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FACTORS AFFECTING HYPERTRIGLYCERIDEMIA
DURING PREGNANCY

By

Helen Jean Palmer

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1979

ABSTRACT

FACTORS AFFECTING HYPERTRIGLYCERIDEMIA
DURING PREGNANCY

BY

Helen Jean Palmer

Rats were ovariectomized and injected daily for 21
days with sesame oil (control, C) or 1 ug estradiol (E)
and/or 2 mg progesterone (P). Plasma triglycerides (TG)
were greater (P <.0.05) in E or E+P treated rats than C.
Parametrial adipose lipoprotein lipase activity (LPLA) was
lower in the E and E+P groups than C on a total tissue ba-
sis. When LPLA was expressed on a DNA basis, no differ-
ences were found.

In experiment 2, ovariectomized rats injected daily
with E+P were injected with saline or 0.2 mg 2-bromo-oc-
ergocryptine (CB-154) and/or 50 I.U. prolactin. 03-154 is
an inhibitor of prolactin secretion. No differences were
found in plasma TG, adipose LPLA or liver TG synthesis
rate after a 12-hour fast.

In experiment 3, pregnant rats were injected with
saline (I), 08-154 (II) or CB-154 and prolactin (III).
Food intake was restricted after day 12 to the amount con-

sumed by 12-day pregnant rats. Fasting plasma TG on days

Helen Jean Palmer

19 and 21 were 300% (P«< 0.05) of days 0 and 12 while adi-
pose LPLA per unit DNA on days 19 and 21 were 50% (P ‘-0.0S)
of days 0 and 12. Liver TG synthesis rates were not dif-
ferent between any of the times. Placental LPLA was
greater on days 19 and 21 than on day 12. Groups I and

III had similar plasma TG which were greater than those of
group II on days 19 and 21. No treatment differences in
the pregnant rats were found for any tissue LPLA or for
liver TG synthesis. The results indicate hypertriglyceri-
demia occurs during pregnancy even when energy is restrict-
ed to the amount consumed by 12—day pregnant rats and that
E promotes increased plasma TG by decreasing adipose LPLA.
Inhibition of prolactin secretion causes an increase in
plasma TG which returns to the control level when exogenous
prolactin is injected with 03-154. The change in plasma
triglycerides caused by prolactin did not correlate with

tissue LPLA or liver TG synthesis.

ACKNOWLEDGMENTS

I want to thank:

Dr. Tucker, Dr. Chenoweth and Dr. Bond for their
suggestions and information;

Dr. Romsos for the learning experiences and finan-
cial help that came from working on the dog
project with him;

Family and friends who were supportive; and

A special thanks to Dr. Bennink for his suggestions,

guidance and encouragement.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . . . . . . . . .

Hypertriglyceridemia During Pregnancy . . . .

Influence of Sex Steroid Hormones on Blood
Triglyceride Concentration . . . . . . . .

MATERIALS AND METHODS . . . . . . . . . . . . . .
Animals and Study Design . . . . . . . . . .
Tissue Preparation . . . . . . . . . . . . .
Assay Methods . . . . . . . . . . . . . . . .

RESULTS . . . . . . . . . . . . . . . . . . . . .

Effect of Sex-Steroid Hormones on Plasma Tri-
glyceride Concentration and Adipose LPLA .

Effect of 03-154 and/or Prolactin in Ovari-
eCtomized Rats I O O O O O I O O O O O O O

Triglyceridemia Changes During Pregnancy . .

Effect of CB-154 and Prolactin on Pregnant Rats

DISCUSSION . . . . . . . . . . . . . . . . . . . .
SUMMARY . . . . . . . . . . . . . . . . . . . . .
REFERENCES . . . . . . . . . . . . . . . . . . . .
APPENDIX A . . . . . . . . . . . . . . . . . . . .
APPENDIX B . . . . . . . . . . . . . . . . . . . .

Page

iv

v1 \N l4 4

18
18
21
22
24

24

26
26
27
51
36
37
43

LIST OF TABLES

Table Page

1 Sex Steroid Hormone Effects on Lipase Activ-
ity and Plasma Insulin . . . . . . . . . l2

2 Oral Contraceptive Effect on Factors Deter-
mining Serum Triglyceride Concentrations. l4

5 Sex Steroid Hormone Effects on Plasma Tri-
glyceride Concentration and Adipose
Tissue LipOprotein Lipase Activity . . . 25

4 Effect of Inhibition or Addition of Prolac-
tin on Enzymes Affecting Plasma Tri-
glyceride (TG) levels in Fasted Ovari-
ectomized Rats . . . . . . . . . . . . . 28

5 Changes in Enzyme Activities Affecting
Plasma Triglyceride (TG) Concentrations
of Fasted Pregnant Rats . . . . . . . . . 29

6 Effect of Reduced Secretion and Ex0genous
Supplementation of Prolactin on Tri-
glyceridemia During Pregnancy . . . . . . 50

B-l Food Consumption, Body Weights and Adipose
Tissue Wet Weights of Estradiol and/or
Progesterone Treated Ovariectomized Rats. 44

B-2 Food Consumption, Body Weights and Adipose
Tissue Wet Weights of CB-l54 and/or
Prolactin Treated Ovariectomized Rats . . 45

B-5 Plasma Triglyceride Concentration (mg/100

ml) of Energy Restricted Fasted and Ad
Libitum Fed Pregnant Rats . . . . . . . . 46

iv

LIST OF FIGURES

Figure Page

1 Synthesis of Triglycerides from Fatty Acids
and Glycerol-B-Phosphate . . . . . . . . 6

INTRODUCTION

Mortality and morbidity due to premature coronary
heart disease is a major health problem in America. Hyper-
triglyceridemia associated with hypercholesterolemia has
been indicated as a risk factor in coronary heart disease
(1). Premenopausal women have a lower risk of coronary
heart disease than post—menOpausal women. It is believed
that physiolOgical levels of estrogen have a protective
effect against coronary heart disease, however high levels
of estrogen promote an increase in the blood triglyceride
concentration. Women taking oral contraceptive pills have
increased blood triglyceride concentrations and have an
increased risk of myocardial infarctions (2). The current
concern with preventing coronary heart disease has lead to
much discussion on what changes need to be made in the
American lifestyle to lower the risk of coronary heart
disease. Dietary manipulation, physical activity and phar-
maceutical intervention have received the main emphasis in
lowering blood cholesterol and triglycerides and presumably
coronary heart disease. For women using oral contracep-
tives, the influence of hormonal preparations on triglycer-

ide metabolism should be considered also.

Hypertriglyceridemia during the last half of preg-
nancy appears to be a normal physiological response. How-
ever, the mechanism(s) involved in producing hypertrigly-
ceridemia is (are) not well understood. Hormonal changes
and/or an increase in food consumption may be causing
changes in lipid metabolism. The research for this thesis
was conducted to investigate the extent of the contribution
of hormonal influence and diet to hypertriglyceridemia dur-
ing pregnancy. The knowledge might also be applied to re-
ducing the hypertriglyceridemia risk factor of coronary

heart disease.

LITERATURE REVIEW

Hypertriglyceridemia has been observed during the
last half of pregnancy in the human (5), rat (4,5,6,7),
and dog (8). Increased serum triglycerides are seen from
the fifth month of pregnancy until parturition in humans.
In the rat, increased triglyceride concentrations begin on
day 12 and continue through day 20 just prior to parturi-
tion on day 21 or 22.

Increased blood triglycerides could result from de-
creased triglyceride removal from the blood and/or in-
creased triglyceride mobilization into the blood. Removal
of triglycerides from the blood occurs when tissue lipo-
protein lipase (LPL) hydrolyzes the triglycerides carried
in chylomicrons and very low density lipOproteins (VLDL).
The fatty acids are then taken up by the tissue and used
for energy or stored as triglycerides. Tissue LPL is asso-
cisted with endothelial cells of capillary walls in the
heart, lung, mammary gland, spleen, muscle, and adipose
tissue. Liver contains hepatic lipase, which is distinct
from other tissue LPL.

An intravenous injection of heparin releases LPL

and hepatic lipase into the blood. Post-heparin lipase

3

4

activity (PHLA) has been used as a measurement of lipase
activity. The amount of enzyme released is proportional
to the heparin dosage, however it is not clear if the re-
leasable pool of enzyme reflects in vivo activity.

A technical problem with measuring PHLA is the dif-
ference in enzyme specificity for the substrate used in
the assay. Knopp et a1. (7) measured PHLA after sham op-
erating or partially hepatectomizing rats 55-58% or
59-69%. By extrapolating to 100%.hepatectomy, they found
liver PHLA was greater when Intralipid, a soybean oil-egg
lecithin emulsion similar to chylomicrons, was used as the
substrate than when Ediol, a coconut oil emulsion was the
substrate. Unless an inhibitor is used in the assay for
hepatic lipase or LPL, it is not possible to attribute a
decrease in PHLA to either enzyme. This problem is impor-
tant to consider when interpreting data in the literature.

Increased mobilization of triglycerides into the
blood, which could produce hypertriglyceridemia, may occur
when substrate availability for triglyceride synthesis is
high and the activity of enzymes involved in triglyceride
synthesis increase. The increase in food consumption dur-
ing the last half of pregnancy could provide more substrate
for triglyceride synthesis.

Triglyceride synthesis occurs in the liver when
energy intake is high or when fasting has increased the

influx of fatty acids mobilized from the adipose tissue.

Carbohydrate is converted to acetyl CoA which is used to
synthesize fatty acids. ,These fatty acids and fatty acids
from hydrolyzed triglycerides are esterfied to glycerol-5-
phosphate in a series of steps to produce triglycerides
(Figure l). The triglycerides combine with phospholipids,
cholesterol and apOproteins synthesized in the liver to

form VLDL which are secreted into the blood stream.

Hypertriglyceridemia During Pregnancy

PHLA has been used to measure triglyceride removal
rates during pregnancy. Fabian et a1. (9) reported women
pregnant 55-58 weeks had lower PHLA than non-pregnant women.
No distinction was made between hepatic lipase and LPL in
the assay system since Infonutrol (a fat emulsion contain-
ing 15% cottonseed oil) was used as a substrate. There was
no distinct enzyme specificity for this substrate. The in-
creased blood volume which normally occurs during pregnancy
was not considered either. KnOpp et a1. (7) found lower
PHLA, which was corrected for the increase in plasma volume,
in the fed rat on day 21 of pregnancy compared to day 12 or
19 when the substrate was Ediol. When Intralipid was used
as the substrate, a significant increase in PHLA was found
on day 12. By day 19, the activity had declined and was
significantly less than the PHLA of 12-day pregnant rats
but not non-pregnant rats. PHLA of 2l-day pregnant rats

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was significantly lower than l9-day pregnant and non-
pregnant rats. From the differences found in enzyme speci-
ficity for the two substrates, the hepatic lipase appears
to increase during the first half of pregnancy while LPL
remains constant. During the second half of pregnancy
both enzyme activities decrease. The adipose tissue LPLA
of 19- and 21-day pregnant rats did parallel the decrease
in PHLA.

Otway and Robinson (5) and Hamosh et al. (6) meas—
ured adipose tissue LPLA of rats during pregnancy and found
significantly less activity on days 20 or 21 of pregnancy
compared to virgin rats. Both investigators reported a
steady decrease in LPLA from day 15 to 19, but the activity
was never significantly different from virgin rats. Hamosh
et a1. (6) did find a significant increase in the activity
on day 12, which agrees with the results of the PHLA re-
ported by Knopp et a1. (7) when Intralipid was used as the
substrate. Both Otway and Robinson (5) and Hamosh et al.
(6) reported an increase in the serum triglyceride level
occurring with the decrease in LPLA. However, the serum
triglyceride level rapidly declined after day 20 even
though LPLA was very low. At this time food intake was re-
ported to decrease significantly. When food intake de-
clines and substrate for triglyceride synthesis is decreased,
serum triglyceride concentration would be expected to de-

crease. The increase in mammary gland LPLA at this time

8

reported by Hamosh et a1. (6) may also contribute to the
decrease in serum triglyceride concentration.

There are few reports on triglyceride production
during pregnancy. Dannenburg et al. (10) found increased
incorporation of l4C-acetate into fatty acids of fed preg-
nant rat liver slices compared to incorporation of non-
pregnant rat liver slices. However, the calculations were
based on the specific activity of exogenous acetate and did
not account for any differences between endogenous pool
sizes which were probably present in the fed state. Otway
and Robinson (5) measured the rate of triglyceride influx
after a Triton WR1559 injection, which inhibits LPL and the
removal of triglycerides from the blood. The rate of tri-
glyceride entry into the plasma gradually increased from
days 10-15 to days 21-22 when expressed per rat. When the
rate was expressed per 200 grams of body weight, the size
of a non-pregnant rat, no differences were found. The in-
crease in triglyceride mobilization reported indicates that
hypertriglyceridemia occurs as a result of the increased
body size during pregnancy.

A relationship between dietary intake and hypertri-
glyceridemia during pregnancy has been reported. Scow et
a1. (4) reported that increased food consumption paralleled
increased triglyceride concentrations after day 12. The
effect of fat content in the diet was also investigated.
When rats were switched to a "fat-free" diet (0.15% methyl

9

linoleate) on day 16 the blood triglyceride concentration
decreased 75 mg % by day 20, while rats on the stock diet
(4.5% fat) had a 125 mg % increase. In contrast, Otway
and Robinson (5) reported similar increases in serum tri-
glycerides in rats fed a "fat-free" diet (0.5% corn oil)

as those fed a stock diet (5% digestible fat) after day 12.
In both of these studies, other components of the diet were
varied and could have affected lipid metabolism.

From the reports on triglyceride removal from the
blood and liver triglyceride synthesis during pregnancy,
hypertriglyceridemia may be the result of increased synthe-
sis when food consumption is increased. Decreased LPLA ap-
pears to contribute to the hypertriglyceridemia during the

latter part of pregnancy.

Influence of Sex Steroid Hormones on Blood Triglyceride

Concentration

During pregnancy, the hormonal balance is changing.
The growing placenta produces estrOgen, Progesterone and
placental lactogen in increasing amounts as pregnancy pro-
gresses both in humans (11,12,15) and rats (14,15). The
change in blood concentration of these hormones corresponds
with the hypertriglyceridemia. Several investigators have
looked at the influence of estrogen and progesterone on the

mechanism of hypertriglyceridemia.

10

The effect of estrogen and progesterone in the con-
traceptive pill on PHLA has been studied. Ham and Rose
(16) reported decreased PHLA in women taking the pill con-
taining both estrogen and progesterone for at least 6
months. Hazzard et a1. (17) measured PHLA in women taking
ethinyl estradiol and medroxyprogesterone for 2 weeks. A
significant decrease in PHLA occurred while plasma trigly-
cerides were significantly increased. Plasma insulin was
also increased in the women taking the oral contraceptive.
Administration of exogenous insulin, which stimulates adi-
pose LPLA, did not increase the PHLA. From the decreased
PHLA, they concluded the increase in triglycerides may be
caused by a decrease in the removal or the hyperinsulinemia
may promote an increase in synthesis of triglycerides or
both.

PHLA may not be a valid way to assess LPLA and the
relationship to triglyceride removal from the blood.
Hazzard et a1. (18) found no differences in oral fat toler-
ance tests conducted on six women before and during therapy
with a contraceptive pill containing estrogen and proges-
terone. At the same time PHLA was depressed while taking
the pill. Further experiments were conducted to provide an
explanation for decreased PHLA without a change in fat tol-
erance. When increasing doses of heparin were administered,
decreased PHLA during therapy was attributed to decreased

LPL release.

11

More recent reports have separated the LPLA and
hepatic lipase activity in post-heparin plasma. Glad et
a1. (19) measured PHLA, hepatic lipase activity and LPLA
after increasing amounts of heparin were injected in women
on a combined oral contraceptive pill. They found no dif-
ferences in LPLA but did measure decreased hepatic lipase
activity in all but one heparin dose in women taking the
pill. Total PHLA was significantly reduced at the higher
heparin doses, which were equivalent to the middle of the
dosage range used by Hazzard et al. (18). They concluded
the releasable pool of hepatic lipase is decreased by a
combined oral contraceptive. Applebaum et al. (20) re-
ported a high correlation (0.969) between hepatic trigly-
ceride lipase and PHLA in a paired study of women taking or
not taking ethinyl estradiol for 2 weeks. Extrahepatic
LPLA was not significantly changed. A large variation but
no significant change in adipose tissue LPLA was found.
They concluded the decreased PHLA was due to the effect of
estrogen on hepatic triglyceride lipase. Neither total
PHLA nor hepatic lipase activity were correlated with the
increase in serum triglycerides. From the changes in
hepatic lipase and results of Hazzard et al., it is possi-
ble that decreased PHLA during estrogen therapy is due to
hepatic lipase resistance to heparin release.

The influence of sex steroid hormones on adipose

tissue LPLA has been investigated. Hamosh and Hamosh (21)

 

l2

injected ovariectomized rats for 7 days with 17- p-estradiol
or progesterone. Adipose tissue LPLA was decreased in
estradiol treated rats while no change was found in pro-
gesterone treated rats compared to ovariectomized rats.
Plasma triglycerides were significantly elevated in estra-
diol treated rats. There were no differences in heart or
lung LPLA. Kim and Kalkhoff (22) injected rats with estra-
diol and/or progesterone for 21 days. They found an in-
crease in triglyceride entry into the plasma in estradiol
and estradiol plus progesterone treated rats which corre-
lated with the hypertriglyceridemia. The lipase activities
which they reported (Table 1) did not correlate with the

serum triglyceride concentration.

Table 1. Sex Steroid Hormone Effects on Lipase Activity
And Plasma Insulin

 

 

Estrogen-and-

 

Progesterone Estrogen Progesterone
Total PHLA f A l
Hepatic Lipase I A I
Adipose LPL ’l J 1‘
(per ug DNA)
Plasma Insulin I T T

 

From Kim and Kalkhoff (22).

Kim and Kalkhoff (22) concluded that sex steroids

primarily influence plasma triglyceride concentrations by

 

13

their action on hepatic synthesis. It is difficult to in-
terpret the sex steroid effects on hepatic lipase and adi-
pose LPL from their data. Total PHLA was decreased by the
E+P treatment, yet hepatic lipase and the tissue lipases,
adipose and mammary gland, were increased. Unless a signif-
icant amount of tissue lipase other than parametrial adi-
pose tissue and mammary gland were contributing to the PHLA,
the results are contradictory.

Two reports have used triglyceride turnover rate,
determined during a steady state, as a measurement of tri-
glyceride production. Kekki and Nikkila (25) measured
triglyceride turnover by injecting 3H-glycerol into women
taking a combined oral contraceptive and compared the rates
to those of women who had never taken oral contraceptive
pills. Both the triglyceride concentration and production
rate were significantly greater in women taking the oral
contraceptive. Data from the control group were used to
develop an equation relating triglyceride concentration to
the turnover rate in order to predict the triglyceride con-
centration of women taking the pill from their turnover
rates. The increase in triglycerides in women taking oral
contraceptives was not as great as would be expected from
the twofold increase in production rate. Enzyme kinetic
analysis of the data resulted in a calculated Ki for the
treated group which was significantly less than the control.

The Kn was used as a measure of triglyceride removal

 

14

efficiency. Since the substrate concentration at which the
turnover was % of Vmax was lower for the treated group, the
removal efficiency was said to be greater. They concluded
that women taking estrogen and progesterone had an increase
in the production of triglycerides and greater removal effi-
ciency from the plasma. Kissebah et a1. (24) compared women
taking a combined preparation, or a progestin, or an estro-
gen to women who had never taken oral contraceptive ster-

oids. The results are shown in Table 2.

Table 2. Oral Contraceptive Effect on Factors Determining
Serum Triglyceride Concentrations

 

 

Serum Triglyceride

 

 

Turn-
Concen- over Km PHLA Intralipid
Treatment tration Rate Clearance
Estro en +
Progegterone T T ‘L — T
Progesterone ¢» " i I T
Estrogen T T “ l ‘—

 

From Kissebah et al. (24).

Triglyceride turnover results (measured after infu-
sion of 14C-palmitate) were similar to those of Kekki and
Nikkila (25). Clearance of exogenous triglycerides was
measured by the rate of Intralipid removal. The clearance
rate was higher in women taking progestins alone or in

combination with estrogen. The data indicate that estrogen

 

15

mainly causes an increased production of triglycerides
while progestin results in an increased triglyceride re-
moval efficiency.

The chicken has been used as a model to study hepa-
tic lipid synthesis during estrogen induced hyperlipidemia.
Chickens are an appropriate model for studying hypertrigly-
ceridemia induced during pregnancy since plasma trigly-
ceride and cholesterol levels and lip0protein composition
are similar to humans (Kudzma et a1. 25). They also mea-
sured changes in lipOprotein composition after 18 days of
diethylstilbestrol (DES) treatment. Chickens treated with
DES had a dose related increase in plasma triglyceride and
cholesterol concentrations found primarily in the VLDL,
similar to women taking estrogen (26). l‘iC-acetate incor-
poration into triglycerides and phospholipids of liver
slices from DES treated chickens was greater than control
birds. No differences were found in the incorporation
into cholesterol or free fatty acids. It was not clear if
any differences in pools of endogenous acetate were con-
sidered. The chicks were fasted for one hour before kill-
ing. Coleman et a1. (27) measured the enzymes of trigly-
ceride synthesis in chicks injected with 2 mg DES for 5
days. Fatty acid CoA ligase, g1ycerol-5—phosphate acyl-
transferase and diacyl glycerol acyltransferase activities
per liver were increased 250 to 500% of the activity of
control chicks. Specific activities of these enzymes,

 

16

however, were not different with treatment. The increase
in total activity did not seem to be a general effect due
to increased protein synthesis but rather to increased
synthesis of these enzymes since diacylglycerol choline
phosphotransferase and succinic dehydrogenase, a mito-
chondrial marker activity, were increased only 55% and 24%
respectively. Specific activity of these two enzymes de-
creased slightly. Chan et a1. (28) found an increase in
mRNA for one of the apOproteins of VLDL after a single in-
jection of 2.5 mg of DES. In another report (29), they
measured VLDL production in vitro and in vivo after a
single injection of estrogen (1 mg) or estrogen (1 mg) plus
progesterone (2 mg). Plasma VLDL concentration increased
to a maximum at 48 hours after the injection of estrogen.
Estrogen or estrogen plus progesterone treatment also in-
creased VLDL synthesis rates in liver slices. Nafoxidine-
HCl, an estrogen antagonist, suppressed VLDL production.
The work in chickens indicates that estrogen promotes VLDL
synthesis in the liver by increasing enzymes for triglycer-
ide and apOprotein synthesis.

From the literature, it seems that both hepatic tri-
glyceride synthesis and decreased triglyceride removal from
the blood are involved in hypertriglyceridemia during preg-
nancy. The reports indicate that sex steroids have an ef-
fect on enzymes involved in both mechanisms. Estrogen may

or may not be affecting LPLA. It is difficult to separate

17

the hormonal effects from the dietary state of the animal
in the investigations so far. Estrogen does have an ef-
fect on enzymes involved in triglyceride synthesis. The
influence of estrogen on hepatic synthesis when substrate
is limited to the non-pregnant level of food intake has
not been considered.

Prolactin has been considered a hormone primarily
involved in lactation, but increased prolactin concentra-
tions seen just prior to parturition and the stimulation of
prolactin secretion by estrogen, are reasons to consider
prolactin when hormonal influences on blood triglyceride
concentrations during pregnancy are studied. The experi-
ments in this thesis were conducted to: (1) determine the
mechanism of the increase in blood triglycerides during
pregnancy, (2) separate the hormonal and dietary influence
on hypertriglyceridemia during pregnancy, and (5) determine

if prolactin has any effect on hypertriglyceridemia during

pregnancy.

 

MATERIALS AND METHODS

Animals and Study_Design

For all experiments, Sprague-Dawley rats (Spartan
Research Animals, Haslett, MI) were housed in a temperature-
humidity controlled room lighted from 7 AM to 7 PM and were
fed the same diet.l

Experiment I was conducted to study the effects of
physiological doses of estrogen and progesterone on plasma
triglycerides and adipose tissue LPLA in ovariectomized
rats. Rats weighing 190-210 g were kept individually in
hanging wire cages. The above diet was fed and food intake
was recorded starting three days after ovariectomy. Treat-
ments were started one week after the Operation. Daily
subcutaneous injections of the following were given for 21
days between 9 AM and 10 AM: (1) 0.1 ml of sesame oil
(control, C); (2) 2.0 mg progesterone (P); (5) 1.0 ug fi -
estradiol-5-benzoate (E); (4) 2.0 mg progesterone and 1.0
ug (3-estradiol-5-benzoate (E+P) (Sigma Chemical Co., St.

Louis, MO). This dosage of estrogen and progesterone

 

1The diet contained (metabolizable energy basis):
20% casein, 59% cornstarch, 1% glucose, 5% corn oil, and
15% lard. Commercial mineral and vitamin mixes were added
to meet NRC requirements (See Appendix A).

18

 

19

causes mammary growth similar to that observed during
pregnancy (50). On days l2, l9, and 21, six rats from

each group were decapitated, blood was collected in heparin-
ized tubes and the parametrial adipose tissue was removed
and placed in ice-cold 0.15 M NaCl.

In EXperiment II, the effect of prolactin and inhi-
bition of its secretion on plasma triglycerides and adipose
tissue LPLA was studied. Since estrogen stimulates prolac-
tin secretion in rats, the animals were ovariectomized to
control endogenous estrogen levels. Rats were housed indi-
vidually in hanging wire cages and food intake was measured.
Groups I, III, IV, and V received 1 ug E and 2.0 mg P in
0.1 m1 sesame oil and Group II received 0.1 m1 sesame oil
daily by subcutaneous injections starting one week after
ovariectomy. Rats were injected twice the day before and
once the morning of killing with: Group I and II--0.2 ml
20% ethanol in saline; Group III--0.1 mg 2-bromo-cc-
ergocryptine (CB-154), an inhibitor of prolactin secretion,
(provided by Sandoz, Inc., E. Hanover, NJ); Group IV-—0.l
mg CB-154 and 25 I.U. of prolactin (NIH-P-B5, provided by
National Institute of Arthritis, Metabolism and Digestive
Diseases) and Group V--25 I.U. of prolactin. This dosage
of CB-154 and prolactin is twice the amount used to inhibit
and restore lactation in the rat (personal communication
from D. E. Bauman). The CB-l54 and prolactin were first
dissolved in ethanol and a drop of l N NaOH. NaCl (0.15M)

 

20

was added to give a final concentration of 20% ethanol.
One-half of the total dose was injected subcutaneously
twice the day before and once the morning of killing on
days 8 and 21. Twelve-hour fasted rats were decapitated,
blood was collected in heparinized tubes and parametrial
adipose tissue was removed and placed in ice-cold 0.15 M
NaCl. Livers for triglyceride synthesis rate determina-
tions were removed and placed in ice-cold tris-sucrose
buffer (pH 7.2; 50 mM tris, 0.5 M sucrose, 1 mM GSH, 1 mM
EDTA).

Experiment III was conducted to investigate the ef-
fects of prolactin and inhibition of its secretion on tri-
glyceridemia during pregnancy. Sprague-Dawley rats (200-
220 g) were mated. The day sperm were seen in the vaginal
smear was designated day l of pregnancy. Food intake was
recorded and restricted after day 12 to the amount consumed
by 12-day pregnant rats in order to control substrate avail-
ability for triglyceride synthesis. Twenty-four rats were
assigned to 5 treatment groups: (1) 0.2 ml 20% ethanol in
saline, (2) 0.1 mg CB—l54 or (5) 0.1 mg CB-l54 and 25 I.U.
of bovine prolactin. The injections were given subcutan—
eously between 9 AM - 11 AM and 5 PM - 6 PM the day before
killing and between 9 AM - 11 AM the day of killing. Rats
were fasted 12 hours and killed 2 hours after the morning
injection on days 0, l2, l9, and 21 of pregnancy. Virgin

rats were on the diet at least 2 weeks prior to killing.

 

21

After decapitation, blood was collected in heparinized
tubes and parametrial adipose tissue, placentas, and the
left mammary gland were removed and placed in ice-cold 0.15
M NaCl. Uterine contents of 12-day pregnant rats were
scraped out of the uterine lining and analyzed as placentas.

Livers were removed and placed in ice-cold tris-sucroselnfifia:

Tissue Preparation

Adipose tissue and placentas were blotted, weighed
and homogenized in 2 volumes per gram of tissue with 0.025
N (NH4)2SO4 - 0.15 M NaCl; pH 8.6 containing 1 unit of
heparin per ml. Acetone-ether powders were prepared as
described by Robinson (51) and modified by Hamosh et al.
(6). Powders were stored in a dessicator at 4° C until
analyzed for LPLA. Mammary glands were blotted dry,
weighed, sliced with a scalpel, frozen on dry ice and ground
with dry ice in a motor-driven mill (Quaker City Mill,
Philadelphia, PA). The frozen particles were scraped into
acetone and acetone-ether powders were prepared.

Livers were homogenized in 2 volumes of ice-cold
tris—sucrose buffer for 50 seconds with a Lourdes homogen-
izer (Vernitron Medical Products, Inc., Carlstadt, NJ) fol-
lowed by 5 passages on a teflon pestle tissue grinder.

The supernatant formed by centrifuging for 20 minutes at
10,000 x g at 4° C was frozen in aliquots for enzyme and

protein analysis.

 

22
Assay Methods

Plasma was analyzed for triglycerides according to
the method of Biggs et a1. (52). Free fatty acids were ex-
tracted according to Dole (55) and analyzed by Itaya and
Ui's method (54).

Adipose tissue, placentas and mammary glands were
analyzed for LPLA using Corey and Zilversmit's method (55).
Adipose tissue and mammary gland powders were homogenized
in 5 volumes per gram of tissue (wet weight) with 0.025 N
(NI-1,92304 - 0.15 M NaCl; pH 8.6 with 1 unit of heparin per
ml in ground glass homogenizing tubes. Placentas were
homogenized with 6 volumes per gram of tissue. Activity
was linear for 60 minutes of incubation at 57° C. All tis-
sues of one experiment were assayed at the same time using
the same rat serum as a source of apOproteins to avoid vari-
ations due to activating factor(s). Aliquots of the extracts
containing fatty acids were neutralized before liquid scin-
tillation counting.

Liver triglyceride synthesis rates were measured by
l4C(U)-_s__r_i_-glycerol-5-phosphate (New England Nuclear, Boston,
Mass.) incorporation into glycerides (Bennink, 56). Opti-
mum pH was 7.2 and incubations were for 45 minutes at 57° C.

Other methods used were: DNA in adipose tissue-—
Setaro and Morley (57); DNA in placenta and mammary gland--
Dische (58); and protein-~Lowry (59).

 

25

0ne~way analysis of variance was used for Experiment
I and two-way analysis of variance was used for Experiments
II and III to determine treatment differences. When the F
value was greater than the critical value, Tukey's, Stu-
dent's t or Scheffe's test were used where indicated to

compare the means (40).

 

RESULTS

Effect of Sex-Steroid Hormones on Plasma Triglyceride Con-
centration and Adipose LPLA
Rats treated with estradiol (E) or estradiol and
progesterone (E+P) had significantly greater (P <..05)
plasma triglyceride concentrations than the control group
(Table 5). Adipose tissue LPLA was lower for E and E+P
treated groups when expressed on a total tissue basis, but
no differences were found when the activity was based on
DNA content. The E and E+P treated groups also ate sig-
nificantly less than the control group and were signifi-
cantly smaller (P <7.05) (Appendix 3-1). The adipose tis-
sue wet weight of the E treated group was significantly
less than the control group, while the E+P treated group
was no different from the control group. Progesterone alone
did not significantly affect the plasma triglyceride con-
centration or adipose tissue LPLA compared to the control
values. Plasma-free fatty acid concentrations were not dif-

ferent in any treatment group.

24

 

25

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26
Effect of CB-154 and/or Prolactin in Ovariectomized Rats

Inhibition of prolactin secretion or the addition
of prolactin did not affect plasma triglyceride concentra-
tion, adipose tissue LPLA or liver triglyceride synthesis
rate after 8 or 21 days of injection (Table 4). Rats
treated for 8 days had lower plasma triglyceride concentra-
tions and lower adipose tissue LPLA than rats treated for
21 days, however the trend in differences between groups was
the same. The animals given estrogen and progesterone had
higher, but not significantly different, plasma triglycer-
ides than those given sesame oil. The fasted state of
these animals reduced the magnitude of the differences
found in the first experiment. They were significantly
lighter and ate significantly less than rats injected with

sesame oil (Appendix B-2).

Triglyceridemia Changes During Pregnancy

Pregnant rats had significantly greater plasma tri-
glyceride concentrations on days 19 and 21 compared to day
12 and non-pregnant rats when food intake was controlled
(Table 5). The in vitro removal capacity of the tissues
did not entirely reflect the differences in plasma trigly-
ceride concentration. Adipose tissue LPLA (per total tis-

sue) was significantly lower on days 12, 19, and 21 of

27

pregnancy than LPLA of non-pregnant rats. Liver trigly-
ceride synthesis rates were not different at any time.
When LPLA was expressed on a DNA basis, period of gesta-
tion did not affect LPLA except for adipose tissue. Adi-
pose LPLA progressively declined from the non-pregnant
state to day 21 of pregnancy, but l2-day pregnant rats
were not significantly different from non-pregnant or 19-

day pregnant rats.

Effect of 03-154 and Prolactin on Pregnant Rats

Inhibiting prolactin secretion with CB-154 increased
plasma triglyceride concentrations of rats pregnant 19 or
21 days but had no effect on non-pregnant or l2-day preg-
nant rats. When prolactin and CB-l54 were co-injected,
plasma triglyceride concentrations returned to the control
level. Neither treatment produced changes in LPLA or liver

triglyceride synthesis rates (Table 6).

28

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DISCUSSION

These studies were conducted to determine the mechan-
ism of hypertriglyceridemia during pregnancy. It is gener-
ally recognized that food intake increases during the last
half of pregnancy simultaneously with increases in serum
triglyceride concentrations (4,5,7). In this study, when
rats pregnant 19 and 21 days were restricted to energy con-
sumption equal to 12 day pregnant rats, elevated serum tri-
glyceride concentrations were still found (Table 5), indi-
cating that more than food intake is involved in hypertri-
glyceridemia during pregnancy.

In the restricted energy state, increased substrate
for liver triglyceride synthesis could come from free fatty
acids hydrolyzed from triglycerides in adipose tissue. An
increase in plasma free fatty acids during pregnancy has
been reported (41). Free fatty acids may be released from
adipose tissue in response to placental lactogen, which in-
creases during pregnancy (42). The free fatty acids could
then be taken up by the liver and re-esterified to glycerol-
5-phosphate to form triglycerides. In this study, however,
liver triglyceride synthesis rates were not increased

(Table 5).

51

 

52

Others have reported that elevated serum triglycer-
ides result from increased synthesis during pregnancy. In-
creased triglyceride mobilization was found during preg-
nancy when based on the whole rat (5). However, no differ-
ences were found when the mobilization was based on unit
body size, suggesting that the increase is related to pro-
portional increases in body tissues such as the liver dur-
ing pregnancy. Increased fatty acid synthesis has also
been reported in the fed pregnant rat (10). The increased
synthesis was measured by l“‘C-acetate incorporation into
triglycerides and did not account for endogenous pools of
acetate, however.

Effects of hormones on triglyceride synthesis have
shown that estrogen increases VLDL synthesis in the chicken
(28,29), triglyceride mobilization in women taking oral
contraceptive pills (25,24), and triglyceride entry into the
blood of ovariectomized rats (22). No hormonal effects on
triglyceride synthesis were found in this study (Table 4).

The lack of difference in triglyceride synthesis
rates in this study may indicate that substrate availability
is more important in determining the rate of triglyceride
synthesis by the liver during pregnancy. Ad libitum fed
pregnant rats have higher serum triglyceride concentrations
(4,5,7,Appendix B-5) than restricted rats in this study.
When substrate supply is limited, increased blood triglyceride

concentrations may result from the estrogenic effect on

53

triglyceride removal. The interaction of hormones such as
estrogen and the increased substrate supply during the lat-
ter half of pregnancy may further enhance the hypertriglycer-
idemia.

Changes in triglyceride removal capacity of LPL in
some tissues during pregnancy have been reported. The adi-
pose tissue LPLA was found to decrease late in pregnancy in
this study (Table 5) and others (5,6,7). A decrease was
seen as early as day 12 of pregnancy when expressed on a
total tissue basis, however the activity per ug of DNA on
day 12 was not different from non-pregnant rats in this in-
vestigation. Others (5,6) have reported no change in adi-
pose LPLA (per g of tissue) until after day 12 of pregnancy.

Hypertriglyceridemia and decreased adipose tissue
LPLA found in ovariectomized rats treated with estradiol or
estradiol and prOgesterone (21, Table 5), suggests that the
contribution of estradiol to increased serum triglycerides
during pregnancy is through the hormone's effect on trigly-
ceride removal by adipose tissue LPL. The mechanism of
estradiol's effect on triglyceride removal from blood is not
clear. One possibility is a direct effect of estradiol on
the tissue to stimulate synthesis of LPL. The general me-
chanism of estrogens' action on tissues is through the for-
mation of an estrogen-protein receptor complex in the cytosol
which moves to the nucleus and interacts with chromatin to

initiate mRNA synthesis. Another possibility is through the

54

hormones stimulation of apoprotein synthesis in the liver
(28). At least one apoprotein (apo C-II) has been found
to activate LPL (45,44) and is a necessary factor in the
assay system for the enzyme. Other apoproteins (apo
C-III's) have been reported to inhibit LPL (45). When
serum from estrogen plus progesterone treated rats was used
as a source of activating factor(s), adipose tissue LPLA
was depressed compared to control serum (46,47). The ef-
fect of apOproteins has been demonstrated on heart (48,49)
and adipose LPL (50). Since both activators and inhibit-
ors are present in serum, the ratio of the apOproteins
would be the determining factor on enzyme activity. The
mechanism seems plausible, however, Hillman et a1. (51)
reported no changes in the ratio of apo C-II and apo C-III
in VLDL of pregnant women.

From the increased activity of mammary (6, Table 5)
and placental LPLA (52, Table 5) on days 19 and 21 of preg-
nancy, it would seem that either apOproteins are not effect-
ors of the enzyme in these tissues as they are in adipose
tissue or that other hormonal interactions are influencing
the activity. Prolactin was found to increase mammary
gland LPLA and decrease adipose tissue LPLA of lactating
rats with no effect on the plasma triglyceride concentra-
tion (55). During pregnancy serum prolactin concentrations
are low until just before parturition (54). It would seem

possible that the large increase in mammary gland LPLA found

 

 

35

just prior to parturition (6, Table 5) could be due to
prolactin. However, in this study, prolactin or inhibi-
tion of its secretion did not affect LPLA of adipose tis-
sue, mammary gland or placenta. Plasma triglyceride con-
centrations were increased on days 19 and 21 when prolac-
tin secretion was inhibited by CB-154, however, the in-
crease could not be attributed to changes in adipose, mam-
mary or placental LPLA or liver triglyceride synthesis
rates (Table 6).

The role of placental lactogen, which has some bio-
logical effects similar to prolactin, has not been fully
investigated due to the difficulty in obtaining a pure
source of the hormone.

More research on placental lactogen and other factors
that could potentially affect the blood triglyceride con-
centration during pregnancy needs to be conducted in order

to fully understand the mechanism of hypertriglyceridemia.

SUMMARY

The influence of hormones and increased food con-
sumption on hypertriglyceridemia during the last half of
pregnancy was investigated in this study. Plasma trigly-
ceride concentrations were increased even when food intake
was restricted after day 12 to the amount consumed by 12-
day pregnant rats. Adipose tissue LPLA decreased; placen-
tal and mammary gland LPLA increased and liver triglyceride
synthesis rates did not change when food intake was con-
trolled during pregnancy. From the effects of estradiol on
the ovariectomized rat adipose LPLA, estradiol is responsi-
ble for the decreased adipose LPLA in pregnant rats. Pro-
lactin decreases plasma triglyceride concentrations late
in pregnancy, but the enzyme activities measured did not
correlate with the changes in plasma triglyceride concentra-
tion which occurred when prolactin secretion was inhibited

and exogenous prolactin was administered.

56

 

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APPENDIX

APPENDIX A

Diet Composition

Basal Mix Diet
Casein 265 g Basal Mix 596 g
Mineral Mix1 40 g Cornstarch 511 g
Vitamin Mix2 20 g Fat (lard) 64 g
Cellulose 40 g
Corn Oil 22 g
Glucose 6 g
DL-methionine 5 g

Choline Chloride 2 g
596 s

 

lMineral Mix No. 4164 (Teklad Test Diets, Madison,
Wisconsin).

2Vitamin Supplement 25450 (U. S. Biochemical Corp,
Cleveland, Ohio).

43

 

APPENDIX B

Table B-1. Food Consumption, Body Weights and Adipose
Tissue Wet Weights of Estradiol and/or fro-
gesterone Treated Ovariectomized Rats

 

 

 

Food Body Adipose

Consumption Weight Tissue
(8) (i) (87
Sesame Oil 19 297 1.9
Progesterone 19 295 1.7
Estradiol 16‘ 249‘ 1.2‘

Progesterone +

Estradiol 16‘ 261‘ 1.4
s.e.2 0.5 4 0.1

 

lAn ‘ denotes a significant difference from the
sesame oil (control) treated group (p ‘¥.O5) by Tukey's
test (18 rate per group).

2s.e. is the pooled standard error of the means.

 

45

Table B-2. Food Consumption, Body Weights and Adipose
Tissue Wet Weights of CB-l54 and/or Prolactin
Treated Ovariectomized Rats

 

 

 

Food Body Adipose

Treatment Consumption2 Weight Tissue
(s) (s) (s)

E+P
Saline 7a 244a 2.6
8.0.
Saline 9b 264b 2.7
E+P
CB-154 7a 256a 2.4
E+P
CB-154
Prl 6a 2428 2.7
E+P
Prl 7a 245a 2.9
s.e.5 0.4 5 0.3

 

1Values with different superscripts are different
(p <1.05) by Tukey's test (12 rats per group).

2During the 24 hours before being killed, the rats
consumed the amount indicated during the first 12 hours
and were fasted for the second 12 hours.

5s.e. is the pooled standard error of the means.

46

Table B-5. Plasma Triglyceride Concentration (mg/100 ml)
of Energy Restricted Fasted and Ad Libitum Fed
Pregnant RatslI2

 

 

 

 

Day Ad Libitum Fed Energy Restricted
0 27a 333

12 77b 388

19 1240 74b

21 111c 86b

s.e.3 15 6

n 5 6

1

Values in the same column with different super-
scripts indicate a significant difference (p ‘3.05) by
Tukey's test.

2Ad libitum fed rats had access to the same diet as
the energy restricted rats until the time they were killed.

3

s.e. is the pooled standard error of the means.