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Evidence for different mechanisms in the circadian and glucocorticoid control of rat liver ornithine aminotransferase synthesis
Life Sciences Vol . 19, pp . 1435-1438, 1976 . Printed in the U .S .A .
Pergamon Presa
EVIDENCE FOR DIFFERENT MECHANISMS IN THE CIRCADIAN AND GLUCOCORTICOID CONTROL OF RAT LIVER ORNITHINE AMINOTRANSFERASE SYNTHESIS Carl Peraino, J . Emory Morris, l and Surendra T . Shenoy Division of Biological and Dledical Research Argonne National Laboratory Argonne, Illinois 60439 (Received in final form September 20, 1976 Summary In untreated rats fed a 60$ casein diet ad libitum the rate of ornithine aminotransferase synthesis, measured immunochemically, decreased 50$ between 9 AM and 9 PM . (Previous studies showed that this decrease represents the descending phase of a circadian cycle for ornithine aminotransferase synthesis .) This decrease was not affected by actinomycin D administration . When triamcinolone was given at 9 AM the rate of synthesis was reduced to 25$ of the 9 AM value but this additional reduction was completely blocked by actinomycin D or a-amanitin treatment . These results suggest that the repression of ornithine aminotransferase synthesis by triamcinolone requires mRNA synthesis, and that the repression may occur at a posttranscriptional stage in the synthesis of the enzyme . However, the circadian decline in ornithine aminotransferase synthesis in untreated rats is apparently not dependent on the synthesis of a post-transcriptional repressor . Previous work in this laboratory showed that, in rats fed a 60$ casein diet, the synthesis of liver ornithine aminotransferase (ED 2 .6 .1 .13) oscillates in a circadian rhythm (1) In addition, treatment of the rats with the glucocorticoid, triamcinolone, 2 markedly represses the synthesis of the enzyme (1) . The present study examines the effects of inhibitors of RNA synthesis on these responses . Experimental Procedure Thirty-five male Sprague-Dawley rats weighing 150 to 160 g were fed _ad libi tum a diet containing 60$ casein for 5 days . The rats were housed five per cage under conditions of constant temperature (23 ° ) and regulated lighting (light, 7 AM to 7 PM ; dark, 7 PM to 7 AM) . On Day 6 the rats were divided into six groups and treated as follows : Group 1, no treatment, killed 9 AM ; Group 2, no treatment, killed 9 PAt ; Group 3, intraperitoneal injections of 150 ug actinomycin D at 8 AM and 3 PM ; Group 4, 5 mg triamcinolone by intramuscular injection at 9 AM ; Group 5, combination of treatments for Groups 3 and 4 ; Group 6, intraperitoneal injections of 150 u8 a-amanitin at 8 AM and 3 PM, triamcinolone as in Group 4 . Groups 3 to 6 were killed at 9 PM . Each rat was given an intraperi1 Present address : Department of Ch~uistry, State University College at Brockport, Stockport, New York 14420 . 2 Triamcinolone, 9a-fluoro-11 ,16 ,17,21-tetrahydroxypregna-l,4- diene -3,20- diene . 1435
1436
Control of Ornithine Aminotraasferase
Vol . 19, No . 9
toneal injection of 100 uCi of [ 3 H]leu 40 min prior to its sacrifice . All livers were analyzed for ornithine aminotransferase activity, the incorporation of [ 3H]leu into total liver protein, and the incorporation of [ 3 H]leu into immunochemically precipitated ornithine aminotransferase . Additional details of the experimental procedure have been published (1) . Results The studies to be described were confined to the 9 AM - 9 PM interval because food intake is at a minimum during the light phase of the circadian cycle (1) . Therefore the interpretation of the effects of RNA inhibitors on enzyme synthesis was not complicated by considerations of inhibitor mediated impairment of food intake and intestinal absorption . In agreement with prior observations on the circadian cycling of ornithine aminotransferase synthesis (1), Table I shows that in untreated rats the synthesis of this enzyme decreased approximately 50$ between 9 AM and 9 PM . This de cline in synthesis was not affected by the administration of actinomycin D at a dosage (2 mg/kg) sufficient to inhibit RNA synthesis (2) . Treatment with triamcinolone (Group 4) caused a reduction of ornithine aminotransferase synthesis to a level 50$ below that seen in untreated rats or those given actinomycin D alone . This effect of triamcinolone was completely blocked by the administration of actinomycin D or a-amanitin . a-Amanitin is a specific inhibitor of mRNA synthesis at the dosage given (2) . TABLE I Effects of Inhibitors of RNA Synthesis on Liver Ornithine Aminotransferase Synthesis between 9 AM and 9 PM in Untreated Rats and Rats Given Triamcinolone at 9 AM .a Group
Treatment
Enzyme Synthesisb
Enzyme Activityc
15 .00 1 2 .70
33 ± 4
1
Control, killed 9 AM
2
Control, killed 9 PM
7 .61 ± 0 .96d
29 ± 4
3
Actinomycin D 8 AM and 3 PM, killed 9 PM
7 .24 1 0 .87 4
30 ± 2
4
Triamcinolone 9 AM, killed 9 PM
3 .74 t 0 .26e
29 ± 1
5
Actinomycin D 8 AM and 3 PM, triamcinolone 9 AM, killed 9 PM
8 .23 t 1 .23~
29 ± 2
6
a-Amanitin 8 AM and 3 PM, triamcinolone 9 AM, killed 9 PM
9 .51 t 1 .61~
33 ± 4
aEach value is the mean ± standard error . maining groups contained 5 rats each .
Group 5 contained 10 rats ; the re-
b dpm (times 10 4 ) in antibody precipitatee protein per g liver divided by total acid insoluble dpm per g liver . cumol of product (A'-pyrroline-5-carboxylate) formed per min per liver per 100 g body weight . d Differs from Group 1, p < 0 .05 . eDiffers from Groups 2 and 3, p < 0 .005 . ( Differs from Group 4, p < 0 .01 .
Vol . 19, No . 9
Control of Ornithine Aminotransferase
1437
Although the rate of ornithine aminotransferase synthesis showed marked changes, the enzyme activity - which is directly related to the amount of enzyme protein (1) - remained relatively constant . This is a reflection of the slow turnover of the enzyme (1 .9-day half-life, refs . 3 and 4), which obscures changes in synthetic rate occurring over a 12-hour interval (1) . Discussion The observation that triamcinolone administration reduced the rate of ornithine aminotransferase synthesis below that seen in rats in whicj~ RNA synthesis had been inhibited implicates post-transcriptional processes as potential sites for the triamcinolone effect . The ability of actinomycin D or a-amanitin to antagonize the repressive action of triamcinolone suggests that this repression requires mRNA synthesis . [Inhibition of RNA synthesis has no effect on steroid uptake or binding to receptors (5) .] The mechanism by which glucocorticoid represses ornithine aminotransferase synthesis, therefore, apparently involves the induction of a regulator via the stimulation of transcription - an interpretation supported by prior studies of steroid action (6,7) . The regulator then interferes with a post-transcriptional stage in the synthesis of ornithine aminotransferase by altering the processing or translation of the mRNA for this enzyme . The failure of actinomycin D to antagonize the circadian decline in ornithine aminotransferase synthesis suggests that in untreated rats this decline does not depend on the synthesis of a post-transcriptional repressor, as de scribed above for the triamcinolone effect . Possible alternative sources for the circadian oscillations in ornithine aminotransferase synthesis not eliminated by the present data include : (a) circadian changes in the synthesis of ornithine aminotransferase mRNA ; (b) circadian changes in the level of a,hypothetical inducer of ornithine aminotransferase mRNA translation . Acknowledgments This work was supported by the United States Energy Research and Development Administration . Excellent technical assistance was provided by Ms . Aldona Prapuolenis . We are indebted to Dr . B . N . Jaroslow for valuable discussions . References 1. 2. 3. 4. 5. 6. 7.
J . E . MORRIS and C . PERAINO, J . Biol . Chem . 251 2571-2578 (1976) . S . T . JACOB, M . B . SCHARF, and E . S . VESSEL, Proc . Nat . Acad . Sei . _71 704707 (1974) . M . M . IP, P . Y . CHEE, and R . W . SWICK, Biochem . Biophys . Aeta _354 29-38 (1974) . P . Y . CHEE and R . W . SWICK, J. BioZ . Chem . 251 1029-1034 (1976) . B . BAGGETT and G . M . STONE, Perspectives inBiological Chemistry, (P .201) Marcel Dekker, New York (1970) . H . A . YOUNG, E . M . SCOLNICK, and W . P . PARKS, J . BioZ . Chem . _250 3337-3343 (1975) . M . J . TSAI, H . C . TOWLE, S . E . HARRIS, and B . W . O'MALLEY, J . Biol . Chem . 251 1960-1968 (1976) .
Pergamon Presa
EVIDENCE FOR DIFFERENT MECHANISMS IN THE CIRCADIAN AND GLUCOCORTICOID CONTROL OF RAT LIVER ORNITHINE AMINOTRANSFERASE SYNTHESIS Carl Peraino, J . Emory Morris, l and Surendra T . Shenoy Division of Biological and Dledical Research Argonne National Laboratory Argonne, Illinois 60439 (Received in final form September 20, 1976 Summary In untreated rats fed a 60$ casein diet ad libitum the rate of ornithine aminotransferase synthesis, measured immunochemically, decreased 50$ between 9 AM and 9 PM . (Previous studies showed that this decrease represents the descending phase of a circadian cycle for ornithine aminotransferase synthesis .) This decrease was not affected by actinomycin D administration . When triamcinolone was given at 9 AM the rate of synthesis was reduced to 25$ of the 9 AM value but this additional reduction was completely blocked by actinomycin D or a-amanitin treatment . These results suggest that the repression of ornithine aminotransferase synthesis by triamcinolone requires mRNA synthesis, and that the repression may occur at a posttranscriptional stage in the synthesis of the enzyme . However, the circadian decline in ornithine aminotransferase synthesis in untreated rats is apparently not dependent on the synthesis of a post-transcriptional repressor . Previous work in this laboratory showed that, in rats fed a 60$ casein diet, the synthesis of liver ornithine aminotransferase (ED 2 .6 .1 .13) oscillates in a circadian rhythm (1) In addition, treatment of the rats with the glucocorticoid, triamcinolone, 2 markedly represses the synthesis of the enzyme (1) . The present study examines the effects of inhibitors of RNA synthesis on these responses . Experimental Procedure Thirty-five male Sprague-Dawley rats weighing 150 to 160 g were fed _ad libi tum a diet containing 60$ casein for 5 days . The rats were housed five per cage under conditions of constant temperature (23 ° ) and regulated lighting (light, 7 AM to 7 PM ; dark, 7 PM to 7 AM) . On Day 6 the rats were divided into six groups and treated as follows : Group 1, no treatment, killed 9 AM ; Group 2, no treatment, killed 9 PAt ; Group 3, intraperitoneal injections of 150 ug actinomycin D at 8 AM and 3 PM ; Group 4, 5 mg triamcinolone by intramuscular injection at 9 AM ; Group 5, combination of treatments for Groups 3 and 4 ; Group 6, intraperitoneal injections of 150 u8 a-amanitin at 8 AM and 3 PM, triamcinolone as in Group 4 . Groups 3 to 6 were killed at 9 PM . Each rat was given an intraperi1 Present address : Department of Ch~uistry, State University College at Brockport, Stockport, New York 14420 . 2 Triamcinolone, 9a-fluoro-11 ,16 ,17,21-tetrahydroxypregna-l,4- diene -3,20- diene . 1435
1436
Control of Ornithine Aminotraasferase
Vol . 19, No . 9
toneal injection of 100 uCi of [ 3 H]leu 40 min prior to its sacrifice . All livers were analyzed for ornithine aminotransferase activity, the incorporation of [ 3H]leu into total liver protein, and the incorporation of [ 3 H]leu into immunochemically precipitated ornithine aminotransferase . Additional details of the experimental procedure have been published (1) . Results The studies to be described were confined to the 9 AM - 9 PM interval because food intake is at a minimum during the light phase of the circadian cycle (1) . Therefore the interpretation of the effects of RNA inhibitors on enzyme synthesis was not complicated by considerations of inhibitor mediated impairment of food intake and intestinal absorption . In agreement with prior observations on the circadian cycling of ornithine aminotransferase synthesis (1), Table I shows that in untreated rats the synthesis of this enzyme decreased approximately 50$ between 9 AM and 9 PM . This de cline in synthesis was not affected by the administration of actinomycin D at a dosage (2 mg/kg) sufficient to inhibit RNA synthesis (2) . Treatment with triamcinolone (Group 4) caused a reduction of ornithine aminotransferase synthesis to a level 50$ below that seen in untreated rats or those given actinomycin D alone . This effect of triamcinolone was completely blocked by the administration of actinomycin D or a-amanitin . a-Amanitin is a specific inhibitor of mRNA synthesis at the dosage given (2) . TABLE I Effects of Inhibitors of RNA Synthesis on Liver Ornithine Aminotransferase Synthesis between 9 AM and 9 PM in Untreated Rats and Rats Given Triamcinolone at 9 AM .a Group
Treatment
Enzyme Synthesisb
Enzyme Activityc
15 .00 1 2 .70
33 ± 4
1
Control, killed 9 AM
2
Control, killed 9 PM
7 .61 ± 0 .96d
29 ± 4
3
Actinomycin D 8 AM and 3 PM, killed 9 PM
7 .24 1 0 .87 4
30 ± 2
4
Triamcinolone 9 AM, killed 9 PM
3 .74 t 0 .26e
29 ± 1
5
Actinomycin D 8 AM and 3 PM, triamcinolone 9 AM, killed 9 PM
8 .23 t 1 .23~
29 ± 2
6
a-Amanitin 8 AM and 3 PM, triamcinolone 9 AM, killed 9 PM
9 .51 t 1 .61~
33 ± 4
aEach value is the mean ± standard error . maining groups contained 5 rats each .
Group 5 contained 10 rats ; the re-
b dpm (times 10 4 ) in antibody precipitatee protein per g liver divided by total acid insoluble dpm per g liver . cumol of product (A'-pyrroline-5-carboxylate) formed per min per liver per 100 g body weight . d Differs from Group 1, p < 0 .05 . eDiffers from Groups 2 and 3, p < 0 .005 . ( Differs from Group 4, p < 0 .01 .
Vol . 19, No . 9
Control of Ornithine Aminotransferase
1437
Although the rate of ornithine aminotransferase synthesis showed marked changes, the enzyme activity - which is directly related to the amount of enzyme protein (1) - remained relatively constant . This is a reflection of the slow turnover of the enzyme (1 .9-day half-life, refs . 3 and 4), which obscures changes in synthetic rate occurring over a 12-hour interval (1) . Discussion The observation that triamcinolone administration reduced the rate of ornithine aminotransferase synthesis below that seen in rats in whicj~ RNA synthesis had been inhibited implicates post-transcriptional processes as potential sites for the triamcinolone effect . The ability of actinomycin D or a-amanitin to antagonize the repressive action of triamcinolone suggests that this repression requires mRNA synthesis . [Inhibition of RNA synthesis has no effect on steroid uptake or binding to receptors (5) .] The mechanism by which glucocorticoid represses ornithine aminotransferase synthesis, therefore, apparently involves the induction of a regulator via the stimulation of transcription - an interpretation supported by prior studies of steroid action (6,7) . The regulator then interferes with a post-transcriptional stage in the synthesis of ornithine aminotransferase by altering the processing or translation of the mRNA for this enzyme . The failure of actinomycin D to antagonize the circadian decline in ornithine aminotransferase synthesis suggests that in untreated rats this decline does not depend on the synthesis of a post-transcriptional repressor, as de scribed above for the triamcinolone effect . Possible alternative sources for the circadian oscillations in ornithine aminotransferase synthesis not eliminated by the present data include : (a) circadian changes in the synthesis of ornithine aminotransferase mRNA ; (b) circadian changes in the level of a,hypothetical inducer of ornithine aminotransferase mRNA translation . Acknowledgments This work was supported by the United States Energy Research and Development Administration . Excellent technical assistance was provided by Ms . Aldona Prapuolenis . We are indebted to Dr . B . N . Jaroslow for valuable discussions . References 1. 2. 3. 4. 5. 6. 7.
J . E . MORRIS and C . PERAINO, J . Biol . Chem . 251 2571-2578 (1976) . S . T . JACOB, M . B . SCHARF, and E . S . VESSEL, Proc . Nat . Acad . Sei . _71 704707 (1974) . M . M . IP, P . Y . CHEE, and R . W . SWICK, Biochem . Biophys . Aeta _354 29-38 (1974) . P . Y . CHEE and R . W . SWICK, J. BioZ . Chem . 251 1029-1034 (1976) . B . BAGGETT and G . M . STONE, Perspectives inBiological Chemistry, (P .201) Marcel Dekker, New York (1970) . H . A . YOUNG, E . M . SCOLNICK, and W . P . PARKS, J . BioZ . Chem . _250 3337-3343 (1975) . M . J . TSAI, H . C . TOWLE, S . E . HARRIS, and B . W . O'MALLEY, J . Biol . Chem . 251 1960-1968 (1976) .