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Influence of dehydroepiandrosterone (DHEA) on cholesterol metabolism in rats
Pharmacological Research Communications, Vol. 15, No. 9, 1983
797
INFLUENCE OF DEHYDROEPIANDROSTERONE (DHEA) ON CHOLESTEROLMETABOLISM IN RATS* David Kr~tchevskyl, Shirley A. TepperI, David M. Klurfeld I, and Arthur G. ~chwartz ~ The Wistar Institute of Anatoniy and Biqlogy 36th Street at Spruce, Philadelphia, Pennsylvania 19104, and =Fels Research Institute, Te~le University, Philadelphia, Pennsylvania 19140 Received ~final ~rm 8May 1983
SUMMARY Wistar rats were fed semipurified diets containing 0.5% of dehydroepiandrosterone (DHEA) for 14 days. DHEAfeeding resulted in weight loss and slight liver enlargement.
I t did not affect serum triglyceride
levels but increased serum cholesterol levels.
Liver triglyceride levels
of DHEA-fed rats were significantly lower than in controls. cholesterol absorption.
DHEAinhibited
Liver slices from DHEA-fed rats incorporated more
acetate into cholesterol than controls; there were no differences in conversion of mevalonate. INTRODUCTION Dehydroepiandrosterone (DHEA) has been shown to inhibit weight gain in mice despite i t s lack of effect on food intake (Tannen and Schwartz, 1981; Schwartz et a l . , 1983). There have been few studies of the effect of DHEA on lipid metabolism.
I t has been found to have no effect on serum cho-
lesterol levels in normal rats (Ben-David et aI., 1967); to reduce t r i g l y ceride levels in mice (Tannen and Schwartz, 1981); and to inhibit in vitro cholesterogenesis in guinea pig ma~ary gland tissue (Granelly and Turner, 1968). Because of the interest in the carcinostatic properties of DHEA
*Supported, in part, by grants (HL-03299, CA-14661 and AG-00368) and a Research Career Award (HL-00734) from the National Institutes of Health and funds from the Commonwealth of Pennsylvania.
0031-6989/83/090797-07/$03.00/0
© 1983 The Italian Pharmacological Society
Pharmacological Research Communications, Vol. 15, No. 9, 1983
798
(Schwartz, 1979) we f e l t i t would be interesting to examine its effects on aspects of lipid metabolism in intact rats. MATERIALS AND METHODS Male Wistar rats were used in both experiments. The rats were caged individually in air-conditioned rooms maintained on a 12-hr light-dark cycle.
In the f i r s t study rats were fed a semipurified diet containing
54.5% sucrose, 25% casein, 10% corn o i l , 5% cellulose, 5% salt mix (AIN), I% vitamin mix (AIN) and 0.5% cholesterol.
This diet provides 53.4% of
calories as carbohydrate, 24.5% as protein and 22.1% as fat. Dehydroepiandrosterone (0.5%) was added at the expense of sucrose.
In the
second study no cholesterol was added to the diet and the sucrose content was increased by 0.5%. The diets were fed for 14 days at which time sera and livers were taken for determination of cholesterol
(ROschlau et a l . ,
1974; DeHoff et a l . , 1978) and triglycerides (Levy and Keyloun, 1972). For cholesterol absorption studies each rat was given a single oral dose of [4-1hC]cholesterol (0.5 pCi) in O.S m! of propylene g|yco]. Feces were collected for 3 days and neutral and acidic steroids isolated and analyzed for radioactivity (Kritchevsky et a l . , 1973). Cholesterol biosynthesis by liver slices was studied using the system described by Bucher and McGarrahan (1956). Incubations in duplicate were carried out using 0.5 gm of liver and either sodium [l-14C]acetate (0.5 pCi) or 2-14C-mevalonic acid
(0.5
). RESULTS AND DISCUSSION
The first study (Table I) was carried out using rats weighing 152 gm. Rats fed DHEA gained significantly less weight than the controls but had enlarged livers.
Serum cholesterol levels were elevated significantly in
rats fed DHEA but triglyceride levels were unaffected.
Liver cholesterol
Pharmacological Research Communications, Vol. 15, No. 9, 1983
Table 1,
799
Influence of dehydroeplandrosterone (DHEA) on Lipid Metabltsm in Rats* (All data n~an ± SEM) Group j . .
+
DHEA Number
7
p**
Control 7
Weight gain, g
21 ± 4
67 -+3
Liver weight, g
9.6 ± 0.3
9.0 ± 0.8
Relative liver weight (g/1OOg)
5.55 -+ 0.14
4,11 -+ 0.20
0.001 ~S
0,001
Serum lipids, mg/dl 70 * 4
0.001
87 ± 13
70 ± 8
NS
Cholesterol
845 ± 38
865 -+ 53
~S
Triglyceride
196 ± 42
507 -+ 95
U.U2
Serum (dpm x 10-4 )
1.51 ± 0.12
0.99 ± 0.09
U,OI
Liver (dpm x 10-5 )
1.89 ± 0.08
2.29 • 0.27
NS
3.8 ± 0.3
3.2 -+ 0.2
NS
Neutral ste~oid (dpm x 10"~)
5.81 ± 0.63
3.90 -+ 0.35
0.01
Aci di c ster~i d (dpm x I0- )
0,51 ± 0.04
0,51 ~ 0.U3
NS
Absorption (%)
42.5 ± 5.8
59.9 ± 3.1
0.05
Cholesterol Trig]yceride
122 ± 8
Liver lipids, mg/lOOg
Cholesterol absorption
Feces Weight, g
* Rats (152 g) fed semipurified diet containing 0,5% cholesterol + 0.5% DHEA for 14 days, * * Student'S t t e s t .
(see t e x t )
Pharmaco/ogical Research Communications, Vol. ;5. No. 9, 1983
800
Table 2.
Influence of dehydroeplandros'terone (DHEA) on l i p i d ~ a b o l i s m in rats* (All data mea, ± SEM) Group p** DHEA
Number
10
Control
<
i0
Weight gain, g
-23 ± 3
Liver weight, g
9.4 ± 0.3
8.9 ± 0.2
NS
5.22 ± 0.14
3.83 ± 0.05
0,001
131 t 6
9g ± 5
0,005
32 ± 2
30 ± 2
NS
Cholesterol
231 ± i0
338 ± 12
0.001
Tri glyceri des
I00 ± 10
362 ± 31
0.001
Cholesterol absorpti on Serum (dpm x 10"~)
1.22 ± 0.14
0,94 ± 0.02
NS
Liver (dpm x 10-5 )
2.30 ± 0.24
2.24 ± 0.33
NS
2.9 ± 0.2
3.9 ± 0.3
0.Oh
Neutral steroid (dpm x i0 "~)
4.23 + 0.53
3.22 ± 0.55
NS
Acidic steroid (dpm x I0- )
0.13 + 0.04
0.20 + 0.05
NS
60.3 ± 4.8
68.9 ± 5,2
NS
Relative liver weight (g/100 g)
31 ± 2
O.001
Serum ]ipids, mg/dl Cholesterol Tri glyce ri de Liver lipids, mg/lO0 g
Feces Weight, g
Absorption (%)
* Rats (202 g) fed cholesterol-free semipurified diet (see text) containing 0,5% DHEA for 14 days. ** Student's t test. t Five rats per group used for absorption study.
Pharmacological Research Communic~'ons, Vol. 15, No. 9, 1983
801
levels were similar in the two groups but liver triglycerlde levels were reduced significantly in the DHEA-fed rats. Rats fed DHEA and [14C]cholesterol accumulated significantly more cholesterol in their sera but less in their liver.
They absorbed s i g n i f i -
cantly less cholesterol and differed from controls by excreting considerably more neutral steroid in the feces (p
Triglyceride accumulation in blood is unaf-
fected but liver triglyceride levels are affected significantly. The second experiment was carried out using larger (202) gm rats.
As
in the previous study weight gain was inhibited, relative liver weight increased, serum cholesterol was raised significantly and serum t r i glycerides unaffected in DHEA-fed rats.
In this study both liver cho-
lesterol and triglycerides were reduced significantly in the test rats. Cholesterol absorption data were consistent with those seen in the earlier experiment (Table I ) .
Cholesterol absorption was decreased in rats fed
DHEA and the decrease was reflected in 31% greater excretion of fecal neutral steroid.
Serum plus liver pools of both cholesterol and t r i g l y -
cerides were significantly lower (p
The
actual pool sizes (mg) were: cholesterol-DHEA, 27.1+_ 1.2 and control, "36.9 ± 1.9; triglycerides, DHEA, 11.2 ± 1.1 and control -34.5_+ 3.0. Data on cholesterol synthesis are presented in Table 3.
Cholesterol
synthesis from acetate was enhanced by DHEAfeeding as was fatty acid synthesis.
Cholesterol synthesis from mevalonate was unaffected,
Ben-
Pharmacological Research Communications, VoL 15, No. 9, 1983
802
Table 3.
Lipogenesis by liver sllces from rats fed 0.5% DEAH*
Group DHEA
P <
'C~ntrol
From Na-I-14C acetate Cholesterol dpm/100 mg l i v e r x 10-2
6.90 ± 0.87
4.16 ± 0.23
O.01
dpm/liver x 10"~
6.88 ± 0.96
3.87 ± 0.23
0.001
10.22 ± 0.38
8.30 ± 0,37
0.01
dpm/100 mg liver x 10-3
6.09 ± 0.57
4.88 ± 0.57
NS
dpm/liver x 10-5
6.04 + 0.62
4.48 -+ 0,46
NS
Fatty acid dpm/liver x i0 -s From 2-1WC-mevalonic acid
* Five rats per group. NS = not significant David et al. (1967) found that DHEA given at a dose of 5 mg/kg had no effect on serum cholesterol levels in normal rats. be hypercholesterolemic,
We have found DHEA to
but we fed DHEA as 0.5% of the diet.
Assuming an
average daily intake of 14 gm of diet per rat the animals ingested 70 mg of DHEA daily or about 350-460 mg/kg.
I t is possible that DHEA becomes
hypercholesterolemic with increasing dosage. Liver l i p i d levels were significantly lower in DHEA-treated rats and the principal effect appeared to be on triglycerides,
Cholesterol absorp-
tion was inhibited in DHEA rats. In our hands cholesterol synthesis from acetate was enhanced in liver slices from rats fed DHEA; cholesterol synthesis from mevalonate was unaffected.
Granelly andTerner (1968) added DHEA to incubation mixtures of
Pharmacological Research Communications, Vol. 15, No. 9, 1983
guinea pig mammary gland and reported inhibition of cholesterol synthesis. Addition of the test co~ound to the incubation mixture is not con~arable to studies using tissues from animals fed the co~ound. Our data confirm the growth inhibiting effect of DHEA (Schwartz, 1979) and suggest that the principa] focus of action may involve triglycerlde metabolism. DHEAincreases cholesterolemia but also inhibits cholesterol absorption.
These finding suggest effects on lipoprotein metabolism which
might be worth further study. REFERENCES Ben-David, M., Dikstein, S., Bismuth, G., and Sulman, F.G. (1967) Proc. Soc. Exp. Biol. Med. 125, 1136. Bucher, N.L.R., and M~rrahan, K. (1956) J. Biol. Chem. 22__22,I. DeHoff, J.L., Davidson, L.M., and Kritchevsky, D. (1978) Clin. Chem. 2__44, 433. Granelly, AoA., and Terner, C. (1968) Endocrinology 83, 1311. Kritchevsky, D., Casey, R.P., and Tepper, S.A. (1973~-'Nutr. Reports. Int. 7, 61. iTritchevsky, D., Moynihan, J.L., and Sachs, M.L. (1961) Proc. Soc. Exp. Biol. Med. 108, 254. Levy, A. L . ~ n d Keyloun, C. (1972) Adv. Automated Ana|. --i, 497. Ri}schlau, P., Berut, E., and Gruber, W. (1974) Z. Klin. Biochem. 12, 403. Schwartz, A.G. (1979) Cancer Reso 39, 1129. Schwartz, A., Hard, G., Pashko, L., Abou-Ghanbia, M., and Sivern, D. (1983) Nutr. Cancer, in press. Tannen, R.H., and Schwartz, A.G. (1981) Fed. Proc. 40, 347.
803
797
INFLUENCE OF DEHYDROEPIANDROSTERONE (DHEA) ON CHOLESTEROLMETABOLISM IN RATS* David Kr~tchevskyl, Shirley A. TepperI, David M. Klurfeld I, and Arthur G. ~chwartz ~ The Wistar Institute of Anatoniy and Biqlogy 36th Street at Spruce, Philadelphia, Pennsylvania 19104, and =Fels Research Institute, Te~le University, Philadelphia, Pennsylvania 19140 Received ~final ~rm 8May 1983
SUMMARY Wistar rats were fed semipurified diets containing 0.5% of dehydroepiandrosterone (DHEA) for 14 days. DHEAfeeding resulted in weight loss and slight liver enlargement.
I t did not affect serum triglyceride
levels but increased serum cholesterol levels.
Liver triglyceride levels
of DHEA-fed rats were significantly lower than in controls. cholesterol absorption.
DHEAinhibited
Liver slices from DHEA-fed rats incorporated more
acetate into cholesterol than controls; there were no differences in conversion of mevalonate. INTRODUCTION Dehydroepiandrosterone (DHEA) has been shown to inhibit weight gain in mice despite i t s lack of effect on food intake (Tannen and Schwartz, 1981; Schwartz et a l . , 1983). There have been few studies of the effect of DHEA on lipid metabolism.
I t has been found to have no effect on serum cho-
lesterol levels in normal rats (Ben-David et aI., 1967); to reduce t r i g l y ceride levels in mice (Tannen and Schwartz, 1981); and to inhibit in vitro cholesterogenesis in guinea pig ma~ary gland tissue (Granelly and Turner, 1968). Because of the interest in the carcinostatic properties of DHEA
*Supported, in part, by grants (HL-03299, CA-14661 and AG-00368) and a Research Career Award (HL-00734) from the National Institutes of Health and funds from the Commonwealth of Pennsylvania.
0031-6989/83/090797-07/$03.00/0
© 1983 The Italian Pharmacological Society
Pharmacological Research Communications, Vol. 15, No. 9, 1983
798
(Schwartz, 1979) we f e l t i t would be interesting to examine its effects on aspects of lipid metabolism in intact rats. MATERIALS AND METHODS Male Wistar rats were used in both experiments. The rats were caged individually in air-conditioned rooms maintained on a 12-hr light-dark cycle.
In the f i r s t study rats were fed a semipurified diet containing
54.5% sucrose, 25% casein, 10% corn o i l , 5% cellulose, 5% salt mix (AIN), I% vitamin mix (AIN) and 0.5% cholesterol.
This diet provides 53.4% of
calories as carbohydrate, 24.5% as protein and 22.1% as fat. Dehydroepiandrosterone (0.5%) was added at the expense of sucrose.
In the
second study no cholesterol was added to the diet and the sucrose content was increased by 0.5%. The diets were fed for 14 days at which time sera and livers were taken for determination of cholesterol
(ROschlau et a l . ,
1974; DeHoff et a l . , 1978) and triglycerides (Levy and Keyloun, 1972). For cholesterol absorption studies each rat was given a single oral dose of [4-1hC]cholesterol (0.5 pCi) in O.S m! of propylene g|yco]. Feces were collected for 3 days and neutral and acidic steroids isolated and analyzed for radioactivity (Kritchevsky et a l . , 1973). Cholesterol biosynthesis by liver slices was studied using the system described by Bucher and McGarrahan (1956). Incubations in duplicate were carried out using 0.5 gm of liver and either sodium [l-14C]acetate (0.5 pCi) or 2-14C-mevalonic acid
(0.5
). RESULTS AND DISCUSSION
The first study (Table I) was carried out using rats weighing 152 gm. Rats fed DHEA gained significantly less weight than the controls but had enlarged livers.
Serum cholesterol levels were elevated significantly in
rats fed DHEA but triglyceride levels were unaffected.
Liver cholesterol
Pharmacological Research Communications, Vol. 15, No. 9, 1983
Table 1,
799
Influence of dehydroeplandrosterone (DHEA) on Lipid Metabltsm in Rats* (All data n~an ± SEM) Group j . .
+
DHEA Number
7
p**
Control 7
Weight gain, g
21 ± 4
67 -+3
Liver weight, g
9.6 ± 0.3
9.0 ± 0.8
Relative liver weight (g/1OOg)
5.55 -+ 0.14
4,11 -+ 0.20
0.001 ~S
0,001
Serum lipids, mg/dl 70 * 4
0.001
87 ± 13
70 ± 8
NS
Cholesterol
845 ± 38
865 -+ 53
~S
Triglyceride
196 ± 42
507 -+ 95
U.U2
Serum (dpm x 10-4 )
1.51 ± 0.12
0.99 ± 0.09
U,OI
Liver (dpm x 10-5 )
1.89 ± 0.08
2.29 • 0.27
NS
3.8 ± 0.3
3.2 -+ 0.2
NS
Neutral ste~oid (dpm x 10"~)
5.81 ± 0.63
3.90 -+ 0.35
0.01
Aci di c ster~i d (dpm x I0- )
0,51 ± 0.04
0,51 ~ 0.U3
NS
Absorption (%)
42.5 ± 5.8
59.9 ± 3.1
0.05
Cholesterol Trig]yceride
122 ± 8
Liver lipids, mg/lOOg
Cholesterol absorption
Feces Weight, g
* Rats (152 g) fed semipurified diet containing 0,5% cholesterol + 0.5% DHEA for 14 days, * * Student'S t t e s t .
(see t e x t )
Pharmaco/ogical Research Communications, Vol. ;5. No. 9, 1983
800
Table 2.
Influence of dehydroeplandros'terone (DHEA) on l i p i d ~ a b o l i s m in rats* (All data mea, ± SEM) Group p** DHEA
Number
10
Control
<
i0
Weight gain, g
-23 ± 3
Liver weight, g
9.4 ± 0.3
8.9 ± 0.2
NS
5.22 ± 0.14
3.83 ± 0.05
0,001
131 t 6
9g ± 5
0,005
32 ± 2
30 ± 2
NS
Cholesterol
231 ± i0
338 ± 12
0.001
Tri glyceri des
I00 ± 10
362 ± 31
0.001
Cholesterol absorpti on Serum (dpm x 10"~)
1.22 ± 0.14
0,94 ± 0.02
NS
Liver (dpm x 10-5 )
2.30 ± 0.24
2.24 ± 0.33
NS
2.9 ± 0.2
3.9 ± 0.3
0.Oh
Neutral steroid (dpm x i0 "~)
4.23 + 0.53
3.22 ± 0.55
NS
Acidic steroid (dpm x I0- )
0.13 + 0.04
0.20 + 0.05
NS
60.3 ± 4.8
68.9 ± 5,2
NS
Relative liver weight (g/100 g)
31 ± 2
O.001
Serum ]ipids, mg/dl Cholesterol Tri glyce ri de Liver lipids, mg/lO0 g
Feces Weight, g
Absorption (%)
* Rats (202 g) fed cholesterol-free semipurified diet (see text) containing 0,5% DHEA for 14 days. ** Student's t test. t Five rats per group used for absorption study.
Pharmacological Research Communic~'ons, Vol. 15, No. 9, 1983
801
levels were similar in the two groups but liver triglycerlde levels were reduced significantly in the DHEA-fed rats. Rats fed DHEA and [14C]cholesterol accumulated significantly more cholesterol in their sera but less in their liver.
They absorbed s i g n i f i -
cantly less cholesterol and differed from controls by excreting considerably more neutral steroid in the feces (p
Triglyceride accumulation in blood is unaf-
fected but liver triglyceride levels are affected significantly. The second experiment was carried out using larger (202) gm rats.
As
in the previous study weight gain was inhibited, relative liver weight increased, serum cholesterol was raised significantly and serum t r i glycerides unaffected in DHEA-fed rats.
In this study both liver cho-
lesterol and triglycerides were reduced significantly in the test rats. Cholesterol absorption data were consistent with those seen in the earlier experiment (Table I ) .
Cholesterol absorption was decreased in rats fed
DHEA and the decrease was reflected in 31% greater excretion of fecal neutral steroid.
Serum plus liver pools of both cholesterol and t r i g l y -
cerides were significantly lower (p
The
actual pool sizes (mg) were: cholesterol-DHEA, 27.1+_ 1.2 and control, "36.9 ± 1.9; triglycerides, DHEA, 11.2 ± 1.1 and control -34.5_+ 3.0. Data on cholesterol synthesis are presented in Table 3.
Cholesterol
synthesis from acetate was enhanced by DHEAfeeding as was fatty acid synthesis.
Cholesterol synthesis from mevalonate was unaffected,
Ben-
Pharmacological Research Communications, VoL 15, No. 9, 1983
802
Table 3.
Lipogenesis by liver sllces from rats fed 0.5% DEAH*
Group DHEA
P <
'C~ntrol
From Na-I-14C acetate Cholesterol dpm/100 mg l i v e r x 10-2
6.90 ± 0.87
4.16 ± 0.23
O.01
dpm/liver x 10"~
6.88 ± 0.96
3.87 ± 0.23
0.001
10.22 ± 0.38
8.30 ± 0,37
0.01
dpm/100 mg liver x 10-3
6.09 ± 0.57
4.88 ± 0.57
NS
dpm/liver x 10-5
6.04 + 0.62
4.48 -+ 0,46
NS
Fatty acid dpm/liver x i0 -s From 2-1WC-mevalonic acid
* Five rats per group. NS = not significant David et al. (1967) found that DHEA given at a dose of 5 mg/kg had no effect on serum cholesterol levels in normal rats. be hypercholesterolemic,
We have found DHEA to
but we fed DHEA as 0.5% of the diet.
Assuming an
average daily intake of 14 gm of diet per rat the animals ingested 70 mg of DHEA daily or about 350-460 mg/kg.
I t is possible that DHEA becomes
hypercholesterolemic with increasing dosage. Liver l i p i d levels were significantly lower in DHEA-treated rats and the principal effect appeared to be on triglycerides,
Cholesterol absorp-
tion was inhibited in DHEA rats. In our hands cholesterol synthesis from acetate was enhanced in liver slices from rats fed DHEA; cholesterol synthesis from mevalonate was unaffected.
Granelly andTerner (1968) added DHEA to incubation mixtures of
Pharmacological Research Communications, Vol. 15, No. 9, 1983
guinea pig mammary gland and reported inhibition of cholesterol synthesis. Addition of the test co~ound to the incubation mixture is not con~arable to studies using tissues from animals fed the co~ound. Our data confirm the growth inhibiting effect of DHEA (Schwartz, 1979) and suggest that the principa] focus of action may involve triglycerlde metabolism. DHEAincreases cholesterolemia but also inhibits cholesterol absorption.
These finding suggest effects on lipoprotein metabolism which
might be worth further study. REFERENCES Ben-David, M., Dikstein, S., Bismuth, G., and Sulman, F.G. (1967) Proc. Soc. Exp. Biol. Med. 125, 1136. Bucher, N.L.R., and M~rrahan, K. (1956) J. Biol. Chem. 22__22,I. DeHoff, J.L., Davidson, L.M., and Kritchevsky, D. (1978) Clin. Chem. 2__44, 433. Granelly, AoA., and Terner, C. (1968) Endocrinology 83, 1311. Kritchevsky, D., Casey, R.P., and Tepper, S.A. (1973~-'Nutr. Reports. Int. 7, 61. iTritchevsky, D., Moynihan, J.L., and Sachs, M.L. (1961) Proc. Soc. Exp. Biol. Med. 108, 254. Levy, A. L . ~ n d Keyloun, C. (1972) Adv. Automated Ana|. --i, 497. Ri}schlau, P., Berut, E., and Gruber, W. (1974) Z. Klin. Biochem. 12, 403. Schwartz, A.G. (1979) Cancer Reso 39, 1129. Schwartz, A., Hard, G., Pashko, L., Abou-Ghanbia, M., and Sivern, D. (1983) Nutr. Cancer, in press. Tannen, R.H., and Schwartz, A.G. (1981) Fed. Proc. 40, 347.
803