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Modulation of hepatic glucose-6-phosphate dehydrogenase activity in male and female rats by estrogen
Life Sciences, Vol. 45, pp. 1559-1565 Printed in the U.S.A.
Pergamon Press
MODULATION OF HEPATIC GLUCOSE&PHOSPHATE DEHYDROGENASE ACTIVITY IN MALE AND FEMALE RATS BY ESTROGEN.
Sobrasua E. M. Ibims, Robin Randalls, Peter Hans and Paul I Musey§#
Department of Biological Science&, Clark Atlanta University; and VA Medical Research Center #, Atlanta, Georgia 303 10. (Received in final form August 14, 1989)
The effect of estradiol-17l3 on the activity of glucose-6-phosphate dehydrogenase was studied in both male and female rats to further characterize the sex differences in the activity of this enzyme. Four groups of intact and castrated rats were implanted subcutaneously with graded doses (2.4,4.8 and 7.2 l.tgIday) of pelleted estradiol in a physiologically relevant experimental system. After fourteen days the rats were sacrificed and their livers were assayed for G6PD activities. The result indicated that : (i)the enzyme activity was 3-fold higher in normal adult female than in male rats, (ii) low doses of E2 (2.4, 4.8 and 7.2 pg/day) increased the activity of G6PD 6-fold in castrated males and over 2-fold in female castrates as well as intact rats (iii) E2 stimulation of G6PD activity appears to be more effective in castrated males than in female rats (iv) sex difference in the activity of G6PD disappeared after treatment with R in castrated rats. It is concluded that the activity of G6PD in rats is markedly enhanced by low doses of E2, which appears to be largely responsible for the sex differences in the activity of this enzyme in rats.
Sex differences in the activity of rat hepatic glucose-6-phosphate dehydrogenase (G6PD), the enzyme that catalyzes the first step in the pentose phosphate pathway, was first reported by Glock and McLean [l], and this phenomenon has since been confirmed in subsequent investigations [2-71. However, the physiological basis for the sex difference is not well understood, although gonadal factors are invariably implicated. Different investigators have ascribed the observed sex differences in the rat to various gonadal hormones, estrogen [ 1,5] and androgen [8,9] or to a gene-dose effect [lo]. Estradiol, the female sex hormone, has been shown to significantly induce a dose-related increase in the activity of G6PD in the uteri of female rats when daily injection of 0.1-1000 ltg estradiol was administered [ 1 l-131. In male rat hepatocyte cultures, the activity of G6PD was also markedly enhanced by estrogen treatment [14]. Huggins and Yao [ 151 and Berdanier [5] also reported that daily subcutaneous administration of 20-60 ltg of estrogen increased the activity of G6PD enzyme in castrated male rats but in contrast to the males, the activity of this enzyme decreased in the liver of castrated female rats. In these studies, varying pharmacological doses (20-60 pg) of daily injections and different exposure rates were used [5,15]. Thus, the exact role of E2 in the induction of G6PD is unclear and the apparent disconcerted results could also be partly due to such variable factors. In order to further clarify the basis of the sex differences, we have investigated the effects of sustained release of low doses of estrogen on the activity of G6PD in intact as well as gonadectomized male and female rats. Address reprint requests to Paul I. Musey, Ph.D., Department of Biological Sciences, Clark Atlanta University, 223 James P. Brawley Dr., Atlanta, Georgia 30314. 0024-3205189 $3.00 +.oo Copyright (c) 1989 Pergamon Press plc
1560
Modulation of G6PD Activity by Estrogen
Vol. 45, No. 17, 1989
Sprague-Dawley male and female rats (100-l 10 g) were maintained under normal conditions of temperature, humidity and fed rodent chow and water d libitum throughout the experimental period. The rats were divided into two groups. Group A (control) consisted of intact (INT) male and female; castrated (CS) male and female rats. Group B consisted of CS males and females, INT male and female rats that received subcutaneous implants of pelleted E2. Each group of controls or E2 supplemented groups contained at least eight rats per treatment. Food and water intake as well as body weights were determined every two days. The initial body was recorded eight days after castration and the final weight recorded before sacrifice.
Castration was performed under ether anesthesia as previously described [ 161, by cutting the scrotal sac between the testes and making two more cuts through the tunica albigans. The testes were individually isolated and excised after the spermatic cords were ligated. Castration in female rats was also performed under ether anesthesia [ 121 by incisions through the lumber region of the back. The incisions in both male and female rats were closed using wound clips. The rats were allowed to recover from surgery for seven days before study.
In order to ensure a low sustained exposure to hormone, pelleted 178Estradiol propionate (Innovative Research of America, Toledo, Ohio), were implanted in three dose levels, 50, 100 and 1.50 &rat. The pellets are designed for a three-week controlled dose-dependent release rate, with effective exposure of 2.4, 4.8 and 7.2 j.tgJday. Blood samples were collected before and after castration as well as after hormone treatment and serum estrogen levels were determined according to the method of Hotchkiss et al [ 171. Two weeks following implantation, the rats were sacrificed by rapid decapitation after mid-day and their livers removed, weighed and prepared for enzyme assay or stored (-700) until assayed.
The tissues preparation and measurements of G6PD activity were performed essentially as described by Ibim et al [7] and Bergmeryer [16] respectively, at 25C using Beckman spectrophotometer Model ACTA 111, and the activity of G6PD was expressed in milliUnits/mg protein. A unit of an enzyme is defined as the amount that will catalyze one micromole of substrate in one minute. Protein was determined by the Bradford dye-protein binding technique [ 181. All samples were analyzed in duplicate. Substrates and chemical reagents are standard grades purchased from Sigma Chemical Co., St Louis, MO. Statistical analysis: the data was analyzed using the analysis of variance and r-test. The level of significance was set at 5% or higher.
Results Table 1 shows the body and liver weights as well as food intake pattern of rats used in this study. The rats were selected from a homogeneous pool, consequently, the initial body weights of male and female rats were identical and did not reflect sex differences. Castrated male rats decreased weight gain as compared to those of controls (19% control., 12% castrated). However, in contrast to castrated males, intact male rats have a higher weight-gain after E2 supplementation. These differences in body weight correlated with food consumption. Intact male rats also consumed approximately 4 gm/day more than castrated counterparts (32g: int., 28g: cast.). In intact female rats, changes in weight-gain were similar (int.: 9.5%, cast: 9%), but increased slightly after E2 treatment in intact females (log: int. treated, 7.5g: cast.). Estrogen treatment however, resulted in an increased food consumption in intact female rats (29g) as compared to female castrates (23g). Liver weights of intact control males were approximately 2g more than in castrated male counterparts (16g versus 14 g). In ET-treated male rats, however, liver weights were essentially similar, and estrogen treatment did not have a significant effect, though in castrated female rats there was a slight reduction
1561
Modulation of G6PD Activity of Estrogen
Vol. 45, No. 17, 1989
in liver weight. These differences when expressed on the basis of 1OOg body weight were not significantly different from the control. TABLE I The Effect of Estradiol Administration on Body and Liver Weights, Food Intake and G6PD Specific Activity.
Condition MALE Int.Cont.
Sp.Act. + G6PD
Mean Body, Liver wt. (g) + SD INITAIL FINAL %CH LIVER
sDb
E2 (2.4 ) F2 (4.8) E2 (7.2 )
282 274 281 288
+ + -I f
2.6 2.5 2.9 3.4
350 362 375 344
f k + f
5.3 4.6 3.8 4.3
19 24 25 16
17 16 16 15
k f -f +
0.2 0.3 0.3 0.3
32 34 30 32
+ f f f
1.2 1.2 1.0 0.9
28.86 120.88 130.39 160.57
f 3.57 f 9.67 f 11.20 5 9.18
Cast.Cont. E2 (2.4 ) E2 (4.8) ~2 (7.2)
292 300 308 294
+ f f +
3.2 3.1 3.5 3.2
331 335 338 323
+ 4.0 +4.2 f 4.5 + 3.9
12 12 08 09
14 14 15 14
+ + + k
0.1 0.1 0.3 0.2
27 29 28 29
+ f + k
0.6 0.8 0.8 0.8
21.36 158.14 198.28 210.36
It 3.53 f 7.31 + 10.60 f 8.70
FEMALE Int.Cont. E2 (2.4) E2 (4.8) ~2 (7.2)
285 282 280 278
+ + f. +
2.9 3.4 4.3 3.2
315 313 315 309
rf: 3.9 + 2.9 k3.9 + 3.5
9.5 10 11 10
16 14 15 14
+ f + +
0.3 0.2 0.3 0.2
28 29 31 29
f f + +
0.8 0.8 0.8 0.6
93.49 203.53 235.26 250.65
f f + f.
4.50 8.53 7.45 11.72
Cast.Cont. ~2 (2.4 ) F2 (4.8) F2 (7.2 )
274 276 280 273
+ + + f
2.5 2.5 2.8 2.4
300 296 300 298
It 3.2 f 4.2 + 3.3 It: 3.8
09 07 07 08
11 11 10 10
f + + f
0.1 0.1 0.1 0.1
22 20 25 23
f * + +
0.4 0.2 0.6 0.4
71.09 145.60 175.28 185.78
f + f +
9.47 4.10 7.75 6.43
aMean Daily Food Intake k SD per rat, N=8
bMean Specific Activity (mU/mg protein)
The activity of G6PD in EZ-supplemented intact and castrated male rats are also compared in Table 1. The activity of the enzyme decreased slightly (28%) in control-castrated males compared to intact males (21.36 f 3.53 versus 28.86 5 3.57). E2 administration to intact males caused a significantly (Pc.01) graded increase in the activity of G6PD from 28.86 f. 3.57 mU/mg protein, (control) to 120.88 + 9.67, 130.39 + 11.20 and 160.57 f 9.18 corresponding to the dose rates of 2.4, 4.8 and 7.2 @day respectively. Estrogen treatment also caused increases in the activity of G6PD in male castrates from 21.36 f 3.34 (control) to 158.14 f 7.31, 198.28 f 10.60 and 210.36 f 8.70 mU/mg protein in a dose-dependent manner. These increases were higher relative to the increases in intact males at the same dose levels respectively. The enzyme activity following E2 administration to intact and castrated female rats were also compared in Table 1. Castration decreased the activity of this enzyme by 25% in intact females (93.49 f 4.49 control; 71.09 f 9.47 castrate), however, E2-supplementation significantly (Pc.01) increased the enzyme activity in castrated females to 145.60 + 4.10, 175.28 f 7.75 and 185.78 + 6.43 mU/mg protein corresponding to E2 exposure rates of 2.4, 4.8 and 7.2 l.tglday respectively. In contrast to male castrates which showed greater sensitivity to E2 stimulation, the activity of G6PD in response to estrogen stimulation was higher in intact than those of castrated female rats (93.49 f 4.49 control to 203.53 k 8.50, 235.26 f 7.45 and 250.65 + 11.7 mU/mg protein at similar exposure rates.
1562
Modulation of G6PD Activity of Estrogen
Vol. 45, No. 17, 1989
300-
3 ._ z k
fl
Castrated Male
q
Castrated Female
KAntact Controls CC=Castrated Controls
200-
F
2
z P ii
(3
loo-
cc
4.8
2.4
Estradiol
7.2
@g/day)
Fig.1 The effect of Estradiol treatment on the activity of G6PD in intact (IC) male and female as well as castrated (CC) control rats and E2-supplemented male and female rats. Histogram represents mean specific activity of G6PD (mU/mg protein) + SD of 8 rats in each treatment or control. All treatmems were significantly (Pc.01) different from control.
300
1 [;1
Int. Male
q
Int. Female
0
4.0 EstkAol
7.2
@g/day)
Fig.2 The effect of Estradiol treatment on the activity of G6PD in intact male and female controls rats and estrogen-treated male and female rats. Histogram represents mean specific activity of G6PD (mU/mg protein) + SD of 8 rats in each treatment or control. All treatments were significantly (P<.Ol) different from control.
Vol. 45, No. 17, 1989
Modulation of G6PDActivity
of Estrogen
1563
Figure 1 compares the activity of G6PD in intact control (IC) and castrated (CC) male and female rats as well as E2 treated rats. In intact rats the activity of G6PD enzyme is 3-fold higher in females as compared to their male counterparts ( 93.49 f 9.47 versus 28.86 f 3.57). Castration caused a 25% decrease in the activity of this enzyme in female rats from 93.49 f 4.49 (intact) to 7 1.09 f 9.47 (castrate), but did not eliminate the the sex difference. The corresponding values for the males were 28.36 f 3.57 (intact) and 21.36 f 3.34 (castrated). In Ez-treated female rats, the activity of G6PD increased from 71.09 f 9.47 (castrate) to 145.60 f 4.10, 175.28 It 7.75 and 185.78 f 6.43 mU/mg protein corresponding to b dose levels of 2.4,4.8 7.2 pg/day respectively. These increases were significantly (Pc.01) different when compared to the intact or castrated controls. In male castrates the activity of G6PD enzyme also increased following estrogen supplementation but at mid and high dose levels, differences due to sex was abolished (Fig. 1). Figure 2 shows the enzyme activity response to estrogen treatment in the intact male and female rats and non estrogen-treated controls. In control rats, the activity of G6PD enzyme showed the presence of sex differences (Female: 93.49 f 4.50, Male: 28.86 f 3.57). Differences in response to sex were preserved despite hormone treatment. The activity of G6PD in intact male rats following estrogen supplementation increased to 120.88 f 9.67, 130.39 f 11.20 and 160.57 f 9.18 at dose levels of 2.4,4.8 and 7.2 l.tg/day. In female rats the stimulation of G6PD activity markedly increased from 93.49 f 4.50 (control) to 203.53 f 8.53, 235.26 rt 7.45 and 250.654 f 11.72 at similar rates of exposure.
Estrogen stimulation of G6PD activity has generally been carried-out with pharmacological doses of intravenous or subcutaneous injection on daily basis (3,5, 14). The present investigation was conducted to simulate physiological conditions and determine whether low sustained doses of estrogen will induce significant increases in the activity of G6PD enzyme. The result given above showed that pharmacological doses of the steroid are not necessary to induce and show the sex differences in the activity of G6PD. In the present study, E2-induced decrease in food intake was not apparent. Within each group set, food consumption was not different between E2-exposed animals and their controls. The major controlling factor in food consumption was the event of castration though on the whole, male rats consumed more food than their female counterparts across group sets. Thus castration decreased food consumption in all animals, male and female with no recovery or further decrease upon estradiol exposure, consequently, food intake, body and liver weights were not influenced by estradiol. These effects of castration and estrogen treatment held true for liver fresh weights though a slight reduction in liver weights resulted in intact male rats upon estrogen exposure. It is however possible that these effects of E2 were minimal due to the low doses used in this study.
The increase in the activity of G6PD in response to %-stimulation may be due to peripheral conversion of testosterone to estrogen and the presence of receptors in intact male and female rats respectively. Castration decreased the activity of G6PD [ 12, 151 in both male and female rats to a level of about 25-28% but did not eliminate the sex difference. This decrease in the activity of G6PD enzyme may be associated with the loss of circulating E2 [20] present at low levels (7-10 pg/ml) in normal rats [21]. The higher activity of G6PD in female castrates (Zfold) as compared to males may be due to increased sensitivity of liver receptors to peripheral conversion of testosterone to EL and the fact that these rats were younger rats that were still growing.
In Ezsupplemented castrates, the activity of G6PD in response to estrogen treatment were similar if not slightly better than those of the female counterparts. The loss of sex difference in the activity of GBPD in estrogen-supplemented rats may be associated with increased sensitivity of the
1564
Modulation of G6PD Activity of Estrogen
Vol. 45, No. 17, 1989
male liver receptors to E2 stimulation. Estrogen administration has been shown to elicit increases in the activity of G6PD in hepatocyte cultures [6], liver of male rats [5, 141 and in the uteri of female rats [ 12, 191. In this study, the initial dose of & (2.4 pg/day) was considered more effective, since the response in the activity of G6PD elicited by higher levels of E2 (4.8,7.2 pg/day) were minimal increments above the initial activity at 2.4 pg/day (Table 1). A 6-fold increase in the activity of G6PD over the control was elicited by estrogen administration, therefore, this increase may be related to a modulation in the activity of G6PD by E2 at the level of transcription. Serum E2 levels were higher (6-13 %) in rats that received the higher (7.2 pg/day) dose while the low to medium E2 levels were identical to the initial dose (2.4 pg/day) and time-course experiments showed that the onset of G6PD activity at 0,4, 8 and 12 days of exposure followed a dose-response kinetics (data not shown). These data therefore suggested that the hormone was effectively delivered to target tissues and the activity of G6PD was a result of estrogen stimulation.
Throughout this study, the administration of estrogen has been shown to stimulate increases in the activity of G6PD at all levels of treatment in male and female rats. These data are in agreement with the finding by Hansen and Jungermann [6] using rat hepatocyte culture. These data are in conflict with the finding of Berdanier [S], Higgins and Yao [ 141 who observed decreased activity of this enzyme in castrated female rats supplemented with E2. The apparent differences in the observed data may be due to varying rates of E:! exposure, food intake or age of animals, all of which are known to influence the activity of G6PD enzyme. Extra-gonadal hormone such as testosterone [6], and non-gonadal hormone such as thyroxine [l] and insulin [6] have been implicated in the regulatory activity of G6PD. Testosterone has been shown to inhibit while thyroxine and insulin appear to promote stimulation in the activity of this enzyme. In this study, the ObSeNed increases in the activity of this enzyme are principally due to estrogen stimulation which regulate G6PD activity and may act in concert with other hormones [ 1,5,6]. It is concluded that pelleted estrogen administered in low doses but sustained release, stimulated the activity of G6PD in castrated as well as intact male and female rats and that it appears sex differences in the activity of this enzyme may be largely under the control of estrogen.
This investigation was supported by Grant No. lS06RR08247 to PIM and a Research Centers in Minority Institutions award, G12RR03062 from the Division of Research Resources, National Institutes of Health.
:: 3. :. 6: 7. :* 10: 11.
G. E. GLOCK and P. MCLEAN. B&hem. J., 55: 400-408 (1953). T. F. SLATER, B. SAWYER and U. STRAULI. Arch. Int.Physiol. B&him. 2: 427-447 (1964). J. ROUX, E. SWIERCZEWSKI and C. TORDET-CARIDROIT. J. Physiol. (Paris) 64: 333-342 (1972). W. G. WINSTON and R. C. REITZ. B&hem. Pharmacol. 28: 1249-1256 (1979). C. D. BERDANIER. J. Nutrition 111: 14251429, (1981). R. J. HANSEN and K. JUNGERMANN. Biol. Chem. Hoppe-Seyler 368: 955-962 (1987). S. E. M. IBIM, P. F. HAN, N. L. EMMETT, L. B. CABEY and J. JOHNSON. Alcohol 2117-119 (1988). E. FARBER, D. KOCH-WEBER and H. POPPER. Endocrinology 40: 205-212 (195 1). E. FARBER and A. SEGALOFF. J. Biol. Chem. 216: 471-477 (1955). L. E. ROSENBERGE, in Duncan’s Disease of Metabolism (Eds. P. K. Bondy and L. E. Rosenberge). p. 31. W. B. Saunders Co., Philadelphia (1974). B. C. MOULTON and K. L. BARKER. Endocr. 91: 491-498 (1972).
Vol. 45, No. 17, 1989
12.
Modulation of G6PD Activity of Estrogen
R. HILF, E. MCDONALD, J. SARTINI, W. D. RECTOR AND H. RICHARDS. Endocrinology a: 280-286 (1972). 13. E. R. SMITH and K. L. BARKER. J. Biol. Chem. &@: 6541-6547 (1974). 14. C. HIGGINS and F. YAO. J. Expt. Med. 110: 899-919 (1959). R. F. OZOLS and R. HILF. Proc. Sot. Exp. Biol. Med. m: 73-77 (1973). :56: H.U.BERGMEYER, “Glucose-6-phosphate dehydrogenase” In. Methods pf Enzymatic Analvsis (vol.II, 3rd ed.) New York, Academic Press, p. 190. (1965). J. HOTCHKISS., L. E .ATKINS and E. KNOBIL. Endocrinology B: 177, (1971). :‘s. M. M. BRADFORD. Anal. B&hem. 22: 248-254 (1976). D. B. M. SCOTT and A. G. LISI. Biochem. J. II: 52-58 (1960). :; A. C. GUYTON, Textbook of Medical PhysiolGy (3rd ed.j W. I% Saunders Co., Philadelphia, p. 1126, 1968. 21. M. S. SMITH, M. E. FREEMAN and J. D. NEILL. Endocr. p6: 219-226 (1975).
1565
Pergamon Press
MODULATION OF HEPATIC GLUCOSE&PHOSPHATE DEHYDROGENASE ACTIVITY IN MALE AND FEMALE RATS BY ESTROGEN.
Sobrasua E. M. Ibims, Robin Randalls, Peter Hans and Paul I Musey§#
Department of Biological Science&, Clark Atlanta University; and VA Medical Research Center #, Atlanta, Georgia 303 10. (Received in final form August 14, 1989)
The effect of estradiol-17l3 on the activity of glucose-6-phosphate dehydrogenase was studied in both male and female rats to further characterize the sex differences in the activity of this enzyme. Four groups of intact and castrated rats were implanted subcutaneously with graded doses (2.4,4.8 and 7.2 l.tgIday) of pelleted estradiol in a physiologically relevant experimental system. After fourteen days the rats were sacrificed and their livers were assayed for G6PD activities. The result indicated that : (i)the enzyme activity was 3-fold higher in normal adult female than in male rats, (ii) low doses of E2 (2.4, 4.8 and 7.2 pg/day) increased the activity of G6PD 6-fold in castrated males and over 2-fold in female castrates as well as intact rats (iii) E2 stimulation of G6PD activity appears to be more effective in castrated males than in female rats (iv) sex difference in the activity of G6PD disappeared after treatment with R in castrated rats. It is concluded that the activity of G6PD in rats is markedly enhanced by low doses of E2, which appears to be largely responsible for the sex differences in the activity of this enzyme in rats.
Sex differences in the activity of rat hepatic glucose-6-phosphate dehydrogenase (G6PD), the enzyme that catalyzes the first step in the pentose phosphate pathway, was first reported by Glock and McLean [l], and this phenomenon has since been confirmed in subsequent investigations [2-71. However, the physiological basis for the sex difference is not well understood, although gonadal factors are invariably implicated. Different investigators have ascribed the observed sex differences in the rat to various gonadal hormones, estrogen [ 1,5] and androgen [8,9] or to a gene-dose effect [lo]. Estradiol, the female sex hormone, has been shown to significantly induce a dose-related increase in the activity of G6PD in the uteri of female rats when daily injection of 0.1-1000 ltg estradiol was administered [ 1 l-131. In male rat hepatocyte cultures, the activity of G6PD was also markedly enhanced by estrogen treatment [14]. Huggins and Yao [ 151 and Berdanier [5] also reported that daily subcutaneous administration of 20-60 ltg of estrogen increased the activity of G6PD enzyme in castrated male rats but in contrast to the males, the activity of this enzyme decreased in the liver of castrated female rats. In these studies, varying pharmacological doses (20-60 pg) of daily injections and different exposure rates were used [5,15]. Thus, the exact role of E2 in the induction of G6PD is unclear and the apparent disconcerted results could also be partly due to such variable factors. In order to further clarify the basis of the sex differences, we have investigated the effects of sustained release of low doses of estrogen on the activity of G6PD in intact as well as gonadectomized male and female rats. Address reprint requests to Paul I. Musey, Ph.D., Department of Biological Sciences, Clark Atlanta University, 223 James P. Brawley Dr., Atlanta, Georgia 30314. 0024-3205189 $3.00 +.oo Copyright (c) 1989 Pergamon Press plc
1560
Modulation of G6PD Activity by Estrogen
Vol. 45, No. 17, 1989
Sprague-Dawley male and female rats (100-l 10 g) were maintained under normal conditions of temperature, humidity and fed rodent chow and water d libitum throughout the experimental period. The rats were divided into two groups. Group A (control) consisted of intact (INT) male and female; castrated (CS) male and female rats. Group B consisted of CS males and females, INT male and female rats that received subcutaneous implants of pelleted E2. Each group of controls or E2 supplemented groups contained at least eight rats per treatment. Food and water intake as well as body weights were determined every two days. The initial body was recorded eight days after castration and the final weight recorded before sacrifice.
Castration was performed under ether anesthesia as previously described [ 161, by cutting the scrotal sac between the testes and making two more cuts through the tunica albigans. The testes were individually isolated and excised after the spermatic cords were ligated. Castration in female rats was also performed under ether anesthesia [ 121 by incisions through the lumber region of the back. The incisions in both male and female rats were closed using wound clips. The rats were allowed to recover from surgery for seven days before study.
In order to ensure a low sustained exposure to hormone, pelleted 178Estradiol propionate (Innovative Research of America, Toledo, Ohio), were implanted in three dose levels, 50, 100 and 1.50 &rat. The pellets are designed for a three-week controlled dose-dependent release rate, with effective exposure of 2.4, 4.8 and 7.2 j.tgJday. Blood samples were collected before and after castration as well as after hormone treatment and serum estrogen levels were determined according to the method of Hotchkiss et al [ 171. Two weeks following implantation, the rats were sacrificed by rapid decapitation after mid-day and their livers removed, weighed and prepared for enzyme assay or stored (-700) until assayed.
The tissues preparation and measurements of G6PD activity were performed essentially as described by Ibim et al [7] and Bergmeryer [16] respectively, at 25C using Beckman spectrophotometer Model ACTA 111, and the activity of G6PD was expressed in milliUnits/mg protein. A unit of an enzyme is defined as the amount that will catalyze one micromole of substrate in one minute. Protein was determined by the Bradford dye-protein binding technique [ 181. All samples were analyzed in duplicate. Substrates and chemical reagents are standard grades purchased from Sigma Chemical Co., St Louis, MO. Statistical analysis: the data was analyzed using the analysis of variance and r-test. The level of significance was set at 5% or higher.
Results Table 1 shows the body and liver weights as well as food intake pattern of rats used in this study. The rats were selected from a homogeneous pool, consequently, the initial body weights of male and female rats were identical and did not reflect sex differences. Castrated male rats decreased weight gain as compared to those of controls (19% control., 12% castrated). However, in contrast to castrated males, intact male rats have a higher weight-gain after E2 supplementation. These differences in body weight correlated with food consumption. Intact male rats also consumed approximately 4 gm/day more than castrated counterparts (32g: int., 28g: cast.). In intact female rats, changes in weight-gain were similar (int.: 9.5%, cast: 9%), but increased slightly after E2 treatment in intact females (log: int. treated, 7.5g: cast.). Estrogen treatment however, resulted in an increased food consumption in intact female rats (29g) as compared to female castrates (23g). Liver weights of intact control males were approximately 2g more than in castrated male counterparts (16g versus 14 g). In ET-treated male rats, however, liver weights were essentially similar, and estrogen treatment did not have a significant effect, though in castrated female rats there was a slight reduction
1561
Modulation of G6PD Activity of Estrogen
Vol. 45, No. 17, 1989
in liver weight. These differences when expressed on the basis of 1OOg body weight were not significantly different from the control. TABLE I The Effect of Estradiol Administration on Body and Liver Weights, Food Intake and G6PD Specific Activity.
Condition MALE Int.Cont.
Sp.Act. + G6PD
Mean Body, Liver wt. (g) + SD INITAIL FINAL %CH LIVER
sDb
E2 (2.4 ) F2 (4.8) E2 (7.2 )
282 274 281 288
+ + -I f
2.6 2.5 2.9 3.4
350 362 375 344
f k + f
5.3 4.6 3.8 4.3
19 24 25 16
17 16 16 15
k f -f +
0.2 0.3 0.3 0.3
32 34 30 32
+ f f f
1.2 1.2 1.0 0.9
28.86 120.88 130.39 160.57
f 3.57 f 9.67 f 11.20 5 9.18
Cast.Cont. E2 (2.4 ) E2 (4.8) ~2 (7.2)
292 300 308 294
+ f f +
3.2 3.1 3.5 3.2
331 335 338 323
+ 4.0 +4.2 f 4.5 + 3.9
12 12 08 09
14 14 15 14
+ + + k
0.1 0.1 0.3 0.2
27 29 28 29
+ f + k
0.6 0.8 0.8 0.8
21.36 158.14 198.28 210.36
It 3.53 f 7.31 + 10.60 f 8.70
FEMALE Int.Cont. E2 (2.4) E2 (4.8) ~2 (7.2)
285 282 280 278
+ + f. +
2.9 3.4 4.3 3.2
315 313 315 309
rf: 3.9 + 2.9 k3.9 + 3.5
9.5 10 11 10
16 14 15 14
+ f + +
0.3 0.2 0.3 0.2
28 29 31 29
f f + +
0.8 0.8 0.8 0.6
93.49 203.53 235.26 250.65
f f + f.
4.50 8.53 7.45 11.72
Cast.Cont. ~2 (2.4 ) F2 (4.8) F2 (7.2 )
274 276 280 273
+ + + f
2.5 2.5 2.8 2.4
300 296 300 298
It 3.2 f 4.2 + 3.3 It: 3.8
09 07 07 08
11 11 10 10
f + + f
0.1 0.1 0.1 0.1
22 20 25 23
f * + +
0.4 0.2 0.6 0.4
71.09 145.60 175.28 185.78
f + f +
9.47 4.10 7.75 6.43
aMean Daily Food Intake k SD per rat, N=8
bMean Specific Activity (mU/mg protein)
The activity of G6PD in EZ-supplemented intact and castrated male rats are also compared in Table 1. The activity of the enzyme decreased slightly (28%) in control-castrated males compared to intact males (21.36 f 3.53 versus 28.86 5 3.57). E2 administration to intact males caused a significantly (Pc.01) graded increase in the activity of G6PD from 28.86 f. 3.57 mU/mg protein, (control) to 120.88 + 9.67, 130.39 + 11.20 and 160.57 f 9.18 corresponding to the dose rates of 2.4, 4.8 and 7.2 @day respectively. Estrogen treatment also caused increases in the activity of G6PD in male castrates from 21.36 f 3.34 (control) to 158.14 f 7.31, 198.28 f 10.60 and 210.36 f 8.70 mU/mg protein in a dose-dependent manner. These increases were higher relative to the increases in intact males at the same dose levels respectively. The enzyme activity following E2 administration to intact and castrated female rats were also compared in Table 1. Castration decreased the activity of this enzyme by 25% in intact females (93.49 f 4.49 control; 71.09 f 9.47 castrate), however, E2-supplementation significantly (Pc.01) increased the enzyme activity in castrated females to 145.60 + 4.10, 175.28 f 7.75 and 185.78 + 6.43 mU/mg protein corresponding to E2 exposure rates of 2.4, 4.8 and 7.2 l.tglday respectively. In contrast to male castrates which showed greater sensitivity to E2 stimulation, the activity of G6PD in response to estrogen stimulation was higher in intact than those of castrated female rats (93.49 f 4.49 control to 203.53 k 8.50, 235.26 f 7.45 and 250.65 + 11.7 mU/mg protein at similar exposure rates.
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Modulation of G6PD Activity of Estrogen
Vol. 45, No. 17, 1989
300-
3 ._ z k
fl
Castrated Male
q
Castrated Female
KAntact Controls CC=Castrated Controls
200-
F
2
z P ii
(3
loo-
cc
4.8
2.4
Estradiol
7.2
@g/day)
Fig.1 The effect of Estradiol treatment on the activity of G6PD in intact (IC) male and female as well as castrated (CC) control rats and E2-supplemented male and female rats. Histogram represents mean specific activity of G6PD (mU/mg protein) + SD of 8 rats in each treatment or control. All treatmems were significantly (Pc.01) different from control.
300
1 [;1
Int. Male
q
Int. Female
0
4.0 EstkAol
7.2
@g/day)
Fig.2 The effect of Estradiol treatment on the activity of G6PD in intact male and female controls rats and estrogen-treated male and female rats. Histogram represents mean specific activity of G6PD (mU/mg protein) + SD of 8 rats in each treatment or control. All treatments were significantly (P<.Ol) different from control.
Vol. 45, No. 17, 1989
Modulation of G6PDActivity
of Estrogen
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Figure 1 compares the activity of G6PD in intact control (IC) and castrated (CC) male and female rats as well as E2 treated rats. In intact rats the activity of G6PD enzyme is 3-fold higher in females as compared to their male counterparts ( 93.49 f 9.47 versus 28.86 f 3.57). Castration caused a 25% decrease in the activity of this enzyme in female rats from 93.49 f 4.49 (intact) to 7 1.09 f 9.47 (castrate), but did not eliminate the the sex difference. The corresponding values for the males were 28.36 f 3.57 (intact) and 21.36 f 3.34 (castrated). In Ez-treated female rats, the activity of G6PD increased from 71.09 f 9.47 (castrate) to 145.60 f 4.10, 175.28 It 7.75 and 185.78 f 6.43 mU/mg protein corresponding to b dose levels of 2.4,4.8 7.2 pg/day respectively. These increases were significantly (Pc.01) different when compared to the intact or castrated controls. In male castrates the activity of G6PD enzyme also increased following estrogen supplementation but at mid and high dose levels, differences due to sex was abolished (Fig. 1). Figure 2 shows the enzyme activity response to estrogen treatment in the intact male and female rats and non estrogen-treated controls. In control rats, the activity of G6PD enzyme showed the presence of sex differences (Female: 93.49 f 4.50, Male: 28.86 f 3.57). Differences in response to sex were preserved despite hormone treatment. The activity of G6PD in intact male rats following estrogen supplementation increased to 120.88 f 9.67, 130.39 f 11.20 and 160.57 f 9.18 at dose levels of 2.4,4.8 and 7.2 l.tg/day. In female rats the stimulation of G6PD activity markedly increased from 93.49 f 4.50 (control) to 203.53 f 8.53, 235.26 rt 7.45 and 250.654 f 11.72 at similar rates of exposure.
Estrogen stimulation of G6PD activity has generally been carried-out with pharmacological doses of intravenous or subcutaneous injection on daily basis (3,5, 14). The present investigation was conducted to simulate physiological conditions and determine whether low sustained doses of estrogen will induce significant increases in the activity of G6PD enzyme. The result given above showed that pharmacological doses of the steroid are not necessary to induce and show the sex differences in the activity of G6PD. In the present study, E2-induced decrease in food intake was not apparent. Within each group set, food consumption was not different between E2-exposed animals and their controls. The major controlling factor in food consumption was the event of castration though on the whole, male rats consumed more food than their female counterparts across group sets. Thus castration decreased food consumption in all animals, male and female with no recovery or further decrease upon estradiol exposure, consequently, food intake, body and liver weights were not influenced by estradiol. These effects of castration and estrogen treatment held true for liver fresh weights though a slight reduction in liver weights resulted in intact male rats upon estrogen exposure. It is however possible that these effects of E2 were minimal due to the low doses used in this study.
The increase in the activity of G6PD in response to %-stimulation may be due to peripheral conversion of testosterone to estrogen and the presence of receptors in intact male and female rats respectively. Castration decreased the activity of G6PD [ 12, 151 in both male and female rats to a level of about 25-28% but did not eliminate the sex difference. This decrease in the activity of G6PD enzyme may be associated with the loss of circulating E2 [20] present at low levels (7-10 pg/ml) in normal rats [21]. The higher activity of G6PD in female castrates (Zfold) as compared to males may be due to increased sensitivity of liver receptors to peripheral conversion of testosterone to EL and the fact that these rats were younger rats that were still growing.
In Ezsupplemented castrates, the activity of G6PD in response to estrogen treatment were similar if not slightly better than those of the female counterparts. The loss of sex difference in the activity of GBPD in estrogen-supplemented rats may be associated with increased sensitivity of the
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Modulation of G6PD Activity of Estrogen
Vol. 45, No. 17, 1989
male liver receptors to E2 stimulation. Estrogen administration has been shown to elicit increases in the activity of G6PD in hepatocyte cultures [6], liver of male rats [5, 141 and in the uteri of female rats [ 12, 191. In this study, the initial dose of & (2.4 pg/day) was considered more effective, since the response in the activity of G6PD elicited by higher levels of E2 (4.8,7.2 pg/day) were minimal increments above the initial activity at 2.4 pg/day (Table 1). A 6-fold increase in the activity of G6PD over the control was elicited by estrogen administration, therefore, this increase may be related to a modulation in the activity of G6PD by E2 at the level of transcription. Serum E2 levels were higher (6-13 %) in rats that received the higher (7.2 pg/day) dose while the low to medium E2 levels were identical to the initial dose (2.4 pg/day) and time-course experiments showed that the onset of G6PD activity at 0,4, 8 and 12 days of exposure followed a dose-response kinetics (data not shown). These data therefore suggested that the hormone was effectively delivered to target tissues and the activity of G6PD was a result of estrogen stimulation.
Throughout this study, the administration of estrogen has been shown to stimulate increases in the activity of G6PD at all levels of treatment in male and female rats. These data are in agreement with the finding by Hansen and Jungermann [6] using rat hepatocyte culture. These data are in conflict with the finding of Berdanier [S], Higgins and Yao [ 141 who observed decreased activity of this enzyme in castrated female rats supplemented with E2. The apparent differences in the observed data may be due to varying rates of E:! exposure, food intake or age of animals, all of which are known to influence the activity of G6PD enzyme. Extra-gonadal hormone such as testosterone [6], and non-gonadal hormone such as thyroxine [l] and insulin [6] have been implicated in the regulatory activity of G6PD. Testosterone has been shown to inhibit while thyroxine and insulin appear to promote stimulation in the activity of this enzyme. In this study, the ObSeNed increases in the activity of this enzyme are principally due to estrogen stimulation which regulate G6PD activity and may act in concert with other hormones [ 1,5,6]. It is concluded that pelleted estrogen administered in low doses but sustained release, stimulated the activity of G6PD in castrated as well as intact male and female rats and that it appears sex differences in the activity of this enzyme may be largely under the control of estrogen.
This investigation was supported by Grant No. lS06RR08247 to PIM and a Research Centers in Minority Institutions award, G12RR03062 from the Division of Research Resources, National Institutes of Health.
:: 3. :. 6: 7. :* 10: 11.
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