone
Table 2. Plasma Residence Times for Apo E Injected on Either HDL or VLDL Particles in Baboons VLDLApo E* Treatment
PlasmaApo E*
PlasmaApo E*
Group/
Residence
FSDt
Production
Baboon No.
Time(h)
1%)
(w/kg/d)
x7054
85.1
3.7
x7057
81.9
3.3
X7061
81.7
5.2
Mean
82.9
ZSD
+ 1.95ll
Rate
HDLApo E*
HDL Apo E
VLDL Apo Et
Production
Residence
FSDt
Production
Residence
FSDt
Rate in Plasma
Time (h)
W)
Rate (mglkgid)
Time(h)
I%1
(w/kg/d1
1.30
84.5
4.1
1.08
69.5
4.1
1.59
1.28
77.2
2.2
0.97
75.2
5.4
1.39
1.32
78.0
1.5
1.03
60.1
5.0
1.80
1.30
79.9
1.03
68.2
+-4.05
zO.O6/1
Control
20.22
1.59
+7.6§#
kO.21
Estrogen x7055
76.5
3.6
0.97
69.4
2.2
0.80
56.2
4.2
1.32
X7056
84.9
7.9
0.87
75.4
3.0
0.70
61.2
5.6
1.21
X7060
78.9
2.6
1.Ol
73.3
2.2
0.84
60.4
2.9
1.32
Mean
80.1
0.95
72.7
0.78
59.3
1.28
*SD
?4.3§8#
22.75
i-0.06tt
LO.07
*3.0§**
kO.07
Estrogen + progesterone X7058
34.0
4.9
1.62
39.8
6.9
0.94
36.5
5.0
1.51
X7062
44.0
9.2
2.38
62.4
3.3
1.06
59.3
13.4
1.77
X7064
45.2
6.7
1.64
44.9
2.5
1.25
42.1
5.7
1.76
Mean
41.1
1.88
55.5
1.08
45.9
*SD
k6.1
-0.43/l
+9.2
+0.1611
1.68
k11.9
to.15
Progesterone x7059
45.7
4.5
2.56
65.1
3.5
1.36
50.9
6.8
2.30
X7063
38.3
7.3
2.25
54.7
3.9
1.35
39.7
4.4
2.17
X7065
34.9
5.5
2.89
46.8
5.5
1.60
48.3
6.5
2.03
Mean
39.6
2.57
55.5
1.44
45.3
2.17
?SD
25.5
+0.32**
+9.2#
+0.14/j
~5.8
20.14tt
*Labeled apo E was incubated with HDL prior to reinjection. tFractional standard deviation, expressed as %. *Labeled apo E was incubated with VLDL prior to reinjection. These values describe the residence time and production rate of apo E within plasma. SValues are significantly different from progesterone and estrogen + progesterone groups (P < .05). Walue for plasma apo E residence time when injected in HDL was significantly (P < ,051 higher than that when injected in VLDL in control and estrogen groups. I/Production rates for plasma and HDL apo E (when injected as HDL) in progesterone groups ware significantly (P < .05) higher than that in other groups. Production rate in plasma in the progesterone group was also significantly higher (P < .05) than that in estrogen + progesterone group. Production rate in plasma and HDL in the control group was significantly (P < .05) higher than that in estrogen group. #Values are significantly different from estrogen VLDL residence time (P < .Ol). *Walues ttApo
are significantly different from plasma (HDL) residence time (P < .05).
E production rate in plasma (when injected as VLDL) in the estrogen group was significantly (P < .05) lower than that in other groups. Apo E
production rate in the progesterone group was significantly higher (P < .Ol) than that in control or estrogen + progesterone groups.
98
KUSHWAHA
ll....I....1....1....1.... 0 50 loo
Il....I....l....l....I....,
I
150
200
250
0
50
progesterone group were significantly higher than those in other groups. Plasma apo E production rate in the estrogen + progesterone group was also significantly (P < .05) higher than that in the control and estrogen groups. Effect of Estrogen and Progesterone on the Metabolism of Apo E in I/LDL
The decay of radioactivity from apo E in VLDL in plasma from representative animals from the control, estrogen, estrogen + progesterone, and progesterone groups is given in Figs 1A and B, and 2A and B, respectively. The data were consistent for VLDL apo E in all animals in each group. Immediately after injection, most of the radioactivity from VLDL apo E was transferred to HDL. The disappearance of radioactivity in the VLDL fraction from representative animals from the controi, estrogen, estrogen + progesterone, and progesterone groups is given in Figs 3A and B, and 4A and B, respectively. Due to a continuous exchange of apo E radioactivity between VLDL and HDL, the VLDL
100
IS0
Z(K)
250
Hours
Fig 3. Disappearance of apo E radioactivity in VLDL (O---O) and HDL (a---a) groups when injected in VLDL (‘3’1) and HDL (“I).
SO
I 100
Hours
0
ET AL
150
200
in representative
animals from the (A) control and (8) estrogen
apo E decay curves were complex, and therefore, residence times for VLDL apo E were measured for plasma only and not for the VLDL fraction. The residence times for VLDL apo E in plasma (Table 2) were considerably different among treatment groups: they were longest in the control group (68.2 5 7.6 hours), followed by the estrogen (59.3 2 2.7 hours), estrogen + progesterone (45.9 k 11.9 hours), and progesterone (45.3 + 5.8 hours) groups. VLDL apo E residence times in the control group were significantly (P < .05) longer than those in other groups. Similarly, VLDL apo E residence times in the estrogen group were significantly (P < .05) longer than those in the estrogen + progesterone and progesterone groups. VLDL apo E residence times in plasma in the control and estrogen groups were significantly (P < .05) shorter than those of plasma and HDL apo E (when apo E was injected in HDL). However, VLDL and HDL apo E residence times in plasma were similar in the estrogen + progesterone and progesterone groups.
250
0
so
Hours Fig 4. Disappearance of apo E radioactivity in VLDL (O---O) and HDL (A-A) and (B) progesterone groups when injected in VLDL (“‘I) and HDL (‘?).
in representative
I(WI Hours
150
animals from the (A) estrogen +
2.50
progesterone
HORMONAL
REGULATION
99
OF APO E METABOLISM
As with plasma HDL apo E, VLDL apo E production rates in plasma were significantly (P < .OS)different among the groups. Progesterone administered alone or with estrogen significantly (P < .05) increased VLDL apo E production rate compared with that in the control or estrogen group. On the other hand, estrogen significantly decreased (P < .05) VLDL apo E production rate in plasma compared with that of the control group. Production rates of VL,DL apo E and HDL apo E in plasma were not significantly different. However, there was a significant (P < .05) interaction with treatments. VLDL apo E production rates in plasma were slightly but consistently higher than HDL apo E production rates in plasma from baboons in 1he control and estrogen groups. In contrast, VLDL apo E production rates in plasma were slightly but consistently lower than HDL apo E production rates in plasma from baboons in the estrogen + progesterone and progesterone groups. DISCUSSION
Summary of Results
These studies present evidence that, like apo B,” apo A-I, and apo A-II metabolism,‘4 the metabolism of apo E is also influenced by estrogen and progesterone administered in pharmacological doses. Estrogen treatment alone or combined with progesterone decreased apo E content in plasma and HDL. Progesterone treatment had a profound effect on the catabolic rate of apo E. Progesterone alone or in combination with estrogen decreased residence times of apo E injected in HDL or in VLDL. On the other hand, progesterone treatment increased the apo E production rate. Since both the production and catabolism of apo E were increased by progesterone treatment, baboons in the progesterone group had levels of apo E in plasma and HDL similar to those in the control group. Estrogen treatment decreased the residence time of VLDL apo E compared with that in the control group. Estrogen treatment also decreased the production rate of apo E. Thus, increased catabolism and decreased production of apo E were responsible for the reduced level of apo E in the estrogen-treated group. Ro/e of Apo E and Its Isoform in Lipoprotein Metabolism
Ape E plays an important role in the metabolism of apo B-containing lipoproteins in the plasma.’ Since apo E is recognized by the chylomicron remnant and apo B/E receptors, it mediates the catabolism of apo E-rich chylomicron and VLDL remnants.8 Apo E has also been suggested to play an important role in the reverse cholesterol transport process by directing cholesteryl ester-enriched large HDL particles to the liver; in some cases, these particles contain only apo E and have been shown to be rapidly removed.” Apo E is also believed to be involved in the conversion of VLDL remnants into LDL.*’ The latter role for apo E has been suggested because subjects with type III hyperlipoproteinemia who lack functional isoforms of apo E (apo E-III or E-IV) cannot convert VLDL remnants to LDL and consequently VLDL remnants accumulate in
their plasma.” Apo E exists in polymorphic forms in humans.’ Apo E polymorphism in humans has been shown to regulate lipoprotein cholesterol in the plasma.h In contrast to that in humans, baboon apo E is not polymorphic, but is like apo E-IV, which does not contain cysteine.” The present studies, therefore, may explain the role of estrogen and progesterone in the regulation of the functional form of apo E and the receptors that recognize it. Effect of Estrogen and Progesterone on Metabolic Heterogeneity of Apo E in VLDL and HDL
Most of the apo E in baboons was present in HDL, consistent with the fact that baboons have very little plasma VLDL.‘*,‘j Apo E injected in HDL or VLDL particles had different residence times in plasma and lipoproteins. Apo E radioactivity in VLDL or in HDL rapidly exchanged with other plasma lipoproteins and the difference in residence times may be due to differential catabolism of apo E in various plasma lipoproteins. Similarly, it has been reported that apo E injected in vivo rapidly exchanged among plasma lipoproteinszx Most such studies have reported the removal of apo E from whole plasma.2y.3”Our studies were conducted using labeled apo E incorporated into both HDL and VLDL to determine if apo E was metabolically heterogeneous in these lipoproteins and if this was affected by estrogen and progesterone treatment. Apo E in HDL had higher residence times than that in VLDL in plasma of all animals. This observation suggests that the metabolism of apo E in VLDL and HDL is heterogeneous. Apo E is more susceptible to catabolism when it is in the VLDL particle; it is relatively more protected when it is incorporated in HDL. Estrogen treatment decreased plasma VLDL apo E residence time compared with that in the control group. As reported earlier, estrogen treatment also decreased the residence time of LDL apo B in these animals.‘” Estrogen treatment in rats has been suggested to increase LDL receptor activity.“,” Thus, the decrease in VLDL apo E residence time in estrogen treated animals may be due to the effect of estrogen on LDL receptor activity. The estrogen + progesterone and progesterone groups had much shorter residence times for apo E in VLDL and HDL than did the estrogen and control groups. However. progesterone treatment did not affect the catabolism of apo B” and, therefore, the decrease in apo E residence time in VLDL and HDL could not be due to LDL receptor activity. The effect of progesterone on VLDL apo E catabolism may be explained by one of two mechanisms. First, progesterone may induce another receptor, possibly the chylomicron receptor, which removes the larger, apo E-enriched VLDL particles, thus causing apo E to be lost rapidly by the VLDL compartment. Second, progesterone may increase VLDL production causing more apo E to be lost as a component of the VLDL remnant. Progesterone treatment increased catabolism of apoproteins of HDL in baboons.“’ Thus, the increase in catabolism of apo E in HDL may be due to the increased catabolism of HDL caused by progesterone treatment. Production of apo E was also affected by the estrogen and progesterone treatment. In general. most of the apo E
100
KUSHWAHA
ET AL
was secreted into HDL. Estrogen decreased production rate but did not affect the route of secretion into the plasma. Like in the control, most (> 80%) of the plasma apo E was secreted through HDL. In contrast, progesterone increased the production of apo E in the plasma and affected the route of secretion. Even though a greater proportion of apo E was secreted in HDL, it was considerably lower ( < 60%) than that in the estrogen or the control group. Thus, progesterone increased the production of apo E via the apo B-containing lipoproteins. The decrease of
the estrogen + progesterone group. This observation suggests that effect of estrogen on apo E production is counterbalanced by progesterone.
apo E production
of the data.
in plasma
and HDL was not observed
in
ACKNOWLEDGMENT
We thank Maggie Garcia and William Ehler for providing technical assistance for these studies. We also thank Thomas Cooper and David Weaver for bleeding and maintaining the tethered baboons. We gratefully acknowledge Jo Fletcher for editorial assistance and Dr Douglas S. Lewis for statistical analysis
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