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Response of sucrose-fed BHE rats to dehydroepiamdrosterone
NUTRITION RESEARCH, Vol. 4, pp. 485-494, 1984 0271-5317/84 $3.00 + .00 Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.
RESPONSE OF SUCROSE-FED BIIE RATS TO DEHYDROEPIANDROSTERONE
Margot P. Cleary, Ph.D., Sue S. Hood, R.D., M.S., Cheryl Chando, R.D., M.S., Carl T. Hansen, Ph.D. and Jeffrey T. Billheimer, Ph.D. D e p a r t m e n t s of Nutrition and Food Sciences and Biological Sciences, Drexel University, Philadelphia, Pennsylvania 19104, Small Animal Section, National Institutes of Health, Bethesda, Maryland 20205 and The Hormel Institute, University of Minnesota, Austin, Minnesota 55912
ABSTRACT Previous studies have shown that administration of dehydroepiandrosterone (DHEA) results in decreased weight gain in several strains of rat. In the present study, sucrose-fed female BHE rats treated with DHEA also had a decrease in weight and food efficiency ratio. In addition, a decrease in glucose-6-phosphate dehydrogenase activity was found in liver. Adipose tissue cellularity was also decreased in DHEA treated rats. DHEA treatment did not alter serum cholesterol, liver cholesterol levels or ACAT activity but did decrease total body cholesterol. An increase in long-chain fatty acyl-CoA hydrolase activity further supported the hypothesis that a futile or substrate cycle may be induced by DHEA. Results of this study suggest that DHEA's ability to inhibit glucose-6-phosphate dehydrogenase may only be of biological significance in v~vo when the activity of the enzyme is elevated. Key Words: BHE rat, glucose-6-phosphate dehydrogenase, cholesterol, l o n g c h a i n f a t t y a c y l - C o A h y d r o l a s e , acyl C o A : c h o l e s t e r o l acyltransferase INTRODUCTION Chronic administration of dehydroepiandrosterone (DHEA) to mice and rats has been shown to result in decreased body weight (I-5). In most cases this occurs without alterations in food intake and a decreased food efficiency ratio is found. To date the m e c h a n i s m of action of this effect on body weight is unknown. DHEA is a known inhibitor of glucose-6-phosphate dehydrogenase (G6PD) (6). The findings that DHEA treatment in mice resulted in decreased lipogenesis (I) and fat cell size (3) appeared to support the possibility that this inhibition m a y play a role in the a n t i o b e s i t y effect of DHEA through l i m i t a t i o n of NADPB production. However, attempts to document an effect on this enzyme in tissues of treated animals have given mixed results. F o l l o w i n g a f a s t i n g - r e f e e d i n g (with a high sucrose diet) regimen, decreases in liver G6PD activity were found in DHEA treated rats (7,8). However, treatment of either chow fed SpragueDawley or lean Zucker rats with DHEA did not result in decreases in G6PD activity (4,9). On the other hand, genetically obese Zucker rats and high fat fed DHEA treated rats (Sprague-Dawley strain) were found to have decreased G6PD activity. In both cases elevations in G6PD were found in comparison to either lean Zucker rats or Sprague-Dawley rats fed a commerical diet. The above data suggested that DHEA's effect on G6PD activity may only be m a n i f e s t w h e n the activity of the enzyme is above some basal level. In the present investigation we a t t e m p t e d to investigate this hypothesis by using a 485
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M.P. CLEARY et a l .
strain of rats, BHE rats, in which glucose-6-phosphate dehydrogenase activity and lipogenesis have been shown to be sensitive to dietary changes (10, 11). In addition, several aspects of cholesterol metabolism were determined as it has been s h o w n that DHEA d e c r e a s e s c h o l e s t e r o l s y n t h e s i s in vitro (12, 13). If DHEA's effect is m e d i a t e d t h r o u g h the r e d u c t i o n of the a v a i l a b i l i t y of NADPH then one w o u l d e x p e c t a c o n c o m i t a n t d e c r e a s e in sterol s y n t h e s i s w h i c h also requires the pyridine nucleotides.
METHODS AND MATERIALS Rats: Female BHE rats were obtained from the Small Animal Section at NIH, Bethesda, Maryland when they were 4-6 weeks old. The rats were housed individually in a temperature controlled room (22-24~ A 12 hour light-12 hour dark cycle w a s used. The r a t s w e r e fed the p o w d e r e d rat chow (Purina #5001) ad l i b i t u m for an initial 2 - w e e k period. All the rats w e r e then s w i t c h e d to a s e m i - p u r i f i e d h i g h s u c r o s e diet (Table I). All rats w e r e a c c l i m a t e d to this diet for an additional 2-week period. The rats were then randomly assigned to either a control (n=6) or the DHEA t r e a t e d (n:7) group. DHEA was i n c l u d e d in the s u c r o s e diet at a level of 0.6% as p r e v i o u s l y d e s c r i b e d (4). D H E A t r e a t m e n t c o n t i n u e d for 20 weeks. At this t i m e the r a t s were k i l l e d by d e c a p i t a t i o n b e t w e e n 8:30 a.m. and 10:00 a.m. Blood was drained from the carcasses, allowed to clot and centrifuged at 1200 rpm for 15 minutes. The serum was stored at -70~ until it was analyzed. Serum c h o l e s t e r o l d e t e r m i n a t i o n w a s m a d e using an e n z y m a t i c assay as d e s c r i b e d in Sigma Technical Bulletin No. 350 (1982). Livers were removed and processed as d e s c r i b e d b e l o w for d e t e r m i n a t i o n of glucose-6-phosphate dehydrogenase, malic enzyme, fatty acid synthetase and long-chain fatty acyl-CoA hydrolase and acyl C o A : c h o l e s t e r o l acyl t r a n s f e r a s e (ACAT) activities. Retroperitoneal and parametrial fat depots were also removed for cellularity determination (see below). Bodies were frozen for later body composition determination.
TABLE I Composition of' Semi-Purified
Sucrose a High Nitrogen Casein a dl Methionine a Celufil a Corn Oil a Total Vitamin Supplement a Salt Mixture a USB XVII Dehydroepiandrosterone b
Sucrose Diet
Sucrose gm/100 gm
Sucrose with DHEA gm/100 gm
65.0 19.5 0.5 5.0 5.0 1.0 4.0 0.0
65.0 19.5 0.5 5.0 5.0 1.0 4.0 0.6
aFrom U.S. Biochemical Corp., Cleveland, Ohio. bFrom Searle Chemicals Inc., Chicago, Illinois.
Liver Preparation: A 3 gram sample of liver was removed and homogenized in a 0.25 M sucrose-1 m M EDTA, pH 7.4, solution. Homogenates were centrifuged at 10,500 g for 15 minutes at 4~ The supernatants were then further centrifuged at 100,000 g for 60 minutes at 4~ The supernatant was removed and stored at -
DEHYDROEPIANDROSTERONE
and BHE RATS
487
70~ for assay of G6PD (14), malic e n z y m e (15) and fatty acid synthetase (16) activities. Mlcrosomes were also prepared for determination of ACAT and longchain fatty acyl-CoA hydrolase activities (17). All enzyme activity was expressed per mg protein as d e t e r m i n e d by the m e t h o d of L o w r y et al. (18). A one gram sample of liver was removed and frozen at -70~ for lipid, cholesterol and protein analyses. Liver cholesterol levels were measured using a procedure described by Rudel and Morris (19). This method uses ~-phthalaldehyde reagent in glacial acetic acid (1:2, w/v) to assay cholesterol content. This particular cholesterol assay was employed because of its reproducibility and the small amount of cholesterol (as little as 5 ug) that can be identified. Adipose Tissue Preparation: Left and right retroperitoneal fat pads and left and right p a r a m e t r i a l fat pads were r e m o v e d and s a m p l e s from each site pooled per rat. Total weight was determined per depot and small samples were taken for fat cell number determination (20). Fat oell number was determined electronically using a Coulter Counter Model ZB. A one gram sample was removed from each fat site sample and frozen. Total lipid in both depot s a m p l e s was determined by the method of Folch et al. (21). Cholesterol levels were determined in the parametrial depot by the method described above for liver. Carcass Composition: Preparation of carcass for lipid extraction included r e m o v a l of internal organs, intestines, paws, tail and fur. The r e m a i n i n g carcass was homogenized in water equaling four times the carcass weight for 15 minutes. Aliquots of the h o m o g e n a t e s were dried in an oven at 60Oc until weights were constant. Lipid extraction was performed using a Soxhlet apparatus with 150 ml chloroform:methanol (2:1, v/v) and the addition of 0.01% butylated hydroxy-toluene to retard fatty acid oxidation. Following lipid extraction, an aliquot was removed for cholesterol determination as described above. Statistic Analysis: Data are presented as means ~ S.D. Data were statistically analyzed using the Student's t test. Significance was at a level of p < 0.05.
RESULTS At the termination of this experiment the sucrose-fed + DHEA female rats weighed 40% less than the sucrose-fed female rats (Figure I). This was a significant difference. In fact, all w e e k l y body weight m e a s u r e m e n t s were significantly decreased in the sucrose-fed + DHEA female rats throughout the experiment. C u m u l a t i v e food intakes were similar for the two groups of rats (Figure 2A). The food efficiency ratio (weight gain [gm]§ intake [gm]) was also calculated. A significant decrease was found for the sucrose-fed + DHEA versus sucrose-fed female rats (Figure 2B). This indicated that the DHEA treated rats used more calories in order to gain an equal amount of body weight as the sucrose-fed rats. Liver Weight and ComPosition (Table II): DHEA treatment increased absolute liver weight (gm) significantly compared to the sucrose-fed female rats. When the contribution of the liver weight, as a percent body weight, was calculated there was also a significant increase in the sucrose-fed + DHEA female rats compared to sucrose-fed control female rats. No difference was found between sucrose-fed and sucrose-fed + DHEA groups in lipid, cholesterol or protein concentration in liver tissue. When total liver protein, lipid and cholesterol were calculated no differences were found. Similarly, no difference was observed in the serum cholesterol levels between the two groups.
488
M.P. CLEARY et al.
300 -K-
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200
,
I-'n-
O
0
1313 I
o
5
I
!
1
10 15 WEEKS on TREATMENT
2O
FIG. 1. Body w e i g h t curve for s u c r o s e fed (: :) and s u c r o s e - f e d + D H E A (m~-i) female BHE rats. Data are means • S.D. *Indicates significant difference between the two groups.
,{TAKE
B. FOOD EFFICIENCY RATIO T
2000
[
U9
9
06
_ L
_
.04; I000
Sucrose fed
Sucrosefed §
kJaose fed
Sucrosefed + DHEA
FIG. 2. C u m u l a t i v e food intake (A) and food e f f i c i e n c y ratio (F.E.R.) for (B) sucrose-fed and sucrose-fed + DHEA female BHE rats. Data are means • S.E.M. *Indicates significant differences between the two groups.
DEHYDROEPIANDROSTERONE and BHE RATS
489
TABLE II Serum Cholesterol, Weight and Composition of Livers of Control and DHEA a Treated Female Rats (x • S.D.)
Sucrose-Fed (n=6)
Liver Liver Liver Liver Liver Liver Liver Liver Serum
Weight (gm) % Body Weight Protein (mg/gm) Protein (total mg) Lipid (mg/gm) Lipid (total mg) Cholesterol (mglgm) Cholesterol (total mg) Cholesterol (mg/dl)
8.62 3.1 20.5 177.0 53.1 475.0 3.61 30.8 93.0
• 0.75* • 0.2* • 1.5 • 2.4 • 0.9 • 80.6 • 0.4 • 4.1 • 12.0
Sucrose-Fed + DHEA (n:7)
11.9 5.6 18.7 210.6 49.3 588.7 3.5 40.9
• 3.3 _+ I .0 • 2.4 • 40.0 • 5.4 • 175.8 +_ 0.3 + 7.6
9T.O +__ 20.0
aDHEA included in the diet at 0.6% for 20 weeks. *Sucrose-fed significantly different from sucrose-fed § DHEA at p < 0.05. Liver Enzyme Determinations: (Table III) DHEA treatment resulted in a significant decrease in hepatic glucose-6-phosphate dehydrogenase activity compared to the sucrose-fed control female rats. Malic enzyme activity was increased by DHEA and fatty acid synthetase decreased with p values in both cases, found to be b e t w e e n 0.1 and 0.05. Long-chain fatty acyl-CoA hydrolase was found to be significantly increased by DHEA treatment but ACAT activity was not affected. TABLE III Liver Enzymes in Female BHE Rats Treated With Dehydroepiandrosterosea (x • S.D.)
Sucrose-Fed (n=6)
Glucose-6-phosphate Dehydrogen~se D Malic enzyme ~ Fatty Acid Synthetase c Long-chain F a t t y . Ac~l CoA hydrolase c ACAT ~
84.26* • 37.88 49.70 # • 4.14 12.60 # • 0.69 42.8* • 9.2 31.9 • 9.1
Sucrose-Fed + DHEA (n=7)
13.01 • 12.20 85.19 • 41.82 9.78 • 4.83 67.7 • 9.7 22.2 • 6.0
aDHEA included in the diet at a level of 0.6% for 20 weeks. bActivity expressed as NADP + reduced/min/mg protein. CActivlty expressed as dpm/mg protein. dAetivity expressed as nmol free fatty acid f o r m e d / 1 5 m i n / m g mic~osomal protein. Sucrose-fed significantly different from sucrose-fed + DHEA at p < O.O5. #Sucrose-fed compared to sucrose-fed + DHEA p < 0.1 > 0.05.
490
M.P. CLEARY et a l .
Adipose Tissu~ (Table IV): Both parametrial and retroperitoneal fat pad w e i g h t s were significantly lower in the sucrose-fed + DHEA f e m a l e group in c o m p a r i s o n to the sucrose-fed control group. When the fat pad w e i g h t s w e r e combined and expressed as a percent of total body weight, the fat pads accounted for a significantly decreased proportion of body weight in the DHEA treated rats (data not shown).
TABLE IV Parametrial and R~troperitoneal Fat Pad Weights a and Cellularity in DHEA Treated Female BHE Rats (x • S.D.)
Sucrose-Fed (n:6)
Sucrose-Fed + DHEA (n:7)
8.97 • 2.80* 5.20 • 1.80" 1.32 • 0.48*
2.43 • 1.50 3.40 • 1.15 0.49 • 0.31
4.29 • 1.87" 2.42 • 1.19 1.22 ~ 0.37*
1.47 • 0.92 1.35 • 0.83 0.52 • 0.33
Parametrial Fat Pads Weight (gm) Fat Cell Number x 106 Fat Cell Size (~g lipid/cell) Retroper~otneal Fat Pads Weight (gm) Fat Cell Number x 106 Fat Cell Size (~g lipid/cell)
acombined both right and left pads. bDHEA included in the diet at 0.6% for 20 weeks. *Sucrose-fed significantly different from sucrose-fed at p < 0.05.
+ DHEA
This decrease in fat pad w e i g h t of the DHEA treated rats was found to be due to a decrease in both fat cell size and n u m b e r in the p a r a m e t r i a l depot. Although both values were decreased in the retroperitoneal fat pad, only fat cell size was significantly different. Carcass Composition (Table V): In all instances, calculations for carcass composition were made without the addition of the removed organs and adipose tissue. On an absolute (gm) basis, carcass weight, carcass water, carcass lipid weight and cholesterol weight were significantly reduced in the sucrose-fed + DHEA female group compared to the sucrose-fed female group. Carcass fat-free dry matter on an absolute basis was not significantly decreased in the sucrosefed + DHEA f e m a l e rats w h e n c o m p a r e d to the sucrose-fed f e m a l e rats. When computed as a percentage of carcass weight, significant decreases were found for both the carcass lipid and cholesterol in the DHEA rats. The percentage of carcass water was increased in the DHEA group. There were no significant differences in organ weights of sucrose-fed + DHEA female rats in comparison to sucrose-fed control rats whether expressed on an absolute (gram) basis or relative to body weight (data not shown).
DEHYDROEPIANDROSTERONE and BHE RATS
491
TABLE V Carcass Composition of DHEA a Treated Female BHE Rats (x • S.D.)
Sucrose-Fed
(n=6) Carcass Weight (gm) Carcass Water (gm)
(%) Carcass Fat Free Dry Matter (gm)
(%) Carcass Lipid (gm) (%) Carcass Cholesterol
(gm) (%)
Sucrose-Fed § DHEA (n:7)
185 ~ 14"
128
~ 34
98 ~ 10" 53 ~ 5*
77 60
• 22 ~ 4
39 ~
9
21 •
4
29 22
+ 9 +_ 3
43 ~ 23 ~
9* 4*
20 15
_+ 7 +_ 3
2.0 ~ 1.0 ~
5.0* 0.2*
0.6• 0.5~
0.4 0.2
aDHEA included in the diet at a level of 0.6% for 20 weeks. *Sucrose-fed significantly different from sucrose-fed + DHEA rats at p < 0.05.
DISCUSSION The results of this study indicate that DHEA a d m i n i s t r a t i o n to BHE rats resulted in a decrease in body weight, confirming earlier observations in two other strains of rats (4,5). The response of this strain of rat was s o m e w h a t intermediate between those found for either Sprague-Dawley and lean Zucker rats or obese Zucker rats. Previously, "lean" rats had responded with small but significant decreases in body weight with essentially no effect on food intake. Obese Zucker rats were found to have quite a large decrease in body weight compared to nontreated obese rats, i.e. 40% (5). Although not an obese rat, the response of the BHE rat was more like that of the obese rat since a large decrease in body weight was found. Precisely why the BHE rat is more responsive to DHEA treatment than other lean rats is not clear. This may be due to the use of a semipurified diet that a l l o w s more DBEA to be absorbed r e s u l t i n g in a higher dosage. Rats fed a semipurified-high fat diet also had a greater response to DHEA treatment than did chow fed rats (4). On the other hand, g e n e t i c a l l y obese rats fed chow had quite a large response to DHEA (5). It may be that there is some combination of dietary as well as genetic factors that are involved in the responses found to DHEA in different rat strains. Determining absorption of DHEA from the gut and circulating levels of DHEA and/or its metabolites are studies to be conducted to clarify this. The decrease in fat pad w e i g h t s and fat cell size and n u m b e r in the sucrose-fed + DHEA rats c o m p a r e d to the sucrose-fed rats is similar to that described for lean and obese Zucker rats (5). These data plus the body composition results indicate a specific effect for DHEA on decreasing body fat with little effect on development of other tissues. Although we had anticipated a p o s s i b l e effect of DHEA on cholesterol metabolism in the sucrose-fed BHE rat, in
492
M.P. CLEARYet al.
general there was no effect on the various m e a s u r e m e n t s made. A decrease in carcass cholesterol was found but this is probably a reflection of the decrease in carcass lipid found. As previously described in the introduction, DHEA is a known inhibitor of the enzyme glucose-6-phosphate dehydrogenase (6). This inhibition has been described in homogenates and supernatants from a variety of mammalian tissues. It has also been found in liver tissue following fasting-refeeding regimens (7, 8). Following long-term DHEA treatment, decreased G6FD activity has been found only in situations where an increase in activity was present in comparison to that of the "control" group. This included a study where high-fat fed rats were found to have increased hepatic G6PD activity compared to chow-fed rats. In this case DHEA treatment resulted in decreased G6PD activity in liver samples from the high fat fed DHEA treated rats but not from the chow-fed DHEA treated rats (4). Zucker obese rats have increased G6PD activity compared to control lean rats and DHEA treatment in obese rats resulted in decreased G6PD activity (9). Thus, BHE rats appeared to respond in a similar fashion; when G6PD activity was increased by sucrose feeding , decreased activity of G6PD was found in the DHEA treated rats. Thus it may be that i n v i v o inhibition of this enzyme may only be of significance w h e n the activity is elevated by some stimulus. Possibly this is due to the presence of specific isoenzymes of G6PD. DHEA has been s h o w n to have different effects on i s o e n z y m e s of G6FD (22-25). Whether this inhibition contributes to DHEA's antiobesity action r e m a i n s to be determined. We have r e c e n t l y proposed that DHEA's ability to decrease body weight without an effect on food intake may be better explained by an induction of a futile cycle of fatty acid m e t a b o l i s m (26). An increase in the activity of long-chain fatty acyl-CoA hydrolase was found in lean and obese DHEA treated rats. This enzyme is responsible for removal of the CoA group from fatty acids. Prior to the fatty acids being utilized by m e t a b o l i c pathways, the CoA group must be reattached in a step requiring ATF hydrolysis. In the BHE rats, DHEA treatment resulted in a 100% increase in long-chain fatty acyl-CoA hydrolase activity. In the control sucrose-fed rats, similar values were found as those described for adult Sprague-Dawley (27) rats and lean and obese Zucker rats (26) indicating that diet and genetics do not appear to alter this enzyme. In general, these data support our hypothesis t h a t inhibition of G6PD probably does not account for the "antiobesity" characteristics of DHEA treatment but may play a role in contributing to the enhanced response to DHEA found when "basal" activity of G6PD is elevated. A more plausible explanation for the p r i m a r y antlobesity effect is that some kind of energy wasting process is induced by DHEA treatment. The increased activity of the enzyme long-chain fatty acyl-CoA hydrolase found with DHEA treatment here and in a previous study suggests a futile cycle of fatty acid metabolism could be induced resulting in decreased weight without effects on food intake.
ACKNOWLEDGMENTS Support of this r e s e a r c h was provided by a grant-in-aid from the Southeastern Pennsylvania Chapter of the A m e r i c a n Heart Association, The Hormel Foundation and National Institutes of Health Grant AM 32965.
IA preliminary study using male BHE rats showed that sucrose-fed male rats had twice the G6PD activity as chow-fed male rats.
DEHYDROEPIANDROSTERONE and BHE RATS
493
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Accepted for publication March 4, 1984.
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using ~-
RESPONSE OF SUCROSE-FED BIIE RATS TO DEHYDROEPIANDROSTERONE
Margot P. Cleary, Ph.D., Sue S. Hood, R.D., M.S., Cheryl Chando, R.D., M.S., Carl T. Hansen, Ph.D. and Jeffrey T. Billheimer, Ph.D. D e p a r t m e n t s of Nutrition and Food Sciences and Biological Sciences, Drexel University, Philadelphia, Pennsylvania 19104, Small Animal Section, National Institutes of Health, Bethesda, Maryland 20205 and The Hormel Institute, University of Minnesota, Austin, Minnesota 55912
ABSTRACT Previous studies have shown that administration of dehydroepiandrosterone (DHEA) results in decreased weight gain in several strains of rat. In the present study, sucrose-fed female BHE rats treated with DHEA also had a decrease in weight and food efficiency ratio. In addition, a decrease in glucose-6-phosphate dehydrogenase activity was found in liver. Adipose tissue cellularity was also decreased in DHEA treated rats. DHEA treatment did not alter serum cholesterol, liver cholesterol levels or ACAT activity but did decrease total body cholesterol. An increase in long-chain fatty acyl-CoA hydrolase activity further supported the hypothesis that a futile or substrate cycle may be induced by DHEA. Results of this study suggest that DHEA's ability to inhibit glucose-6-phosphate dehydrogenase may only be of biological significance in v~vo when the activity of the enzyme is elevated. Key Words: BHE rat, glucose-6-phosphate dehydrogenase, cholesterol, l o n g c h a i n f a t t y a c y l - C o A h y d r o l a s e , acyl C o A : c h o l e s t e r o l acyltransferase INTRODUCTION Chronic administration of dehydroepiandrosterone (DHEA) to mice and rats has been shown to result in decreased body weight (I-5). In most cases this occurs without alterations in food intake and a decreased food efficiency ratio is found. To date the m e c h a n i s m of action of this effect on body weight is unknown. DHEA is a known inhibitor of glucose-6-phosphate dehydrogenase (G6PD) (6). The findings that DHEA treatment in mice resulted in decreased lipogenesis (I) and fat cell size (3) appeared to support the possibility that this inhibition m a y play a role in the a n t i o b e s i t y effect of DHEA through l i m i t a t i o n of NADPB production. However, attempts to document an effect on this enzyme in tissues of treated animals have given mixed results. F o l l o w i n g a f a s t i n g - r e f e e d i n g (with a high sucrose diet) regimen, decreases in liver G6PD activity were found in DHEA treated rats (7,8). However, treatment of either chow fed SpragueDawley or lean Zucker rats with DHEA did not result in decreases in G6PD activity (4,9). On the other hand, genetically obese Zucker rats and high fat fed DHEA treated rats (Sprague-Dawley strain) were found to have decreased G6PD activity. In both cases elevations in G6PD were found in comparison to either lean Zucker rats or Sprague-Dawley rats fed a commerical diet. The above data suggested that DHEA's effect on G6PD activity may only be m a n i f e s t w h e n the activity of the enzyme is above some basal level. In the present investigation we a t t e m p t e d to investigate this hypothesis by using a 485
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M.P. CLEARY et a l .
strain of rats, BHE rats, in which glucose-6-phosphate dehydrogenase activity and lipogenesis have been shown to be sensitive to dietary changes (10, 11). In addition, several aspects of cholesterol metabolism were determined as it has been s h o w n that DHEA d e c r e a s e s c h o l e s t e r o l s y n t h e s i s in vitro (12, 13). If DHEA's effect is m e d i a t e d t h r o u g h the r e d u c t i o n of the a v a i l a b i l i t y of NADPH then one w o u l d e x p e c t a c o n c o m i t a n t d e c r e a s e in sterol s y n t h e s i s w h i c h also requires the pyridine nucleotides.
METHODS AND MATERIALS Rats: Female BHE rats were obtained from the Small Animal Section at NIH, Bethesda, Maryland when they were 4-6 weeks old. The rats were housed individually in a temperature controlled room (22-24~ A 12 hour light-12 hour dark cycle w a s used. The r a t s w e r e fed the p o w d e r e d rat chow (Purina #5001) ad l i b i t u m for an initial 2 - w e e k period. All the rats w e r e then s w i t c h e d to a s e m i - p u r i f i e d h i g h s u c r o s e diet (Table I). All rats w e r e a c c l i m a t e d to this diet for an additional 2-week period. The rats were then randomly assigned to either a control (n=6) or the DHEA t r e a t e d (n:7) group. DHEA was i n c l u d e d in the s u c r o s e diet at a level of 0.6% as p r e v i o u s l y d e s c r i b e d (4). D H E A t r e a t m e n t c o n t i n u e d for 20 weeks. At this t i m e the r a t s were k i l l e d by d e c a p i t a t i o n b e t w e e n 8:30 a.m. and 10:00 a.m. Blood was drained from the carcasses, allowed to clot and centrifuged at 1200 rpm for 15 minutes. The serum was stored at -70~ until it was analyzed. Serum c h o l e s t e r o l d e t e r m i n a t i o n w a s m a d e using an e n z y m a t i c assay as d e s c r i b e d in Sigma Technical Bulletin No. 350 (1982). Livers were removed and processed as d e s c r i b e d b e l o w for d e t e r m i n a t i o n of glucose-6-phosphate dehydrogenase, malic enzyme, fatty acid synthetase and long-chain fatty acyl-CoA hydrolase and acyl C o A : c h o l e s t e r o l acyl t r a n s f e r a s e (ACAT) activities. Retroperitoneal and parametrial fat depots were also removed for cellularity determination (see below). Bodies were frozen for later body composition determination.
TABLE I Composition of' Semi-Purified
Sucrose a High Nitrogen Casein a dl Methionine a Celufil a Corn Oil a Total Vitamin Supplement a Salt Mixture a USB XVII Dehydroepiandrosterone b
Sucrose Diet
Sucrose gm/100 gm
Sucrose with DHEA gm/100 gm
65.0 19.5 0.5 5.0 5.0 1.0 4.0 0.0
65.0 19.5 0.5 5.0 5.0 1.0 4.0 0.6
aFrom U.S. Biochemical Corp., Cleveland, Ohio. bFrom Searle Chemicals Inc., Chicago, Illinois.
Liver Preparation: A 3 gram sample of liver was removed and homogenized in a 0.25 M sucrose-1 m M EDTA, pH 7.4, solution. Homogenates were centrifuged at 10,500 g for 15 minutes at 4~ The supernatants were then further centrifuged at 100,000 g for 60 minutes at 4~ The supernatant was removed and stored at -
DEHYDROEPIANDROSTERONE
and BHE RATS
487
70~ for assay of G6PD (14), malic e n z y m e (15) and fatty acid synthetase (16) activities. Mlcrosomes were also prepared for determination of ACAT and longchain fatty acyl-CoA hydrolase activities (17). All enzyme activity was expressed per mg protein as d e t e r m i n e d by the m e t h o d of L o w r y et al. (18). A one gram sample of liver was removed and frozen at -70~ for lipid, cholesterol and protein analyses. Liver cholesterol levels were measured using a procedure described by Rudel and Morris (19). This method uses ~-phthalaldehyde reagent in glacial acetic acid (1:2, w/v) to assay cholesterol content. This particular cholesterol assay was employed because of its reproducibility and the small amount of cholesterol (as little as 5 ug) that can be identified. Adipose Tissue Preparation: Left and right retroperitoneal fat pads and left and right p a r a m e t r i a l fat pads were r e m o v e d and s a m p l e s from each site pooled per rat. Total weight was determined per depot and small samples were taken for fat cell number determination (20). Fat oell number was determined electronically using a Coulter Counter Model ZB. A one gram sample was removed from each fat site sample and frozen. Total lipid in both depot s a m p l e s was determined by the method of Folch et al. (21). Cholesterol levels were determined in the parametrial depot by the method described above for liver. Carcass Composition: Preparation of carcass for lipid extraction included r e m o v a l of internal organs, intestines, paws, tail and fur. The r e m a i n i n g carcass was homogenized in water equaling four times the carcass weight for 15 minutes. Aliquots of the h o m o g e n a t e s were dried in an oven at 60Oc until weights were constant. Lipid extraction was performed using a Soxhlet apparatus with 150 ml chloroform:methanol (2:1, v/v) and the addition of 0.01% butylated hydroxy-toluene to retard fatty acid oxidation. Following lipid extraction, an aliquot was removed for cholesterol determination as described above. Statistic Analysis: Data are presented as means ~ S.D. Data were statistically analyzed using the Student's t test. Significance was at a level of p < 0.05.
RESULTS At the termination of this experiment the sucrose-fed + DHEA female rats weighed 40% less than the sucrose-fed female rats (Figure I). This was a significant difference. In fact, all w e e k l y body weight m e a s u r e m e n t s were significantly decreased in the sucrose-fed + DHEA female rats throughout the experiment. C u m u l a t i v e food intakes were similar for the two groups of rats (Figure 2A). The food efficiency ratio (weight gain [gm]§ intake [gm]) was also calculated. A significant decrease was found for the sucrose-fed + DHEA versus sucrose-fed female rats (Figure 2B). This indicated that the DHEA treated rats used more calories in order to gain an equal amount of body weight as the sucrose-fed rats. Liver Weight and ComPosition (Table II): DHEA treatment increased absolute liver weight (gm) significantly compared to the sucrose-fed female rats. When the contribution of the liver weight, as a percent body weight, was calculated there was also a significant increase in the sucrose-fed + DHEA female rats compared to sucrose-fed control female rats. No difference was found between sucrose-fed and sucrose-fed + DHEA groups in lipid, cholesterol or protein concentration in liver tissue. When total liver protein, lipid and cholesterol were calculated no differences were found. Similarly, no difference was observed in the serum cholesterol levels between the two groups.
488
M.P. CLEARY et al.
300 -K-
L9
200
,
I-'n-
O
0
1313 I
o
5
I
!
1
10 15 WEEKS on TREATMENT
2O
FIG. 1. Body w e i g h t curve for s u c r o s e fed (: :) and s u c r o s e - f e d + D H E A (m~-i) female BHE rats. Data are means • S.D. *Indicates significant difference between the two groups.
,{TAKE
B. FOOD EFFICIENCY RATIO T
2000
[
U9
9
06
_ L
_
.04; I000
Sucrose fed
Sucrosefed §
kJaose fed
Sucrosefed + DHEA
FIG. 2. C u m u l a t i v e food intake (A) and food e f f i c i e n c y ratio (F.E.R.) for (B) sucrose-fed and sucrose-fed + DHEA female BHE rats. Data are means • S.E.M. *Indicates significant differences between the two groups.
DEHYDROEPIANDROSTERONE and BHE RATS
489
TABLE II Serum Cholesterol, Weight and Composition of Livers of Control and DHEA a Treated Female Rats (x • S.D.)
Sucrose-Fed (n=6)
Liver Liver Liver Liver Liver Liver Liver Liver Serum
Weight (gm) % Body Weight Protein (mg/gm) Protein (total mg) Lipid (mg/gm) Lipid (total mg) Cholesterol (mglgm) Cholesterol (total mg) Cholesterol (mg/dl)
8.62 3.1 20.5 177.0 53.1 475.0 3.61 30.8 93.0
• 0.75* • 0.2* • 1.5 • 2.4 • 0.9 • 80.6 • 0.4 • 4.1 • 12.0
Sucrose-Fed + DHEA (n:7)
11.9 5.6 18.7 210.6 49.3 588.7 3.5 40.9
• 3.3 _+ I .0 • 2.4 • 40.0 • 5.4 • 175.8 +_ 0.3 + 7.6
9T.O +__ 20.0
aDHEA included in the diet at 0.6% for 20 weeks. *Sucrose-fed significantly different from sucrose-fed § DHEA at p < 0.05. Liver Enzyme Determinations: (Table III) DHEA treatment resulted in a significant decrease in hepatic glucose-6-phosphate dehydrogenase activity compared to the sucrose-fed control female rats. Malic enzyme activity was increased by DHEA and fatty acid synthetase decreased with p values in both cases, found to be b e t w e e n 0.1 and 0.05. Long-chain fatty acyl-CoA hydrolase was found to be significantly increased by DHEA treatment but ACAT activity was not affected. TABLE III Liver Enzymes in Female BHE Rats Treated With Dehydroepiandrosterosea (x • S.D.)
Sucrose-Fed (n=6)
Glucose-6-phosphate Dehydrogen~se D Malic enzyme ~ Fatty Acid Synthetase c Long-chain F a t t y . Ac~l CoA hydrolase c ACAT ~
84.26* • 37.88 49.70 # • 4.14 12.60 # • 0.69 42.8* • 9.2 31.9 • 9.1
Sucrose-Fed + DHEA (n=7)
13.01 • 12.20 85.19 • 41.82 9.78 • 4.83 67.7 • 9.7 22.2 • 6.0
aDHEA included in the diet at a level of 0.6% for 20 weeks. bActivity expressed as NADP + reduced/min/mg protein. CActivlty expressed as dpm/mg protein. dAetivity expressed as nmol free fatty acid f o r m e d / 1 5 m i n / m g mic~osomal protein. Sucrose-fed significantly different from sucrose-fed + DHEA at p < O.O5. #Sucrose-fed compared to sucrose-fed + DHEA p < 0.1 > 0.05.
490
M.P. CLEARY et a l .
Adipose Tissu~ (Table IV): Both parametrial and retroperitoneal fat pad w e i g h t s were significantly lower in the sucrose-fed + DHEA f e m a l e group in c o m p a r i s o n to the sucrose-fed control group. When the fat pad w e i g h t s w e r e combined and expressed as a percent of total body weight, the fat pads accounted for a significantly decreased proportion of body weight in the DHEA treated rats (data not shown).
TABLE IV Parametrial and R~troperitoneal Fat Pad Weights a and Cellularity in DHEA Treated Female BHE Rats (x • S.D.)
Sucrose-Fed (n:6)
Sucrose-Fed + DHEA (n:7)
8.97 • 2.80* 5.20 • 1.80" 1.32 • 0.48*
2.43 • 1.50 3.40 • 1.15 0.49 • 0.31
4.29 • 1.87" 2.42 • 1.19 1.22 ~ 0.37*
1.47 • 0.92 1.35 • 0.83 0.52 • 0.33
Parametrial Fat Pads Weight (gm) Fat Cell Number x 106 Fat Cell Size (~g lipid/cell) Retroper~otneal Fat Pads Weight (gm) Fat Cell Number x 106 Fat Cell Size (~g lipid/cell)
acombined both right and left pads. bDHEA included in the diet at 0.6% for 20 weeks. *Sucrose-fed significantly different from sucrose-fed at p < 0.05.
+ DHEA
This decrease in fat pad w e i g h t of the DHEA treated rats was found to be due to a decrease in both fat cell size and n u m b e r in the p a r a m e t r i a l depot. Although both values were decreased in the retroperitoneal fat pad, only fat cell size was significantly different. Carcass Composition (Table V): In all instances, calculations for carcass composition were made without the addition of the removed organs and adipose tissue. On an absolute (gm) basis, carcass weight, carcass water, carcass lipid weight and cholesterol weight were significantly reduced in the sucrose-fed + DHEA female group compared to the sucrose-fed female group. Carcass fat-free dry matter on an absolute basis was not significantly decreased in the sucrosefed + DHEA f e m a l e rats w h e n c o m p a r e d to the sucrose-fed f e m a l e rats. When computed as a percentage of carcass weight, significant decreases were found for both the carcass lipid and cholesterol in the DHEA rats. The percentage of carcass water was increased in the DHEA group. There were no significant differences in organ weights of sucrose-fed + DHEA female rats in comparison to sucrose-fed control rats whether expressed on an absolute (gram) basis or relative to body weight (data not shown).
DEHYDROEPIANDROSTERONE and BHE RATS
491
TABLE V Carcass Composition of DHEA a Treated Female BHE Rats (x • S.D.)
Sucrose-Fed
(n=6) Carcass Weight (gm) Carcass Water (gm)
(%) Carcass Fat Free Dry Matter (gm)
(%) Carcass Lipid (gm) (%) Carcass Cholesterol
(gm) (%)
Sucrose-Fed § DHEA (n:7)
185 ~ 14"
128
~ 34
98 ~ 10" 53 ~ 5*
77 60
• 22 ~ 4
39 ~
9
21 •
4
29 22
+ 9 +_ 3
43 ~ 23 ~
9* 4*
20 15
_+ 7 +_ 3
2.0 ~ 1.0 ~
5.0* 0.2*
0.6• 0.5~
0.4 0.2
aDHEA included in the diet at a level of 0.6% for 20 weeks. *Sucrose-fed significantly different from sucrose-fed + DHEA rats at p < 0.05.
DISCUSSION The results of this study indicate that DHEA a d m i n i s t r a t i o n to BHE rats resulted in a decrease in body weight, confirming earlier observations in two other strains of rats (4,5). The response of this strain of rat was s o m e w h a t intermediate between those found for either Sprague-Dawley and lean Zucker rats or obese Zucker rats. Previously, "lean" rats had responded with small but significant decreases in body weight with essentially no effect on food intake. Obese Zucker rats were found to have quite a large decrease in body weight compared to nontreated obese rats, i.e. 40% (5). Although not an obese rat, the response of the BHE rat was more like that of the obese rat since a large decrease in body weight was found. Precisely why the BHE rat is more responsive to DHEA treatment than other lean rats is not clear. This may be due to the use of a semipurified diet that a l l o w s more DBEA to be absorbed r e s u l t i n g in a higher dosage. Rats fed a semipurified-high fat diet also had a greater response to DHEA treatment than did chow fed rats (4). On the other hand, g e n e t i c a l l y obese rats fed chow had quite a large response to DHEA (5). It may be that there is some combination of dietary as well as genetic factors that are involved in the responses found to DHEA in different rat strains. Determining absorption of DHEA from the gut and circulating levels of DHEA and/or its metabolites are studies to be conducted to clarify this. The decrease in fat pad w e i g h t s and fat cell size and n u m b e r in the sucrose-fed + DHEA rats c o m p a r e d to the sucrose-fed rats is similar to that described for lean and obese Zucker rats (5). These data plus the body composition results indicate a specific effect for DHEA on decreasing body fat with little effect on development of other tissues. Although we had anticipated a p o s s i b l e effect of DHEA on cholesterol metabolism in the sucrose-fed BHE rat, in
492
M.P. CLEARYet al.
general there was no effect on the various m e a s u r e m e n t s made. A decrease in carcass cholesterol was found but this is probably a reflection of the decrease in carcass lipid found. As previously described in the introduction, DHEA is a known inhibitor of the enzyme glucose-6-phosphate dehydrogenase (6). This inhibition has been described in homogenates and supernatants from a variety of mammalian tissues. It has also been found in liver tissue following fasting-refeeding regimens (7, 8). Following long-term DHEA treatment, decreased G6FD activity has been found only in situations where an increase in activity was present in comparison to that of the "control" group. This included a study where high-fat fed rats were found to have increased hepatic G6PD activity compared to chow-fed rats. In this case DHEA treatment resulted in decreased G6PD activity in liver samples from the high fat fed DHEA treated rats but not from the chow-fed DHEA treated rats (4). Zucker obese rats have increased G6PD activity compared to control lean rats and DHEA treatment in obese rats resulted in decreased G6PD activity (9). Thus, BHE rats appeared to respond in a similar fashion; when G6PD activity was increased by sucrose feeding , decreased activity of G6PD was found in the DHEA treated rats. Thus it may be that i n v i v o inhibition of this enzyme may only be of significance w h e n the activity is elevated by some stimulus. Possibly this is due to the presence of specific isoenzymes of G6PD. DHEA has been s h o w n to have different effects on i s o e n z y m e s of G6FD (22-25). Whether this inhibition contributes to DHEA's antiobesity action r e m a i n s to be determined. We have r e c e n t l y proposed that DHEA's ability to decrease body weight without an effect on food intake may be better explained by an induction of a futile cycle of fatty acid m e t a b o l i s m (26). An increase in the activity of long-chain fatty acyl-CoA hydrolase was found in lean and obese DHEA treated rats. This enzyme is responsible for removal of the CoA group from fatty acids. Prior to the fatty acids being utilized by m e t a b o l i c pathways, the CoA group must be reattached in a step requiring ATF hydrolysis. In the BHE rats, DHEA treatment resulted in a 100% increase in long-chain fatty acyl-CoA hydrolase activity. In the control sucrose-fed rats, similar values were found as those described for adult Sprague-Dawley (27) rats and lean and obese Zucker rats (26) indicating that diet and genetics do not appear to alter this enzyme. In general, these data support our hypothesis t h a t inhibition of G6PD probably does not account for the "antiobesity" characteristics of DHEA treatment but may play a role in contributing to the enhanced response to DHEA found when "basal" activity of G6PD is elevated. A more plausible explanation for the p r i m a r y antlobesity effect is that some kind of energy wasting process is induced by DHEA treatment. The increased activity of the enzyme long-chain fatty acyl-CoA hydrolase found with DHEA treatment here and in a previous study suggests a futile cycle of fatty acid metabolism could be induced resulting in decreased weight without effects on food intake.
ACKNOWLEDGMENTS Support of this r e s e a r c h was provided by a grant-in-aid from the Southeastern Pennsylvania Chapter of the A m e r i c a n Heart Association, The Hormel Foundation and National Institutes of Health Grant AM 32965.
IA preliminary study using male BHE rats showed that sucrose-fed male rats had twice the G6PD activity as chow-fed male rats.
DEHYDROEPIANDROSTERONE and BHE RATS
493
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Accepted for publication March 4, 1984.
of in
using ~-