Effects of gonadal hormones on cholesterol metabolism in the rat

Journal of Atherosclerosis Research Elsevier Publishing Company, Amsterdam - Printed in The Netherlands

E F F E C T S O F G O N A D A L H O R M O N E S ON C H O L E S T E R O L M E T A B O L I S M IN THE RAT

S. MUKHERJEE AND S. GUPTA with the technical assistance of ARCHANABHOSE Laboratories of Lipid Research, Department of Applied Chemistry, University of Calcutta, Calcutta (India) (Revised, received February 2nd, 1967)

SUMMARY The g o n a d a l regulation of synthesis a n d catabolism of cholesterol in t h e liver and its r e l a t i o n s h i p with serum cholesterol level have been i n v e s t i g a t e d in i n t a c t , g o n a d e c t o m i z e d a n d h o r m o n e - t r e a t e d rats in the presence and absence of d i e t a r y fat as well as cholesterol. Although, females have higher rates for synthesis a n d b r e a k down of cholesterol in the liver t h a n males, t h e y exhibit higher serum cholesterol values. G o n a d e c t o m y reduces biogenesis of cholesterol in the liver, while rates of c a t a b o l i s m are increased in males and decreased in females following h o r m o n e depletion a n d these effects are reversed b y hormone replacement. E x c l u s i o n of fat from the diet caused m a r k e d lowering of rates of s y n t h e s i s b u t is w i t h o u t a n y influence on sterol d e g r a d a t i o n in the liver. On a cholesterol diet, however, c a t a b o l i s m is stimulated, b u t synthesis reduced. H o r m o n a l influences on catabolism were more enhanced in cholesterol-fed animals, b u t their effects on synthesis in animals on a cholesterol or fat-free diet were not clear-cut due to v e r y low rates of conversion of a c e t a t e to cholesterol in the liver. A positive correlation b e t w e e n serum cholesterol a n d h e p a t i c rates of synthesis a n d b r e a k d o w n in various endocrine states has not been o b t a i n e d , p r o b a b l y due to the influences of hormone a d m i n i s t r a t i o n or w i t h d r a w a l on other endocrine systems which usually function in an i n t e g r a t e d manner.

INTRODUCTION Much interest has recently been centered on the effects of steroid h o r m o n e s on the synthesis, storage, mobilization and d e g r a d a t i o n of lipids, especially of cholesterol, p a r t i c u l a r l y as these m a y relate to the clinical problems of atherosclerosis. There have been n u m e r o u s r e p o r t s in the l i t e r a t u r e concerning the effects of g o n a d a l hormones J. Atheroscler. Res., 7 (1967) 435-452

436

s. MUKHERJEE, S. GUPTA, A. BHOSE

and estrogenic steroids on lipid metabolism in experimental animals and in men, and several review articles have appeared on the subject 1-3. F r o m the investigation of early workers in the field of hormonal regulation of atherosclerosis, it is evident t h a t atherosclerosis has never been produced in an experimental animal simply b y hormonal manipulation and in the absence of dietary fat or cholesterol 4,5. Most studies on hormones and atherosclerosis have dealt with their effects in animals and in h u m a n s after ingestion of an atherogenic diet 6-s. Studies on the regulation of cholesterol metabolism b y gonadal steroid hormones have mostly been confined to observing changes in serum cholesterol, cholesterolphospholipid ratio and/5-1ipoprotein cholesterol subsequent to hormone t r e a t m e n t " - l l . Simultaneous studies on metabolic alterations in rates of biosynthesis and catabolism of cholesterol are fewer in comparison and results are equivocal. Table I summarizes the effect of sex hormones and gonadectomy on cholesterol synthesis in the rat. G o n a d e c t o m y of males has been found to have no effect on cholesterol synthesis in the liverl2, is, or m a y reduce or increase synthesisl4, is. Significant reduction in sterol synthesis in gonadectomized females has been obtained b y some workers 12, while others have failed to substantiate such findings la. An increase or a decrease in hepatic synthesis of cholesterol have been reported following estrogen administration to the r a t 16-1s. Similarly, either a decrease 1~ or an increasela or even an absence of effect of androgen administration on cholesterol synthesis in the rat a p p e a r in published literaturOg. Some of the variations in results between groups of investigators m a y h a v e arisen due to variation in species and strains, differences in experimental conditions, such as dose and duration of hormone t r e a t m e n t , t y p e of diet ingested, and use of different criteria for evaluating rates of synthesis (vide Table 1). Although there appears to be little doubt t h a t b o t h endogenous and exogenous steroids have definite effects on overall lipid metabolism, the exact m e c h a n i s m b y which these effects are brought about remains speculative. A re-investigation of the problem of regulation of cholesterol metabolism in male and female rats b y gonadal hormones has been a t t e m p t e d in the present studies using the following parameters of lipid metabolism: (a) level of serum and liver cholesterol, (b) synthesis of cholesterol from [1-14C]acetate in liver slices and in liver microsomes, and (c) conversion of [4-14C]cholesterol to bile acids, as well as, oxidation of [26-14C] cholesterol in liver mitochondrial preparation. Studies on hormonal influences have been divided in three phases: (1) effect of extirpation of the endocrine gland to observe effects of depletion of hormone; (2) effect of replacement-therapy b y administration of near physiological doses of respective hormones to gonadectomized rats; (3) effect of exaggerated dose of sex hormones of same as well as of opposite physiological action to the normal animal.

j . Atheroscler. Res., 7 (1967) 435-452

EFFECTS OF G O N A D A L HORMONES ON CHOLESTEROL METABOLISM IN THE RAT

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MATERIALSAND METHODS Male and female rats of CDRI strain from inbred UCS colony have been used in the investigation. The animals were maintained on three experimental diets: a control fat diet containing 15 % cottonseed oil, a potentially atherogenic diet containing 1 % cholesterol plus 15 % cottonseed oil diet, and thirdly, a fat-free regimen. All animals were maintained on respective diets for a period of 16 weeks. Gonadectomy was performed on half of the experimental animals after 8 weeks on diet. Replacement-therapy of gonadectomized animals was started on the day following surgery and continued for a period of 8 weeks on a third of the operated a n i m a l s - males received 3 0 0 / , g of testosterone and females 30/zg of estradiol-17 (6 injections/ week/100 g weight rat). A third of the intact animals received intramuscular injections of 1 mg of testosterone or 100 #g of estradiol-17 per 100 g body weight and hormone treatment continued also for 8 weeks prior to the date when they were killed. The hormones were dissolved in propylene glycol and control animals received the same volume of propylene glycol injection. After this time, the animals were killed by removal of blood by cardiac puncture. The livers were immediately excised, trimmed and used for metabolic studies. A part of the liver was saved for cholesterol estimation and kept frozen until used for lipid extraction. The serum of the rats was extracted for cholesterol analysis using a modified SPERRY-BRAND method ~0. The lipids of liver were extracted b y the method of THOMSONe~ al. 2I and cholesterol determined b y a modified S P E R R Y - W E B B method ~ as developed b y ALFIN-SLATER AND DEUEL*. Cholesterol biosynthesis from [1-14C~acetate in rat liver slices was studied according to the procedure of HOTTA et al. 34 as modified b y MUKHERJEE AND ALFINSEATER23, Liver microsomes for synthesis experiments were obtained b y the method of BUCHNER tt al. 24 as adopted by POPJAK25. Oxidation of [26-14C]cholesterol was followed by the method of KRITCHEVSKYet al. 26 and conversion of [4-14Clcholesterol to bile acids was studied by the procedure outlined b y FREDRICKSONaS. Extraction procedure for recovering bile acid was essentially t h a t followed by FREDRICKSON4 with the exception that the purification of the extracted bile acids was carried out according to SJOVALL27 on an alumina column. RESULTS

G o n a d a l h o r m o n e s a n d s e r u m cholesterol

It appears from results given in Table 2 that on all the three experimental diets, average serum cholesterol remained lower in male rats than the corresponding values for females. On a fat-deficient diet the difference between mean values was small but significant (P < 0.05) and when fat or cholesterol is present in the diet, * Unpublished observations. J. Atheroscler. Res., 7 (1967) 435-452

EFFECTS OF GONADAL HORMONES ON CHOLESTEROL METABOLISM IN THE RAT

439

TABLE 2 EFFECT

OF DIETARY

TREATED

LIPID

ON SERUM

CHOLESTEROL

OF INTACT,

GONADECTOMIZED

AND

HORMONE-

RATS

Males

Females

total

free

%free total

(rag~t00 ml) Cottonseed oil diet Intact Intact + androgen I n t a c t + estrogen Gonadectomized Gonadectomized + androgen Gonadectomized + estrogen

56.0 62.0 69.1 90.0

444•

Cholesterol diet Intact Intact + androgen I n t a c t + estrogen Gonadectomized Gonadectomized + androgen Gonadectomized + estrogen

% free

(mg/lO0 ml)

4.8 a 2.7 3.5 2.3

14.0 16.5 16.0 16.1

4- 2.1 4- 3.0 4- 1.5 52 3.3

25.0 26.6 23.1 18.1

1.6

17.2 52 2.1

24.4

67.0 4- 2.0

92.7 4- 3.2

14.0 52 1.7

15.1

111.0 4- 3.6

45.6 41.5 49.0 55.0

13.0 12.0 15.0 I0.0

4- 1.0 52 1.2 52 1.0 4- 0.8

28.0 29.0 30.0 18.1

48.0 32.0 50.0 64.0

47.2 52 3.1

15.0 4- 1.7

31.8

64.0 4- 3.0

12.0 4-

1.2

18.7

62.0 4-

1.9

31.0 4- 0.9

37.7

72.0 4-

12.0 4- 0.6

17.0

4.0 3.0 7.0 9.0

24.0 67.0 48.0 7fi.0

2,0 2.1 3.0 0.7

22.4 41.3 28.9 30.4

132.0 125.0 200.0 296.0

135.0 4- 7.0

34.0 4- 2.5

25.2

274.0 4- 16.0

53.6 4- 8.8

19.7

70.5 4-

Fat-free diet Intact Intact + androgen I n t a c t + estrogen Gonadectomized Gonadectomized + androgen Gonadectomized + estrogen

free

107.0 162.0 166.0 246.0

44452

52 452 4-

1.5 1.5 1.2 1.8

• 444-

80.0 68.0 91.0 108.0

4444-

4• 52 4-

• 444-

3.7 2.2 4.7 6.1

2.5 1.4 1.7 2.2

1.1 6.0 5.0 3.0 16

26 15 27 21

44452

1.0 2.0 1.65 2.8

32.5 22.1 29.6 19.4

1.0

19.7

20.4 4- 4.0

18.3

12.5 6.0 16.5 14.0

26.0 19.0 33.0 21.8

13.2 4-

33.0 36.0 78.0 148.0

4- 1.1 4- 0.8 + 1.4 4- 1.6

4444-

1.5 2.5 2.0 2.8

25.0 28.8 39.0 S0.0

200.0 52 12.3

90.0 4- 10.0

45.0

244.0 4- 7.8

65.0 4- 7.0

26.6

a S t a n d a r d error of t h e mean. there occurs a highly significant difference between

s e r u m c h o l e s t e r o l of m a l e a n d

female rats (P < 0.001). Gonadectomy

a l w a y s r e s u l t e d i n e l e v a t i o n of s e r u m

c h o l e s t e r o l (0.05 > P >

0.001), the rise being mainly in the esterified cholesterol when rats were maintained on a fat-free or a fat diet. On the cholesterol diet, however, a significant increase in both ester (P < 0.001) and free cholesterol was observed. Hormonal gonadectomized

replacements

to

r a t s h a d a t e n d e n c y t o r e v e r s e t h e e f f e c t s i r r e s p e c t i v e of d i e t g r o u p s ,

in both male and female animals, although

i n n o n e of t h e c a s e s w e r e t h e c o n t r o l

values reached, especially when the diet was fortified with fat or cholesterol. In intact

animals

a n d i n t h e a b s e n c e of d i e t a r y f a t , a n d r o g e n

pressed serum cholesterol in female rats. Estrogen administration,

treatment

de-

on the other hand,

elevated serum cholesterol but to an insignificant extent, although the effect was more pronounced

w h e n d i e t a r y l i p i d s a r e p r e s e n t ( P < 0.01). O r c h i d e c t o m i z e d

rats recei-

j . Atkeroscler. Res., 7 (1967) 435-452

440

S. MUKHERJEE, S. GUPTA, A. BHOSE

TABLE 3 EFFECT

OF DIETARY

TREATED

LIPID

ON LIVER

CHOLESTEROL

OF

INTACT,

GONADECTOMIZED

AND

HORMONE-

RATS

dVIales

Females

total

free

%free total

(mg/g)

free

% free

(rag~g)

Cottonseed oil diet Intact I n t a c t + androgen I n t a c t + estrogen Gonadectomized Gonadectomized + androgen Gonadectomized + estrogen

2.7 3.2 1.9 2.5

• 0.17 • 4- 0.10 • 0.15

2.07 2.51 1.60 2.06

4- 0.06 4-0.11 4- 0.07 4- 0.15

76.6 78.4 84.2 82.4

2.17 2.33 1.75 2,01

444+

0.10 0.12 0.05 0.05

2.05 ~_ 0.06 1.81 • 0.12 1.64 -s 0.08 1.78 4- 0.06

94.4 77.6 96.0 88.5

2.8 4- 0.12

2.29 4- 0.10

81.5

2.30 4- 0.05

1.98 4- 0.12

86.1

2.1

4- 0.08

1.80 -k 0.05

87.5

2.10 4- 0.08

1.92 4- 0.06

89.1

5.6 5.81 2.40 3.71

4- 0.18 4- 0.12 • 0.05 4- 0.12

1.9 2.1 1.2 1.85

_4- 0.06 4- 0.10 • 0.05 + 0.06

34.0 36.0 50.0 50.0

2.95 3.15 2.10 3.91

1.45 1.70 1.38 1.87

4- 0.05 4- 0.07 4- 0.05 4- 0.08

49.1 55.0 65.7 47.8

5.64 4- 0.14

2.80 4- 0.10

49.6

3.40 4- 0.06

2.60 4- 0.10

76. l

2.90 4- 0.08

1.75 • 0.10

60.5

2.92 4- 0.10

1.61 4- 0.07

55.1

3.5 4- 0.07 7.2 • 1.0 3.5 -h 0.05 10.6 •

10.8 12.6 18.9 12.0

24.5 19.5 86.0 112.0

! 1.6 4- 1.0 • 5.6 • 10.5

3.6 4- 0.05 3.8 • 0.06 7.6 • 0.50 14.3 • 1.6

14.7 19.5 8.8 12.8

4- 10.5

17.8

• 1.6

14.8

6.2

4- 0.8

16.2

Fat-free diet Intact I n t a c t 4- androgen I n t a c t 4- estrogen Gonadectomized Gonadeetomized + androgen Gonadectomized + estrogen

4444-

0.10 0.10 0.04 0.10

Cholesterol diet Intact I n t a c t 4- androgen I n t a c t + estrogen Gonadectomized Gonadectomized + androgen Gonadectomized + estrogen

32.4 57.0 18.5 88.6

4- 2.0 -- 9.1 4- 2.5 •

37.8

• 3.3

4.1

• 0.05

10.8

120.0

74.9

• 5.0

9.2

• 1.0

12.4

38.1

•

2.5

ring estrogen therapy had higher serum cholesterol levels than gonadectomized

or

u n o p e r a t e d c o n t r o l s o n all t h r e e d i e t s . A n d r o g e n t r e a t m e n t of o v a r i e c t o m i z e d a n i m a l s caused marked

d e c r e a s e in s e r u m c h o l e s t e r o l o n l y i n t h e p r e s e n c e of d i e t a r y l i p i d .

Thus, h o r m o n a l m a n i p u l a t i o n led to c h a r a c t e r i s t i c sex differences d e p e n d i n g on t h e p r e s e n c e of d i e t a r y lipids. M a l e r a t s h a d l o w e r s e r u m c h o l e s t e r o l t h a n f e m a l e s . With respect to a hypercholesterolemic response to a potentially atherogenic diet, f e m a l e s d i s p l a y e d g r e a t e r s u s c e p t i b i l i t y a f t e r 16 w e e k s o n d i e t . E s t r a d i o l t r e a t m e n t r e v e r s e d t h i s e f f e c t a n d o u r o w n f i n d i n g s c o n f i r m t h e e a r l i e r r e p o r t s of MOSKOWlTZ

et al. 1~ PRIEST et a l J 1 a n d AFTERGOOD et al. 28. However, t e s t o s t e r o n e t r e a t m e n t

did

n o t c a u s e a n y l o w e r i n g of s e r u m c h o l e s t e r o l , r e p o r t e d b y I~ILLIOS AND MANN 9; i n f a c t , a rise w a s n o t e d in our studies. S t u d i e s w i t h i n t a c t a n d g o n a d e c t o m i z e d rats, m a i n t a i n e d o n a f a t - d e f i c i e n t d i e t , i n d i c a t e d t h a t t h e p r e s e n c e of d i e t a r y l i p i d s w a s n e cessary to demonstrate

the gonadal hormone effects on serum cholesterol.

j , Atheroscler, t?es., 7 (1967) 435-452

E F F E C T S O F G O N A D A L H O R M O N E S ON C H O L E S T E R O L M E T A B O L I S M I N T H E R A T

441

Gonadal hormones and liver cholesterol On b o t h fat and fat-free diets, males exhibited higher liver cholesterol levels than females (Table 3). Gonadectomy or replacement t h e r a p y had relatively little effect on liver cholesterol of rats. Significant changes, however, were obtained when estrogen was administered to males and androgen to females (P < 0.05). On all dietary regimens estrogen t r e a t m e n t decreased liver cholesterol in males, the effect being m a x i m u m when diets contained cholesterol. In contrast, elevation of liver cholesterol followed androgen treatment of control and gonadectomized females in all diet groups (P < 0.03). Thus, estrogen can effect lowering of liver cholesterol even when rats are maintained on a potentially atherogenic diet.

Cholesterol biosynthesis Incorporation of [l-a4C]acetate in liver slices. The highest rates of synthesis of cholesterol were observed in the presence of dietary fat as evidenced from measurements of radioactivity of digitonin-precipitable steroids (Table 4). Absence of dietary fat or the presence of cholesterol in the diet resulted in m a r k e d depression of synthesis. I t m a y be noticed t h a t normally females h a v e higher rates of synthesis t h a n males. G o n a d e c t o m y depressed synthesis in b o t h sexes when dietary lipid was present. Hormone replacement reversed the effect of gonadectomy, this effect being m a x i m u m also with fat-fed animals. In the absence of dietary fat, an increase of synthesis rate was obtained in orchidectomized rats while ovariectomized animals exhibited decreased incorporation of radio-acetate into cholesterol. Estrogen administration to intact males significantly depressed cholesterol synthesis on all experimental diets. Synthesis in females was stimulated b y androgen treatment. Gonadectomized animals behaved differently from intact rats on various diets when t r e a t e d with hormones of opposite physiological activity: estrogen enhanced synthesis in cholesterol or fat-fed groups, while in the absence of fat decreased synthesis occurred. Testosterone administration, on the other hand, increased the rate of incorporation of acetate into cholesterol in fat-free as well as in fat-fed gonadectomized females, but, on cholesterol feeding decreased synthesis resulted. Cholesterol synthesis in rat liver microsomes. Experiments with washed microsomes and dialyzed soluble enzymes obtained from livers of rats on three experimental diets using intact, gonadectomized and hormone-treated animals showed t h a t the effects of gonadal hormones and diet are similar in most cases to those observed when using a liver slice technique (vide Tables 4 and 5). However, some variation was noticed between microsomal synthesis and liver slice incorporation d a t a a m o n g fatfree animals subjected to gonadectomy. While orchidectomized rats showed increased synthesis rates b y the former technique, microsomal synthesis rates r e m o v e d this a p p a r e n t l y anomalous situation. The expected decrease in rates of synthesis was obtained with gonadectomized males c o m p a r e d with intact controls with respect to c o m p a r a t i v e rates of synthesis of cholesterol in male and female rats. Our d a t a on j. Atheroscler. Res., 7 (1967) 4 3 5 - 4 5 2

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7 (1967) 435-452

S. G U P T A ,

A. BHOSE

EFFECTS OF GONADAL HORMONES ON CHOLESTEROL METABOLISM IN THE RAT

443

TABLE 5 EFFECT

OF

SYNTHESIS

DIET,

GONADECTOMY

AND

HORMONE

SUPPLEMENTATION

IN

RATS

ON

MICROSOMAL

OF CHOLESTEROL

Mate

Cottonseed oil diet Intact Intact + estrogen Gonadectomized Gonadectomized 4- testosterone Fat-free diet Intact Intact + estrogen Gonadectomized Gonadectomized + testosterone Cholesterol diet Intact Intact + estrogen Gonadectomized Gonadectomized + testosterone

Counts~rag protein

Female

Counts~rag protein

(5) (5) (5)

1494 z~ 222 996 4- 180 528 i 61

(5) (5) (5)

1575 ~ 132 1586 ~ 190 597 4- 101

(5)

1270 :k 280

intact intact + testosterone gonadectomized gonadectoraized 4- estrogen

(5)

1481 4- 300

(5) (4) (4)

327 • 85 211 4- 70 143 4- 66

(5) (4) (4)

408 ~ 156 512 4- 141 166 4- 54

(4)

351 4- 55

intact intact + testosterone gonadectomized gonadectolrdzed + estrogen

(4)

587 ~ 125

(5) (5) (5)

270 • 70 139 4- 44 112 4- 36

(5) (5) (5)

310 i 67 488 4- 116 175 ~ 72

(5)

328 4- 81

intact intact + testosterone gonadectomized gonadectomized + estrogen

(5)

176 z~ 90

Figures in parentheses represent number of animals. Each incubation flask contained 200 /z moles phosphate buffer, pH 7.4, 100 /t moles nicotinamide, 7 /* moles MgC12, 30/t moles GSH, 15 p moles ATP, 3.5 /~ moles CoASH, 1 /~ mole NAD, 1 /z mole NADH, 0.5/~ mole NADPH, 30-50 mg of microsomal protein suspended ill buffer, soluble enzymes (containing 20-25 mg protein), 0.5 /, mole [1-14C]acetate (9.0 • 108 cpm) and incubated in air at 37.5~ for 3 hours. m i c r o s o m a l s y n t h e s i s are i n general a g r e e m e n t with those of FILLIOSet al. 15, KRtTCHEVSKu et a l ) 3 a n d DUGAL AND SAUCIER14 in t h a t female r a t s h a v e r e l a t i v e l y highel rates of s y n t h e s i s c o m p a r e d to males. R e g a r d i n g d i e t a r y influences on cholesteror s y n t h e s i s in m i c r o s o m e s t h e lowest rates were observed w i t h a n i m a l s on an a t h e r o g e n i c diet. Some of our f i n d i n g s on m i c r o s o m a l synthesis in response to h o r m o n a l m a n i festations differed from earlier reports w i t h liver slice e x p e r i m e n t s . KRITCHEVSKY et al.la o b t a i n e d s i g n i f i c a n t l y higher rates of s y n t h e s i s i n g o n a d e c t o m i z e d males, while lower rates of s y n t h e s i s have been observed following g o n a d e c t o m y of b o t h sexes. F r o m t h e i r o b s e r v a t i o n s w i t h operated animals, KRITCHEVSKY et al.13 c o n c l u d e d t h a t the effect of a n d r o g e n is to reduce cholesterol s y n t h e s i s i n r a t liver. T h e d a t a p r e s e n t e d here w i t h l i v e r slices a n d with microsomal p r e p a r a t i o n s fail to s u b s t a n t i a t e those o b s e r v a t i o n s . T h e i n c r e a s e d s y n t h e s i s rates o b t a i n e d i n our studies w i t h estrogen s u p p l e m e n t a t i o n of o v a r i e c t o m i z e d rats suggest a r e t u r n to n o r m a l a n d need n o t necessarily be i n t e r p r e t e d as due to s t i m u l a t i o n b y estrogen of cholesterol synthesis. M a n i p u l a t i o n of r a t e s of cholesterol b i o s y n t h e s i s b y h o r m o n e s is m o r e clearly e x h i b i t e d in e x p e r i m e n t s d e s i g n e d to show the effects of the a d m i n i s t r a t i o n of sex h o r m o n e s of opposite physiological a c t i v i t y to i n t a c t male a n d female rats. E s t r o g e n a d m i n i s t r a J. Atheroscler. Res., 7 (1967) 435-452

444

S. MUIr

~

m

~~

~dg oNo

~ ~~ NS-

. ~ •

~'~ ~o o=~..

~ 1 ~-

0 0

~o

O~ ~0

0

~

0

++

~~176

.~.~.~

~-~ ~.~ 0

0

0

~

~oo J. Atheroscler. Res., 7 (1967) 4 3 5 - 4 5 2

S. GUPTA, A. BHOSE

EFFECTS OF GONADAL HORMONES ON CHOLESTEROL METABOLISM IN THE RAT

445

tion d e p r e s s e d s y n t h e s i s in m a l e s on e i t h e r a f a t diet or a fat-free regimen. E v e n in c h o l e s t e r o l - f e d a n i m a l s , w h e r e r a t e s of s y n t h e s i s were significantly lower, s l i g h t depression in s y n t h e s i s w a s n o t e d . O u r findings t h u s agree w i t h earlier f i n d i n g s of ROSENMAN et al. 18 a n d MUKHERJEE AND ALFIN-SLATER29 t h a t estrogen h a s a n i n h i b i t o r y effect on c h o l e s t e r o l s y n t h e s i s in t h e m a l e r a t . A n d r o g e n a d m i n i s t r a t i o n to i n t a c t females, on t h e o t h e r h a n d , caused an i n c r e m e n t in r a t e s of s y n t h e s i s , b o t h in t h e presence a n d t h e a b s e n c e of d i e t a r y fat. U n f o r t u n a t e l y , due to v e r y low r a t e s of s y n thesis in a n i m a l s on a cholesterol diet, t h e h o r m o n a l effects are m a s k e d c o n s i d e r a b l y b y i n h i b i t i o n effects of e x o g e n o u s l y a d m i n i s t e r e d cholesterol. Cholesterol degradation i n the rat liver T h e conversion of [d-14C]cholesterol to bile acids in r a t liver m i t o c h o n d r i a l p r e p a r a t i o n s in p r e s e n c e of soluble co-factors h a s b e e n i n v e s t i g a t e d a n d t h e e x p e r i m e n t a l r e s u l t s are s h o w n in T a b l e 6. F e m a l e r a t s h a d significantly h i g h e r r a t e s of c o n v e r s i o n of c h o l e s t e r o l t o bile acid, w h e t h e r o r n o t f a t or cholesterol is p r e s e n t in t h e diet. T h e c a t a b o l i c r a t e s are m a x i m a l in t h e p r e s e n c e of d i e t a r y c h o l e s t e r o l in b o t h m a l e a n d f e m a l e rats. O r c h i d e c t o m y r e s u l t s in an a p p r e c i a b l e i n c r e a s e in t h e r a t e of t r a n s f o r m a t i o n of cholesterol to bile acids, t h e effect b e i n g m o r e p r o n o u n c e d a g a i n on a c h o l e s t e r o l diet. A n d r o g e n s u p p l e m e n t a t i o n does n o t reverse t h e effect o b s e r v e d in g o n a d e c t o m i z e d males. O v a r i e c t o m y , on t h e o t h e r h a n d , c a u s e d s i g n i f i c a n t lowering of c h o l e s t e r o l c a t a b o l i s m w h e n c o m p a r e d w i t h i n t a c t rats. E s t r o g e n s u p p l e m e n t a t i o n to g o n a d e c t o m i z e d females c o u n t e r a c t e d t h e effect, i r r e s p e c t i v e of d i e t groups. F u r t h e r , c a t a b o l i c r a t e s in g o n a d e c t o m i z e d m a l e s were c o m p a r a b l e t o t h o s e o b s e r v e d in i n t a c t females, a n d s i m i l a r l y , e s t r o g e n - t r e a t e d m a l e s h a d r a t e s of d e g r a d a t i o n like t h o s e of i n t a c t females. I t c a n b e c o n c l u d e d from t h e a b o v e r e s u l t s t h a t cholesterol d e g r a d a t i o n is f a v o u r e d b y e s t r o g e n a n d d e p r e s s e d b y a n d r o g e n , t h e effects b e i n g p e r s i s t e n t w i t h all diets b u t h o r m o n a l influences are m a n i f e s t e d to a m o r e m a r k e d e x t e n t w i t h a n i m a l s on a c h o l e s t e r o l - r i c h diet.

TABLE 7 E F F E C T O F D I E T ON O X I D A T I O N O F [ 9 - 6 - 1 4 C ] C H O L E S T E R O L I N R A T L I V E R M I T O C H O N D R I A

Males

Females

9( % cholesterol counts recovered as 14C0~)

Intact Intact + androgen Intact + estrogen Gonadectomized Gonadectomized + androgen Gondectomized + estrogen

(6) (6J (6) (6) (6) (6)

16.74 II,61 33.70 36.67 9.40 35.20

• 4,10 • 3.75 4- 6.21 • 5,75 4- 3.10 4- 7.0

40,6 20.0 35,1 15.6 15.0 40.4

• 2,96 • 3,33 • 5.42 • 2.86 4- 3.91 4- 5.50

Figures in parentheses represent number of animals. j . Atheroscler. Res., 7 (1967) 435-452

....[ 1 .....

m..... "--

:-:2;

ml

Fig. 1. T o t a l s e r u m c h o l e s t e r o l level of i n t a c t , g o n a ddeeccttoo~m i s e d a n d h o r m o n e - s u p p l e m e n t e d r a t s o n 15 % c o t t o n s e e d oil diet.

a:===,

..........

N

ii

~J :j

/

~-

+!i

r~, .+

Fig. 2. Free s e r u m c h o l e s t e r o l level of i n t a c t , g o n a d e c t o m i s e d a n d h o r m o n e - s u p p l e m e n t e d r a t s o n 15 % c o t t o n s e e d oil diet.

,i84 8o

i~i~~!+!i +Il

t,t~ ;'4 Q

~! [i ~i~,;

iNN

L in J~

Fig. 3. Total serum cholesterol level of intact, g o n a d e c t o m i s e d a n d h o r m o n e - s u p p l e m e n t e d r a t s on fat-free diet.

EFFECTS OF GONADAL HORMONES ON CHOLESTEROL METABOLISM IN THE RAT

447

Our results on cholesterol degradation to bile acids are in excellent agreement with those of KRITCI-IEVSKYet al.aO,31, whose conclusions are based on studies of mitochondrial oxidation of [26-14C3cholesterol. Using the same criterion as a measure of catabolic rate, we have investigated cholesterol degradation on only one experimental diet (vide Table 7). These results confirm our observations on bile acid formation from [4-I4C]cholesterol. One interesting feature, however, emerges from this comparative study of the relative rates of side chain degradation and ring hydroxylation using E26-14C!cholesterol and [4-14Clcholesterol respectively. Side chain degradation b y rat liver mitochondria is always higher than bile acid formation in all comparable groups and the effects of gonadal hormones are more pronounced in degrading the cholesterol side chain. Marked elevation in rates of side chain oxidation over rates of bile acid formation has been obtained following estrogen administration. We are inclined to believe t h a t feminization of males, caused either b y estrogen treatment or b y bilateral gonadectomy, or estrogen therapy of orchidectomized rats, improves the capacity of rat livers to oxidize and degrade cholesterol side chain to the level of intact females. The implication of these results on the physiology of the animal is too premature to predict, but it is probable that enhanced side chain catabolism m a y be necessary to meet an increased physiological demand on the part of the animal for greater production of steroid hormones, for which cholesterol m a y act as a precursor. DISCUSSION

The studies on the gonadal regulation of cholesterol metabolism present a problem in interpreting the possible mode of action of sex hormones. In both sexes, hormonal depletion of the animal b y gonadectomy caused increased serum cholesterol in spite of lower rates of synthesis. In ovariectomized rats, a decrease in the breakdown of cholesterol results, on the other hand, enhanced degradation of sterol is observed in operated males. The biological effects of estrogen in the male rat are to depress synthesis and to promote catabolism of cholesterol in the liver, yet serum cholesterol levels are higher t h a n in untreated animals. Androgen effects in females are to increase synthesis, reduce cholesterol breakdown and depress serum cholesterol level. Serum cholesterol levels in hormone-treated animals must, therefore, be simultaneously under control of factors, endocrine or otherwise, other than gonadal hormones, and cannot be satisfactorily explained in terms of hepatic synthesis and breakdown. The circulating cholesterol m a y primarily be synthesized in the liver and rapidly equilibrated with serum and other tissues. We have little information about factors which dictate partitioning of cholesterol between serum and other tissues. Our evidence indicates significant lowering of liver cholesterol following estrogen administration either to intact or to gonadectomized rats, even on cholesterol diet. It is possible that apart from its effects on alterations in rates of synthesis and breakdown, estrogen mobilizes cholesterol from liver and other tissues to maintain an elevated serum cholesterol level. Simultaneous observations of higher catabolic rates in such animals

j . Atheroscler. Res., 7 (1967) 435-452

i~!~

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~~

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i

:

r

i

Fig. 4. F r e e s e r u m c h o l e s t e r o l level of i n t a c t , g o n a d e c t o m i s e d a n d h o r m o n e - s u p p l e m e n t e d r a t s o n f a t - f r e e diet.

,oot

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..

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=

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A

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........

l

It t

. . . . . . . . . . . .

.

.

.

.

ii

M&LI~

.

: ,:

:..::.

:

....

Fig. 5. T o t a l s e r u m c h o l e s t e r o l level of i n t a c t , g o n a d e c t o m i s e d a n d h o r m o n e - s u p p l e m e n t e d r a t s o n c h o l e s t e r o l d i e t (15 ~o c o t t o n s e e d oil + 1 ~o cholesterol).

9

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.. GONA~163TOMI~FO ~..60N40 # 4110RO~s '~"//AO

§

::::

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r~

Fig. 6. F r e e s e r u m c h o l e s t e r o l level of i n t a c t , g o n a d e c t o m i s e d a n d h o r m o n e - s u p p l e m e n t e d r a t s o n c h o l e s t e r o l d i e t (15 % c o t t o n s e e d oil + 1 % cholesterol) 9

|!Sii,5:::::::

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~.~

,,,

z'o

o.~

Fig. 7. T o t a l l i v e r c h o l e s t e r o l level of i n t a c t , g o n a d e c t o m i s e d a n d h o r m o n e - s u p p l e m e n t e d r a t s on 15 % c o t t o n s e e d oil diet.

T"

!i~~ ;

i

O"5

i M

A

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.Z:

IN Ltl

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7

E

Fig. 8. F r e e l i v e r c h o l e s t e r o l level of i n t a c t , g o n a d e c t o m i s e d a n d h o r m o n e - s u p p l e m e n t e d r a t s o n 15 % c o t t o n s e e d oil diet.

6"0

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"

9

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M

A

-

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~ /aTA~T ~ ANplt~TN

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Fig. 11. Total liver cholesterol level of intact, gonadectomised and h o r m o n e - s u p p l e m e n t e d rats on cholesterol diet (15 % cottonseed oil + 1 % cholesterol).

.......... |:=:,...o...

M

A

I.

1 I[

B=::=

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Fig. 12. Free liver cholesterol level of intact, gonadectomised and h o r m o n e - s u p p l e m e n t e d rats on cholesterol diet (15 % cottonseed oil + 1 % cholesterol).

EFFECTS OF GONADAL HORMONES ON CHOLESTEROL METABOLISM IN THE RAT

may reflect an increased demand

451

o n t h e a n i m a l f o r h i g h e r r a t e s of s t e r o i d h o r m o n e

synthesis from cholesterol itself, and several endocrine glands may function essent i a l l y i n a n i n t e g r a t e d m a n n e r 6 , 32. U n l e s s o u r o b s e r v a t i o n s a r e c o u p l e d w i t h i n d e p e n dent

assessment

of c h o l e s t e r o l

other steroid hormones m o d e of g o n a d a l

disappearance

from other

tissues and synthesis

of

from cholesterol, it would be difficult to interpret the precise

c o n t r o l of s e r u m l i p i d l e v e l s o n l y f r o m

studies

on relative rates

of h e p a t i c s y n t h e s i s a n d d e g r a d a t i o n of c h o l e s t e r o l . ACKNOWLEDGEMENTS

This investigation and Industrial

Research

State Department The authors Koley,

Lecturer,

gonadectomy

was supported

by a grant

f r o m t h e C o u n c i l of S c i e n t i f i c

.Partial financial support was

of A g r i c u l t u r e

wish to acknowledge Department

also obtained

from United

Grant (FG-In-181). t h e s k i l f u l t e c h n i c a l a s s i s t a n c e of D r . B. N.

of P h y s i o l o g y ,

Calcutta

University,

in performing

of r a t s .

REFERENCES

1 CooK, D. P., Steroids a n d lipid metabolism. In: R. I. DORFMAN (Ed.), Hormone Research, Vol. 3, Academic Press, New York, N.Y., p. 185. 2 MALINOW, M. R., H o r m o n e s and atherosclerosis. In: S. GARATTINI AND P. A. SHORE (Eds.), Advances in Pharmacology, Vol. 2, Academic Press, New York, N.Y., 1963, p. 21. 3 MARSHALL, N. B., G o n a d a l h o r m o n e s and lipid metabolism. In: R. PAOLETTI (Ed.), L i p i d Pharmacology, Academic Press, New York, N.Y., 1964, p. 325. 4 HUEI~ER, W. C., Atherosclerosis, Arch. Path., 1944, 38: 162. 5 HUEPER, W. C., Atherosclerosis, Arch. Path., 1945, 39: 51. 6 BOYD, G. S., H o r m o n e s a n d cholesterol metabolism. In: J. K. GRANT (Ed.), The Control of L i p i d Metabolism, Academic Press, New York, N_Y., p. 79. 7 OLIVER, M. F. AND G. S. BOYD, The influence of sex-hormones on t h e circulating lipids and lipoproteins in c o r o n a r y sclerosis, Circulation, 1956, 13: 82. S STAMLER, J., R. PICK AND L. INT. KATZ, Prevention of coronary atheroselerosis b y estrogen a d m i n i s t r a t i o n in t h e cholesterol-fed chick, Circulat. Res., 1953, 1: 94. 9 FILLIOS, L. C. AND G. V. MANN, The importance of sex in t h e variability of the cholesteremic response of r a b b i t s fed cholesterol, Circular. Res., 1956, 4: 406. 10 MOSKOWITZ, M. S., A. A. MOSKOWITZ, W. L. BRADFORD AND R. W. WISSLER, Change~ in serum lipids a n d coronary arteries of t h e r a t in response to estrogens, Arch. Path., 1956, 61: 245. 11 PRIEST, R. E., M. T. SCHROEDER, R. RASMUSSEN AND R. W . WISSLER, Q u a n t i t a t i v e s t u d y of influence of estrogenic substances on serum lipids of rats fed atherogenic diet, Proc. Soc. exp. Biol. ( N . Y . ) , 1957, 96: 208. 12 AFrEEGOOD, L. AND R. B. ALF1N-SLATER, Dietary a n d gonadal h o r m o n e effects on lipid m e t a bolism in the rat, J. L i p i d Res., 1965, 6: 287. 13 KRITCHEVSKY, D., E. STAPLE, J. L. RABINOWITZ AND M. W, WHITEHOUSE, Differences in cholesterol oxidation a n d biosynthesis in liver of male a n d female rats, Amer. J. Physiol., 1961, 200: 519. 14 DUGAL, L. P. AND G. SAUCIER, The effect of castration on t h e incorporation of [1-14C] acetate into cholesterol in t h e albino rat, Canad. J. Biochem., 1957, 35: 391. 15 FILLIOS, L, C., R, KAPLAN, R. S. MARTIN AND F, STARE, G o n a d a l regulation of cholesterol metabolism, Amer. J. Physiol., 1958, 193: 47. 16 COLEMAN, R. D., Y. M. CHEN AND R. B. ALFIN-SLATER, Cholesterol metabolism in gonadectomized rats, Circular. Res., 1958, 6: 172. 17 NOBLE, N. R. AND R. J. BOUCEK, Biochemical studies on cholesterol in vivo cultivated connective tissue, Circulat. Res., 1957, 5: 573. is ROSENMAN, R. H., M. R. FRIEDMAN AND S. O. BYERS, T h e effect of various hormones upon hepatic synthesis of cholesterol in rats, Endocrinology, 1952, 51: 142. j . Atheroscler. Res., 7 (1967) 435-452

452

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