Inhibition of estrogen-induced glycogen synthesis in the rat uterus by cycloheximide

ARCHIVES

OF

BIOCHEMISTRY

Inhibition

AND

BIOPHYSICS

1&&-109

of Estrogen-Induced Uterus HELEiSE

United

119,

States Department

(1967)

Glycogen

Synthesis

in the Rat

by Cycloheximide

C. CECIL

of Agriculture, Beltsville,

AND

Animal Maryland

Received

JOEL BITMAN Husbandry 20705

Research Division,

ARS,

June 29, 1966

The sequence of changes occurring in the uterus with estrogen, estrogen and cycloheximide, and cycloheximide alone was studied. Cycloheximide (actidione) or estradiol caused an increase in uterine water, weight, and free glucose. Sixteen hours after estrogen administration the uterine glycogen had increased 5-fold. When a single dose of actidione was administered 30 minutes before estradiol, glycogen synthesis was suppressed for the first 8 hours, after which estrogen stimulated glycogen synthesis. Three doses of actidione, at 30 minutes before, 535 and 10% hours after estradiol, were necessary to completely suppress the glycogen synthesis for 16 hours. The influence of cycloheximide on glycogen synthesis after it had been initiated was studied by administering actidione after estrogen. Complete suppression occurred when actidione was administered 1% hours after estrogen and only partial suppression at 2% hours, and actidione was ineffective when administered 4 hours after estradiol. Consequently, there was an initial period of about 3 hours after estrogen treatment during which the glycogen synthetic system could be inhibited by cycloheximide. After this time, the presence of cycloheximide w-as unable to suppress glycogen synthesis.

Increased protein synthesis has been implicated as an early consequence of estrogen action and has led to the proposal that the primary step in estrogen action involves a stimulation of some protein synthetic process (1). Experiments with puromycin, (I), actinomycin (a), and cycloheximide (3, 4) demonstrated that when protein synthesis was interrupted, the characteristic tissue responses of estrogen action were abolished. Estrogen stimulates uterine carbohydrate systems and causes a marked increase in uterine glycogen (5-7). The present investigation was initiated to determine whether the estrogen-dependent increase in uterine glycogen synthesis would be altered when protein synthesis was inhibited. MATERIALS Female albino rats, gm), were ovariectomized

AND

ment. The estrogen rats were injected subcutaneously with 1 rg of estradiol in 0.1 ml of 10% ethanol. Rats that were treated with cycloheximide were injected intraperitoneally with 0.25 mg cycloheximide (actidione, Upjohn Co.) in 1 ml of Hz0 at the designated times. Fasted rats had food removed 19 hours before killing but were allowed free access to H20. The animals were killed by decapitation and blood was collected in heparinized tubes. The uterus, liver, and gastrocnemius were quickly excised. Water content was determined by the wet-dry difference in weights after drying overnight in aacuo at 100”. Total free glucose was extracted (8) and glucose was determined enaymically by the glucose oxidase method (9). Glycogen was estimated by the anthrone method of Seifter et al. (10). Statistical comparisons were made with Student’s t test with correction for unequal group size. RESULTS

METHODS

85-100 days old (200-250 one week prior to treat-

Figures l-4 show the time sequence of the changes that occur in the uterus in the pres105

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FIG. 1. Effect of actidione (A), estradiol (E), and actidione + estradiol (A + E) on uterine wet weight and water content. Control value represents the mean value of 29 rats and each experimental point is the mean from a group of 6-10 rats at that time period. Mean standard error for water was f0.4a/,; range, 0.2-0.7%. Mean standard error for uterine weight was f12 mg; range, 3-29 mg.

ence of estradiol alone (E), estradiol and the inhibitor cycloheximide injected 30 minutes before e&radio1 (A + E), and cycloheximide alone (A). Cycloheximide as well as estrogen resulted in an imbibition of uterine water, and the group treated with both showed a synergistic effect, the water content being significantly higher at 8 and 16 hours than that of either treatment alone (p < .OOl) (Fig. 1). Muscle and liver Hz0 were unaffected by any of the treatments. Uterine weight changes paralleled those of uterine water (Fig. 1). Mean blood glucose levels of the 4 groups (control, E, A + E, and A) ranged from 134 to 143 mg/lOO ml and showed little change during the experimental periods. Similarly, muscle glucose levels did not change during treatment. Alterations were observed in

uterine and liver glucose however. Figure 2 demonstrates that actidione or e&radio1 stimulated an increase in free uterine glucose, maximal levels being attained at the 5-hour time period. The uterine glucose for all rats receiving estradiol (E or A + E) was significantly higher than controls. However, in the A group, the glucose level had returned to the control value at 16 hours: E and AE+ at 2,5, 8, and 16 hours vs. controls, p < .OOl; A at 2, 5, and 8 hours vs. control p < .025, .OOl,and .Ol, respectively. When rats were treated with actidione, liver glucose decreasedto about one half the control level at 8 hours (p < .OOl) but showed a tendency to return to higher values at 16 hours. All liver glucose concentrations in the actidione groups, except the 16-hour A+E, were significantly less than the control level (p < .05-.OOl). Estrogen-treated rats showed a smaller decline (about 20 %), which was not different from the controls at the 5 % level of significance (p = .lOO-.200). ~~0 _

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FIG. 2. Effect of actidione (A), estradiol (E), and actidione + estradiol (A + E) on uterine and liver glucose content. Numbers of rats represented are as follows: Liver glucose controls (30), uterine glucose controls (24), A (4 rats/time period), A + E and E (6 rats/time period). Mean standard error for liver glucose was flO8 pg/lOO mg dry weight (DW); range, 39-170. Mean standard error for uterine glucose was 130 ag/lOO mg DW; range, S-66.

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FIG. 3. Effect of actidione (A), estradiol (E), and actidione + estradiol (A + E) on uterine glycogen. Each experimental point, represents the mean value from a group of 4-11 rats at that time interval; the control value represents 28 rats. Mean standard error, f4G pg/lOO mg dry weight; range 10-89.

Estrogen produced a large increase in uterine glycogen that continued during the 16-hour period studied here (Fig. 3). Act’idione alone caused a 50 % increase in uterine glycogen at 2 hours and maintained this level (p < .005-.OOl). The group treated with estradiol and actidione, given 30 minutes before the estradiol, showed a glycogen curve similar to that of A alone for the first 8 hours, but t’he 16-hour uterus had a glycogen content approaching that of the E group. This result suggested that actidione was supressing estrogen action for the first 8 hours, but after this, estrogen stimulated glycogen synthesis. The gastrocnemius muscle glycogen concentration (2269 pg/lOO mg dry weight & 115 XE) was not affected by any of the treatments. However, 5 hours after treatment with actidione, liver glycogen was depleted and remained low at the S- and 16-hour periods (Fig. 4). Estrogen treatment had no effect on liver glycogen concentration. In order to determine if actidione caused a direct breakdown of uterine glycogen, rats were treated with actidione 16 hours postestrogen. At this time uterine glycogen stimulation due to estrogen is maximal (6). Four and one-half hours later the glycogen concentration in uteri from the A+E treated

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rats (2024 pg glycogen/lOO mg dry weight f 61 SE) was the same as the 20% hour E group (1913 pg glycogen/lOO mg dry weight =t 50 SE), thus demonstrating that no glycogenolysis due to actidione occurred. Fasted rats were also treated with estradiol and actidione to compare uterine glycogen synthesis wit,h that occurring in fed rats. Uterine glycogen and glucose concentrations showed the same responses to the various treatments as the fed rats. Liver glycogen was severely depleted by fasting and t’he residual glycogen did not show a further decrease with actidione treatment. Muscle glycogen content was only about 75 % of that of the fed animals and showed no changes during estrogen or acbidione treatment. Water content of the three Cssues studied was unaffected by fast’ing. Since a single dose of actidione appeared to suppress the estrogen-induced uterine glycogen synthesis for about S hours (Fig. 3), the increase in glycogen from the S- to 16-hour time periods could be caused by an insufficient amount of actidione present, or possibly, the action of estradiol was able to overcome the inhibitory actions of actidione. These possibilities were tested by administering mulbiple doses of actidione and meas-

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FIG. 4. Effect of actidione (A), estradiol (E), and actidione + estradiol (A + E) on liver glycogen. Each experimental point represents the mean value from a group of 4-12 rats at, that, time interval (total 96 rats). Mean st,andard error 1735 pg/loO mg dry weight (DW) ; range, 144-2297.

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uring the uterine glycogen 16 hours post estrogen. Figure 5 shows that uterine glycogen attained a level of 1600 pg/lOO mg dry weight at 16 hours with estrogen alone (E) and that a single dose of actidione (lxA+E) resulted in a glycogen concentration of 1100 pg/lOO mg dry weight. A second dose of actidione given 7% hours after estrogen (2xA+E) partially prevented the estrogeninduced glycogen increase (Fig. 5). When three doses of actidione were given at 30 minutes before, 5% and lO>s hours after estradiol, the glycogen concentration was reduced to that of the rats treated with actidione alone (Fig. 5). The preceding experiments demonstrated that cycloheximide, present either before or at the same time as estrogen, could completely suppress the initiation of uterine glycogen synthesis by the estrogen. The possibility exists that once the glycogen enzyme systems are stimulated, they could

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inhibition of glycogen synthesis after prior stimulation by eskadiol. Each bar represents the mean value &standard error. Numbers of rats were as follows: A (20)) E (15)) and AE (6-10). A represents the combined mean for the 4 treatment groups.

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of estradiol and multiple doses of actidione on uterine glycogen. Uterine glycogen was measured 16 hours post-estradiol. E, estradiol alone; (IxA+E), 0.25 mg actidione 30 minutes before 1 pg estradiol; (2xA+E), 0.25 mg actidione 30 minutes before and 7% hours post-estradiol; (3xA+E), 0.25 mg actidione 30 minutes before, 5% hours, and 10% hours after estradiol. Each bar represents the mean &standard error from a group of B-10 rats.

continue and, be relatively insensitive or refractory to inhibition. The influence of cycloheximide on glycogen synthesis after initiation by estradiol was therefore studied by administering actidione at various time periods after estrogen treatment (Fig. 6). Comparisons of the uterine glycogen were then made 10 hours after the estradiol injection. When actidione was administered 1% hours after estradiol, almost complete suppression of the estrogen-induced glycogen synthesis occurred. Actidione administered 2% hours after estradiol resulted in only partial suppression. If actidione was injected more than 4 hours after estrogen, it was ineffective in suppressing glycogen synthesis. These experiments indicated that there was an initial period of about 3 hours after estrogen treatment during which the glycogen synthetic system was sensitive to inhibition by cycloheximide. After this time, the presence of cycloheximide was unable to suppress glycogen synthesis. DISCUSSION

Studies by Mueller et al. (1) have demonstrated that puromycin blocks the protein

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synthesis and water imbibition that estrogen hormoual studies have usually been large stimulates in the rat uterus. Due to the and have caused general toxic effects on the short-lived action of puromycin, repeated animals. Several recent studies have emphainjections were made and puromycin was sized this aspect of antibiot)ic action and given at the same time and every l-2 hours have recommended caution in the interpretation of data involving hormonal action and during estrogen treatment. These findings were confirmed by Hamilton (11). Subse- these inhibitors. Lerner et al. (13) found quently, Ui and Mueller (2) found that the that actinomycin D only inhibited the uteroantibiotic actinomycin D, given at the same trophic responses to est,rogen at dose levels time as estrogen, was able to inhibit estrogen that interfered wit,h normal body growth. action, apparently by inhibiting the syntheAnot,her recent report, by Greif et al. (14), sis of RNA. III other experiments, Gorski suggests that the effe&s of puromycin should and Axman (3) reported that cycloheximide, be interpreted carefully because of the toxgiven 30 minutes before estradiol, inhibit,ed icity of the compound. Toxic symptoms in estrogenstimulated protein synthesis in the animals have been noted previously by rat uterus. All of t’hese results, measuring Young et al. (15), Gorski and Axman (3), other parameters of estrogen action, are and in a previous report from our laboratory thus similar to the data in the present paper w. REFERENCES involving glycogen synthesis, where cycloheximide, given before or at the same time 1. MKELLER, G. C., GORSKI, J., AND A~zawa, Y., as estrogen, blocked the estrogenic response. Proc. Sad. ilcad. Sci. U.S. 47, 104 (1961). Since cycloheximide is a potent inhibitor of 2. 171, II., AND MUELLER, G. C., Pm. ,Yatl. rlcad. Sci. U.S. 60, 256 (1963). protein synthesis, it appears that uterine 3. GORSKI, J., .ixo AXILMN, S. M. C., Llrch. Uioglycogen synthesis is suppressed when prothem. Biophys. 105, 517 (1964). t.ein synthesis is inhibited. The possibility 4. NICOLETTE, J. A., AND GOR~KI, .J., rlrch. exists that these are coincident occurrences Biochem. Biophyys. 107, 279 (1964). and that the suppression of glycogen synthe5. CECIL, 11. C., BITKIN, J., .IND WRENN, T. R., sis may be independent of protein inhibition. Endocrinology 74, 701 (1961). However, the data presented here also 6. BITMAS, J., CECIL, 1%. C., >IENCH, M, L., demonstrate that if estrogen has already AND WREXN, T. Il., Endocrinology 76, 63 stimulated the glycogen enzymic systems, (1965). they cannot then be suppressed by the drug. 7. WILLUMS, H. E., AND PROTINE, H. T., E&ocrinology 78, 786 (1966). Ui and Mueller (2) also tested for time pe8. BITM,W, J., TI~EZISE, L. .4., AND CECIL, I-1. C., riods of unusual sensitivity in the estrogenic Arch. Hiochem. Biophys. 113, 414 (19GS). response by administering actinomycin D 9. WORTHINGTON BIOCHEIVIICAL CORPORATION, after the injection of the estrogen. If actinoI)eseriptiveMarlrlaI IGo. 11, p. 75. Freehold, mycin D was administered at the same time New Jersey (1961). or one hour after estrogen, it was inhibitory 10. SEIBTER, S., DAYTON, P., SOVIC, B., AND and suppressed uterine wet weight and laMUNT\VYLER, E., Bwh. Biochem. Biophys. beled glycine incorporation into protein. If 26, 191 (1950). given 2 hours afterward, it did not restrict 11. ~L~ILTON, T. Il., &oc. Natl. Iliad. sci. the estrogenic response. Our finding that C.S. 49, 373 (19G3). 12. GOLDBERG, II. S. (Ed.), “Antibiotics, Their cycloheximide does not suppress glycogen Chemistry and Non-Medical Uses.” Van synthesis when given 4 hours after estrogen Nostrand, Princeton, Xew Jersey (1959). suggests that once the glycogen synthetic machinery is set in motion, it is not depend- 13. LERNEK, L. J., HILF, R., TURKHEIMEH, A. R., AND MICHET+ I., J. Endoci-inok 33, 531 ent upon further protein synthesis. (1965). Cycloheximide, actinomycin D, and puro14. GREIF, R. L., SONG, C. S., AND CHIPKIN, II., mycin are potent antibiotics and inhibit Edocrinology 77, 223 (1965). mammalian cell growth as well as bacterial 15. YOCNG, C. W., ROBIKSON, P. F., AND SXKTOH, I?.., lkxhem. Pharnzacol. 12, 855 (1963). growth (12). The levels of antibiotic used in