Regulation of mRNA levels for five urea cycle enzymes in rat liver by diet, cyclic AMP, and glucocorticoids

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 256, No. 1, July, pp. 343-353, 198’7

Regulation of mRNA Levels for Five Urea Cycle Enzymes in Rat Liver by Diet, Cyclic AMP, and Glucocorticoids’ SIDNEY M. MORRIS, JR.,*,’ CAROLE L. MONCMAN,*s3 KATHERINE GEORGE J. DIZIKES,? STEPHEN D. CEDERBAUM,f AND WILLIAM

D. RAND,*p4 E. O’BRIEN*

*Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh, Pittsburgh, 15261;TDepartments of Psychiatry and Pediatrics and the Mental Retardation Research Center, UCLA Center for the Health Sciences, Los Angeles, CalZfwnia 90024 and $Institute of Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030

Pennsylvania

Received January

81987, and in revised form March 17,1987

Adaptive changes in levels of urea cycle enzymes are largely coordinate in both direction and magnitude. In order to determine the extent to which these adaptive responses reflect coordinate regulatory events at the pretranslational level, measurements of hybridizable mRNA levels for all five urea cycle enzymes were carried out for rats subjected to various dietary regimens and hormone treatments. Changes in relative abundance of the mRNAs in rats with varying dietary protein intakes are comparable to reported changes in enzyme activities, indicating (a) that the major response to diet occurs at the pretranslational level for all five enzymes and (b) that this response is largely coordinate. In contrast to the dietary changes, variable responses of mRNA levels were observed following intraperitoneal injections of dibutyryl CAMP and dexamethasone. mRNAs for only three urea cycle enzymes increased in response to dexamethasone. Levels of all five mRNAs increased severalfold in response to dibutyryl CAMP at both 1 and 5 h after injection, except for ornithine transcarbamylase mRNA which showed a response at 1 h but no response at 5 h. Combined effects of dexamethasone and dibutyryl CAMP were additive for only two urea cycle enzyme mRNAs, suggesting independent regulatory pathways for these two hormones. Transcription run-on assays revealed that transcription of at least two of the urea cycle enzyme genes-carbamylphosphate synthetase I and argininosuccinate synthetase-is stimulated approximately four- to fivefold by dibutyryl CAMP within 30 min. The varied hormonal responses indicate that regulatory mechanisms for modulating enzyme concentration are not o 1987 Academic press, I~~. identical for each of the enzymes in the pathway.

The urea cycle is comprised of five enzymes-carbamyl-phosphate synthetase I (carbamoyl-phosphate synthetase (ami This work was supported in part by Grant AM 33144 from the National Institutes of Health, a grant from the Health, Research and Services Foundation, and by a Basil O’Connor Starter Research Grant from the March of Dimes Birth Defects Foundation (S.M.M.); and by Grant HD-06576 from the National Institutes of Health and March of Dimes Birth Defects Foundation Grant 6-428 (S.D.C.). ’ To whom correspondence should be addressed.

monia), EC 6.3.4.16), ornithine transcarbamylase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3), argininosuccinate synthetase (L-citrulline: L-aspartate ligase (AMP-forming), EC 6.3.4.5), argininosuccinate lyase (L-argininosuccinate arginine-lyase, EC 4.3.2.1), and arginase (L-arginine ureohydrolase, EC ’ Present address: Department of Biochemistry, Rutgers University, Piscataway, NJ 08854. 4 Present address: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706.

343

0003-9861/87 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

344

MORRIS

3.5.3.1)-which catalyze the net conversion of two molecules of ammonia and one of bicarbonate into urea. The activities of these enzymes in liver exhibit adaptive responses to changes in dietary protein intake (l-9). In fact, the adaptive response of urea production was one of the first to be demonstrated for a metabolic pathway in animals, as shown by Folin’s report of a nearly lo-fold change in urea excretion when humans switched from a normal protein diet to a protein-free diet (10). Modulation of enzyme activities by glucagon (11-16) and glucocorticoids (6,8,12,14, 16-19) suggests that these hormones may be predominant mediators of the adaptive response to diet. Long-term changes in enzyme activity are largely due to changes in enzyme mass (2,20,21,25), which in turn primarily reflect alterations in enzyme synthesis rates (22-24). Although these conclusions have been based on detailed studies of several of the urea cycle enzymes, they have generally been considered to be applicable to all five. A major goal in studies of metabolic regulation has been to identify the regulated steps at which changes in enzyme mass are effected. Despite the fact that both qualitative and quantitative information concerning the possibility of translational and post-translational regulation of the urea cycle enzymes is still incomplete, several reports have nonetheless demonstrated regulation at the pretranslational level (25-29). However, these reports have been limited to studies of mRNAs for the two mitochondrial enzymes. Since adaptive responses of the urea cycle enzymes are generally coordinate in degree, questions arise as to (i) the identity of the regulated pretranslastep(s) (e.g., translational, tional, transcriptional) involved in these responses and (ii) whether coordinate changes in enzyme levels reflect regulatory events at a common step for all five enzymes. Although investigations of this question for metabolic enzymes in animal cells are too few to permit generalized conclusions, there are indications that coordinate regulation may be complex. For example, synthesis rates of malic enzyme and fatty acid synthase, which also exhibit co-

ET AL.

ordinate adaptive responses in liver (3032), differ not only in their response to specific hormones (32) but also in their regulation at translational or pretranslational steps (33). Since different components of the urea cycle are localized within two distinct subcellular compartments and genes for these enzymes are contained within different linkage groups (34), determination of the regulated steps for the individual enzymes under a variety of experimental conditions would be valuable for our understanding of the nature of coordinate regulation of gene expression in animal cells. The availability of cloned cDNAs for all five urea cycle enzymes has made it feasible to begin such an investigation. This study was undertaken to determine whether or not mRNA levels for the urea cycle enzymes exhibit coordinate responses to alterations in diet or to short-term exposure to hormones and whether hormonal regulation occurs at the level of transcription. MATERIALS

AND

METHODS

Materials. [a-?]dCTP, [o-“PJIJTP, and Gene Screen membranes were purchased from New England Nuclear. [5-‘H]CTP was obtained from ICN Radiochemicals. Nick-translation kits (Bethesda Research Laboratories) and oligolabeling kits (Pharmacia) were purchased from the indicated sources. Restriction enzymes were purchased from Bethesda Research Laboratories or New England BioLabs. Guanidine hydrochloride, ribonuclease A, dexamethasone, and dibutyryl CAMP were obtained from Sigma Chemical Co. Ribonuclease inhibitor (RNasin) was a product of Promega Biotec. Protein-free diet (Cat. No. 904666, supplemented with vitamins and trace minerals), normal protein diet (27% casein; Cat. No. 902487), and high protein diet (60% casein; Cat. No. 904669) were purchased from ICN Nutritional Biochemicals. Aminophenylthioether paper was from Schleicher & Schuell. RNase-free DNase was purchased from Cooper Biomedical, Inc. Eschtichia coli RNA polymerase was supplied by New England BioLabs. Nucleoside-5’-diphosphate kinase was purchased from Boehringer-Mannheim. cDNA clones. cDNA clones for carbamyl-phosphate synthetase I (pCPSr4; Ref. (35)) and arginase (p3Bl; Ref. (36)) of rat have been described. The cDNA clone for rat phosphoenolpyruvate carboxykinase (pPCK10; Ref. (37)) was generously provided by Dr. Richard Hanson. A full-length cDNA clone for rat argininosuccinate synthetase (pASrl1) was obtained by screening a rat kidney cDNA library (37) with the

mRNA

LEVELS

FOR UREA

CYCLE

homologous human cDNA (pAS1; Ref. (38)). cDNA clones for ornithine transcarbamylase (39) and argininosuccinate lyase (pAL3; Ref. (40)) were of human origin. Animal care. Male Sprague-Dawley rats (150-175 g) were obtained from Hilltop Lab Animals, Inc. (Scottdale, PA). Rats were maintained on a 12 h lightdark cycle and supplied with standard rat chow and water ud lib. Changes in diet, injections of hormones, and sacrifices of rats were routinely carried out at 9:00-1O:OOAM to minimize diurnal variation. Rats were sacrificed by decapitation. Dietary regimens and hormone treatments are described in the legends to the tables and figures. Experimental procedures involving animals were approved by the Animal Care and Use Committee of the University of Pittsburgh. Isolation of RNA and quadjkation of mRNA levels. Total RNA was isolated by a modification (41) of the method of Chirgwin et al. (42). Samples of total RNA (5 pg) were spotted onto Gene Screen filters for dotblot analysis as previously described (43) and fixed by exposure to ultraviolet light (44). In many cases the results of the dot-blot analysis were verified by quantitative Northern blot analysis. Total RNA (5 Kg) was electrophoresed on 0.9% agarose gels containing formaldehyde (45,46), blot-transferred to Gene Screen using 25 mM sodium phosphate (pH 6.5) as transfer buffer, and fixed by exposure to ultraviolet light (44). “‘P-labeled DNA probes were prepared by nicktranslation (47) or by using random oligonucleotides as primers (48). Hybridization of Northern blots and dot blots was carried out as described previously (43) or by a modification (49) of the hybridization conditions described by Church and Gilbert (44). All hybridizations were performed at 43°C and all posthybridization washes were at 50°C. Blots were subjected to autoradiography at -75°C in the presence of an intensifying screen. Regions of the filters containing hybridized RNA were cut out and analyzed by liquid scintillation counting. Equivalent areas which contained no RNA were cut out in triplicate from each filter and counted to determine filter background, which was subtracted from all hybridized RNA values. .ln vitro transcription assays. Rat liver nuclei were isolated essentially according to published methods (50, 51). The homogenization buffer contained 0.32 M sucrose, 3 mM CaCl2, 2 mM magn&Um SC&ate, 1 mM dithiothreitol, 10 mM Tris-HCl (pH 7.5), 0.1% Triton X-100. Following centrifugation through a buffered 2 M sucrose cushion, nuclei were stored at -75°C in 25% glycerol, 10 mM magnesium acetate, 0.1 mM EDTA, 5 mM dithiothreitol, 100 mM 4-(2-hydroxyethyl)-l-piprrazineethanesulfonate (Hepes),’ pH 7.5, at a concentration of 2 X 10’ nuclei/ml. ,’ Abbreviations used: Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; DPT diphenylthioether; SDS, sodium dodecyl sulfate; CAMP, cyclic AMP.

ENZYMES

IN RAT

LIVER

345

In vitro transcription reactions contained 2 X lo7 nuclei in 25% glycerol, 75 mM Hepes (pH 7.5), 100 mM KCl, 5 mM magnesium acetate, 1 mM MnC12, 50 /LM EDTA, 100 &i [a2P]UTP (3000 Ci/mmol), 1 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 4 mM dithiothreitol, 0.1 mg/ ml heparin, 8.8 mM phosphocreatine, 40 @g/ml creatine phosphokinase, 0.1 mg/ml nucleoside-5’-diphosphate kinase, in a total volume of 200 ~1(50-53). The reaction was carried out at 22°C for 30 min and was terminated by the addition of 10 vol of 1% SDS, 10 mM EDTA (pH 7), 100 pg/ml carrier yeast RNA (52). RNA was isolated by extraction with phenol in the presence of SDS at pH 5.0 and 55°C (51,52). Plasmid DNAs for hybridization were linearized and denatured prior to binding to disks of diazophenylthioether (DPT) paper according to the manufacturer’s protocol. [‘H]cRNA for use as internal standard to determine hybridization efficiency was generated from the PstI cDNA fragment of pPCKl0 according to Lis et al (54). Hybridization efficiency was calculated as the percentage of total cpm [‘H]cRNA added to each hybridization reaction which was specifically bound to the pPCKl0 filter. Each hybridization vial contained four DPT filters with pBR322, pCPSr4, pASrl1, or pPCK10. Prehybridization, hybridization, and washing of the filters were performed as described elsewhere (50). After the RNA was washed it was quantitatively eluted from the individual filters by two extractions with 50 mM NaOH at 37°C and neutralized, and the radioactivity was determined by liquid scintillation counting. Calculation of transcription rates is described in the legend to Table III. RESULTS

Eflect of diet on mRNA levels for urea cycle enzymes. Dietary regimens were chosen to represent a wide range in amino acid catabolism, resulting in a wide variation in amino nitrogen excretion in the form of urea. Time points were selected to assess short- and long-term adaptation to altered dietary protein intake in animals which had been maintained on diets of different protein content. The relative abundance of mRNAs for the urea cycle enzymes is generally lowest on a protein-free diet and increases in response to starvation or a high-protein diet (Table I and Fig. 1). Relative mRNA abundances during starvation or feeding a 60% casein diet were quite similar for the two groups of rats which had initially been maintained on either 27% casein or protein-free diets. Relative abundance for the different urea cycle enzyme mRNAs varied

9.9 * 0.1 11.1 + 0.3 7.5 f 0.3 4.6 f 0.2

155% 4

2

1

179k

216f

204 + 3

60% casein, 6 days

12.6 k 0.6

0.4-9.7 24

0.7-4.5 6

0.4-14.8 37

7.5 f 0.6

2.2 f 0.5

7.5 2 0.9

1.0 0.9 f 0.1

9.5 f 1.3

1.7 2 0.3

9.7 f 3.0

1.6 f 0.5

0.4 f 0.1

Argininosuccinate synthetase

4.4 * 0.3

1.9 + 0.4

3.9 -c 0.3

1.0 1.3kO.2

4.4 2 0.3

1.0 + 0.1

1.1 4.5 f 0.4

0.7 f 0.1

Ornithine transcarbamylase

8.2 AI 0.6

2.8 + 1.1

10.3 f 0.9

1.7 -t_0.2

1.0

10.8 + 0.4

2.4 + 0.1

1.5 + 0.4 14.8 f 2.0

0.4 f 0.1

Carbamylphosphate synthetase I

12

1.0-11.8

6.3 -t 0.5

2.8 f 0.8

11.8 + 1.5

1.0 3.0 f 0.5

5.3 f 1.5

1.8 f 0.5

11.5k2.3

2.5 f 0.5

1.8 f 0.8

Argininosuccinate lyase

0.6-14.2 24

4.6 + 0.8

0.9 f 0.2

14.2 f 1.1

2.0 f 0.7

0.6 f 0.1

Arginase

Note. Rats weighing 150-170 g were divided into two groups which were fed either 0 or 27% casein diets for 6 days. Each group was then further divided into two sets of rats which were either starved or fed 60% casein diet for the times indicated. Total RNA was isolated from livers and analyzed for relative abundance of individual mRNAs by dot-blot hybridization as described under Materials and Methods. Levels of the mRNAs for each group are expressed relative to the mRNA levels in livers of rats which had been maintained on 27% casein diet for 6 days. The number of animals in each group is indicated (n). The results represent a minimum of duplicate determinations for each rat and are expressed as the means f SE. Standard errors less than kO.05 are not listed.

Range Fold difference

252 k 12

60% casein, 6 days

6

161~~ 2

2252

Starve, 5 days

60% casein, 1 day

Starve, 1 day 10.6 + 0.1

7.3 k 0.3

116+- 3

Starve, 5 days 60% casein, 1 day

27% casein, 6 days

3.6 + 0.1

142+

6.2 + 0.2 4.6 -c 0.2

6

6

146k

Liver weight (Ed

Starve, 1 day

n

Body weight k)

0% casein, 6 days

Diet

I

EFFECT OF STARVATION AND VARYING DIETARY PROTEIN CONTENT ON mRNA LEVELS FOR UREA CYCLE ENZYMES IN RAT LIVER

TABLE

mRNA

LEVELS CWbEtfl7yl Phosphate Synthetase I

FOR UREA Ornithine TmnSC8rbamytase

CYCLE Agininosuccinate Synthetase

ENZYMES Argininosuccinate Lyase

IN RAT

LIVER

347

Aginase

FIG. 1. Effect of starvation and varying dietary protein content on mRNA levels for urea cycle enzymes in rat liver. Results are from Table I. Open bars, rats fed protein-free diet for 6 days; solid bars, starved rats; hatched bars, rats fed 60% casein diet. For each pair of bars, the first represents values for rats after 1 day of treatment; the second represents values for rats after 5 days (starved) or 6 days (60% casein diet). Pairs of bars marked by an asterisk represent rats initially maintained on 27% casein diet; the other pairs represent rats initially maintained on protein-free diet. Standard error bars have been omitted for the sake of clarity.

from 6- to 37-fold (Table I), falling into two classes. The “fold” differences in range for carbamyl-phosphate synthetase I, argininosuccinate synthetase, and arginase are not significantly different from one another but are distinct from the fold differences for ornithine transcarbamylase and argininosuccinate lyase, which, in turn, are not significantly different from each other. If overall patterns of response to the different diets are compared, three general patterns are apparent (Fig. 1). The mRNAs for carbamyl-phosphate synthetase I and argininosuccinate synthetase comprise one group, in which starvation and feeding on a high-protein diet increase mRNA abundance to a similar extent. A second group consists of mRNAs for argininosuccinate lyase and arginase, in which levels during starvation increase to the same extent as the first group but response to a high-protein diet is about half that of starvation. The mRNA for ornithine transcarbamylase constitutes a third category in which increases in mRNA abundance are comparable for both starvation and the highprotein diet but the maximum increase is only about half that found for the other four mRNAs.

Effect of dibutyryl CAMP and dexamethasone wn mRNA levels. Activities of the

urea cycle enzymes in rat liver are elevated in response to glucagon (11-16) or glucocorticoids (6,8,12,14,16-19), and decrease in response to adrenalectomy (7,8,1’7,18). Levels of carbamyl-phosphate synthetase I mRNA are increased by relatively longterm treatment with glucagon or CAMP (27-29) and glucocorticoids (27-29). We examined the short-term effects of hormonal treatment in order to minimize indirect effects on mRNA levels. For these experiments rats were initially maintained on a protein-free diet for 5-6 days so that basal mRNA levels for the urea cycle enzymes would be low. The mRNAs for the various urea cycle enzymes differ in the pattern and magnitude of their response to dibutyryl CAMP and dexamethasone (Table II). Messenger RNAs for ornithine transcarbamylase and argininosuccinate lyase show little or no response to dexamethasone, while mRNA levels for the other three enzymes increase markedly, albeit to different degrees. In the absence of a detailed time course of response, however, it cannot be determined whether the different increases in individ-

348

MORRIS

ET AL.

TABLE

II

EFFECT OF DIBUTYRYL CAMP AND DEXAMETHASONE ON RELATIVE ABUNDANCE OF mRNAs FOR UREA CYCLE ENZYMES IN RAT LIVER

Treatment Untreated controls + Dibutyryl CAMP (1 h) + Dibutyryl CAMP (5 h) + Dexamethasone (5 h) + Dexamethasone & dibutyryl CAMP (5 h)

Carbamylphosphate synthetase I

Ornithine transcarbamylase

Argininosuccinate synthetase

Argininosuccinate lyase

Arginase

1.0

1.0

1.0

1.0

1.0

2.5 f 0.1

1.9 f 0.2

4.5 f 0.4

2.2 f 0.1

4.8 f 0.2

5.0 + 0.4

0.8

5.8 + 0.4

2.6 f 0.1

6.7 + 0.3

2.3 + 0.1

0.9

4.4 f 0.7

1.1 f. 0.1

1.7 f 0.1

6.5 f 0.4

0.9 + 0.1

10.2 f 0.9

3.2 f 0.2

6.3 f 0.5

Note. Rats weighing 150-1’70 g were maintained on a protein-free diet for 5-6 days before hormone treatment. Groups of three rats received single intraperitoneal injections of dexamethasone (5 mg/kg), intraperitoneal injections of dibutyryl CAMP (25 mg/kg) at 2-h intervals, or a combination of the two injection schedules. Rats were sacrificed at 1 or 5 h following injection, and liver RNA was isolated and analyzed as described under Materials and Methods. Levels of the mRNAs for each group are expressed relative to the mRNA levels in livers of untreated controls. Results for each condition represent a minimum of duplicate determinations for each of three rats and are expressed as the means -C SE. Standard errors less than kO.05 are not listed.

ual mRNA levels at 5 h reflect intrinsic differences in magnitude of response rather than differences in half-lives of the mRNAs. Four of the five mRNAs are significantly increased by dibutyryl CAMP at both 1 and 5 h following initial injection. The fifth, ornithine transcarbamylase mRNA, shows an increase 1 h after injection and then declines below control values at 5 h. A comparison of mRNA levels at 1 and 5 h after administration of dibutyryl CAMP suggests that the half-lives of these mRNAs are probably on the order of an hour or less. The combined effects of dexamethasone and dibutyryl CAMP are additive for the mRNAs for carbamyl-phosphate synthetase I and argininosuccinate synthetase. In contrast, arginase mRNA is no more responsive to the combined hormones than to dibutyryl CAMP alone, while ornithine transcarbamylase mRNA is not responsive to the combination (Table II). Effect of dibutyryl CAMP on urea cycle enzyme gene transcription. The rapid increase in mRNA levels in response to dibutyryl CAMP prompted us to assess the effects of this agent on transcription rates

of these genes. Transcription run-on assays used nuclei isolated from rats treated as described in Table III. To ensure sufficient hybridizable counts for these assays, transcription rates were determined only for carbamyl-phosphate synthetase I and argininosuccinate synthetase genes. Based on relative signal intensities in Northern and dot-blot analyses, mRNAs for these two enzymes are nearly lo-fold more abundant than for the other three urea cycle enzymes. Transcription rate measurements for the phosphoenolpyruvate carboxykinase gene were also carried out as a positive control for the CAMP response. The transcription rate for carbamylphosphate synthetase I, argininosuccinate synthetase, and phosphoenolpyruvate carboxykinase increased four- to fivefold within 30 min following a single intraperitoneal injection of dibutyryl CAMP (Table III). Our results for transcription rates of the phosphoenolpyruvate carboxykinase gene agree very well with published values not only with regard to the fold increase effected by dibutyryl CAMP, but also in terms of actual transcription rates (Table III).

mRNA

LEVELS

FOR UREA

CYCLE

ENZYMES

IN RAT

LIVER

349

These results show that CAMP affects transcription rates of three different genes within the same tissue to the same extent. Furthermore, the magnitude of the increased transcription rates is sufficient to account for the increased steady-state levels found at 5 h following CAMP treatment (Table II). DISCUSSION

Effects of diet. Activities of the urea cycle enzymes are elevated in response to starvation and high-protein diets and reduced in response to low-protein or protein-free diets (l-9). The conclusion that altered enzyme activities reflect changes in enzyme mass rather than in catalytic efficiency has been verified for only four urea cycle enzymes and only during changes caused by varying content of protein in the diet (2, 20,21,25). All five urea cycle enzymes, under all conditions of dietary or hormonal manipulations, are generally assumed to be regulated by this mechanism. Changes in mRNA abundance as a function of diet largely parallel previously reported changes in enzyme activities (e.g., compare Fig. 1 of the present study with Fig. 1 of Schimke’s report (3)). However, there are some discrepancies. For example, the difference in enzyme activities per milligram protein between rats which had been starved or fed a protein-free diet is approximately lo- to 15-fold, except for arginase which showed only a 2.5fold difference (3). The values of mRNA abundance for ornithine transcarbamylase and argininosuccinate lyase fall within this range, but changes in mRNA abundance for the other three urea cycle enzymes, including arginase, are greater than 20-fold. The discrepancies are likely due to differences in experimental design or to failure in some eases to take into account the long halflives of the urea cycle enzymes (22,23,56). These discrepancies between mRNA levels and reported enzyme activities are relatively minor and do not alter the conclusions that diet-induced changes in enzyme levels primarily reflect regulation at a pretranslational step and that this regulation is largely coordinate for all five mRNAs.

350

MORRIS

Effects of diet on mRNA levels for carbamyl-phosphate synthetase I and ornithine transcarbamylase have previously been examined by two groups (25,26). Our results are in good agreement with studies comparing levels of carbamyl-phosphate synthetase I mRNA in rats fed a low- or protein-free diet versus a high-protein diet (25,26). However, in the only study which examined the effects of diet on levels of both mRNAs, Mori et aL (25) reported that starvation decreased levels of translatable mRNA for carbamyl-phosphate synthetase I and ornithine transcarbamylase-a finding in complete contrast to the increases in enzyme activity and to the results of the present study. The reason for this discrepancy is unclear, although measurement of translatable mRNA levels can be problematic in the absence of independent assessments of mRNA integrity. Effects of diet on mRNA abundance for the other three urea cycle enzymes have not previously been reported. The fact that levels of the urea cycle enzymes and their mRNAs are modulated in response to glucagon and glucocorticoids suggests that these hormones are likely to be important mediators of the adaptive response to diet. However, such findings do not exclude the possibility that certain dietary factors may also act directly as regulatory agents. Arginine mediates repression of argininosuccinate synthetase and argininosuccinate lyase activities in several mammalian cell lines (55-59). The repression of argininosuccinate synthetase occurs at a pretranslational level (59), and cisacting DNA sequences required for arginine-mediated repression have been located near the transcription initiation site of the human argininosuccinate synthetase gene (60, 61). Efects of glucagon or CAMP. Activities of all five urea cycle enzymes increase in response to glucagon treatment of intact rats (13,15). Glucagon also stimulates activities of several urea cycle enzymes in isolated rat hepatocytes (14,16). Messenger RNA levels for carbamyl-phosphate synthetase I (27-29) and ornithine transcarbamylase (29) are elevated by glucagon or CAMP analogs. Our results are consistent with most of

ET AL.

the previously reported observations on enzyme levels, with the exception of the results for ornithine transcarbamylase mRNA. Previous studies have reported increased activity (13,15) or mRNA level (29) for ornithine transcarbamylase in rats subjected to high levels of glucagon over a period of days. Thus, indirect effects due to long-term treatment with high levels of glucagon could not be ruled out. The short time periods in the present study should minimize possible secondary or indirect effects. Moreover, ornithine transcarbamylase activity could not be induced by glucagon alone in either of the studies which employed isolated rat hepatocytes (14,16), a finding consistent with the possibility that the glucagon-induced increases in the intact animal may have been due to indirect effects. The transient increase in ornithine transcarbamylase mRNA levels at 1 h after injection of dibutyryl CAMP, followed by a decline to slightly below control levels at 5 h, resembles the rapid rise and fall in mRNA levels for tyrosine aminotransferase in CAMP-treated rats (62). However, a conflicting report found that tyrosine aminotransferase mRNA levels remain elevated in animals receiving multiple injections of dibutyryl CAMP (63). Thus, the pattern of response for ornithine transcarbamylase remains something of an enigma. Except for ornithine transcarbamylase mRNA, the increase in mRNA level for the other four urea cycle enzymes is about twice the previously reported increases in enzyme activity or mRNA levels due to glucagon (ll-16,27-29). However, rats used in those studies were maintained on 1525% casein diets, resulting in basal levels of enzymes and mRNAs which are nearly twice those in rats maintained on a proteinfree diet, as was done in the present study. Thus, differences in diet could account for the twofold discrepancy. With the exception of ornithine transcarbamylase, our results indicate that the increased enzyme activities in response to glucagon are primarily due to corresponding increases in mRNA abundance. In rat liver, CAMP stimulates transcription of genes for phosphoenolpyruvate car-

mRNA

LEVELS

FOR UREA

CYCLE

boxykinase (50), tyrosine aminotransferase (63), and p33 mRNA (64), while inhibiting transcription of genes for L-type pyruvate kinase (65, 66) and a 5.4-kb mRNA for an unidentified protein (67). However, there are several cases where CAMP regulates mRNA levels at a post-transcriptional step(s). These include mRNAs for lactate dehydrogenase A (68), L-type pyruvate kinase (66, 69), and malic enzyme (70). The present study has demonstrated that transcription of at least two of the genes for urea cycle enzymes is stimulated by CAMP and that the degree of stimulation is approximately the same for carbamylphosphate synthetase I, argininosuccinate synthetase, and phosphoenolpyruvate carboxykinase. Since the increased transcription rates are sufficient to account for the increases in the corresponding mRNA levels seen 5 h after injection of dibutyryl c,4MP, these results argue that increased levels of carbamyl-phosphate synthetase I and argininosuccinate synthetase in response to CAMP are primarily due to regulation at the level of transcription. The rapid stimulation of transcription rate suggests that it is likely a primary effect of CAMP rather than a secondary effect due to synthesis of another gene product. Efects of g2ucocort&ids. Activities of the urea cycle enzymes are lowered in response to adrenalectomy (7, 8, 17, 18) and raised in response to glucocorticoid treatment (6, 8, 12, 14, 16-19). Levels of carbamyl-phosphate synthetase I mRNA are elevated in response to glucocorticoids (27-29), while the level of ornithine transcarbamylase mRNA shows a paradoxical decrease (29). We found that dexamethasone increased mRNA levels only for carbamyl-phosphate synthetase I, argininosuccinate synthetase, and arginase (Table II). This is at variance with reports showing increased activities for all five urea cycle enzymes in rat liver. However, previous investigators have generally measured responses to glucocorticoids a day or more following treatment, so the possibility of indirect effects could not be excluded. The absence of a dexamethasone effect on mRNA levels for ornithine transcarbamylase at 5 h and the actual decline in mRNA levels for this enzyme at 18-20 h

ENZYMES

IN RAT

LIVER

351

following injection (29) suggest that previously reported increases in enzyme levels are due to regulation at the translational or post-translational level. A similar argument applies to the discrepancy between enzyme activity and mRNA level for argininosuccinate lyase, although the possibility remains that mRNA levels for this enzyme may increase after longer duration of treatment due to indirect effects. The apparently additive effect of dexamethasone and dibutyryl CAMP on mRNA levels for two of the urea cycle enzymes raises the possibility that these agents may be acting by independent regulatory mechanisms to elevate the levels of these mRNAs. However, a firm conclusion cannot be drawn in the absence of additional data on time course and dose-response for these two hormones. Previous investigators employing primary cultures of rat hepatocytes have suggested that dexamethasone is primarily a permissive agent for the stimulatory action of glucagon on mRNA levels for the urea cycle enzymes (14). On the other hand, dexamethasone alone is sufficient to elevate activities of argininosuccinate synthetase and arginase (71,72), as well as synthesis rates and mRNA levels for carbamyl-phosphate synthetase I (7375) in rat hepatoma cell lines. Experiments using isolated rat hepatocytes cultured in a chemically defined medium are required to more precisely address the questions of whether glucocorticoids or CAMP are necessary and sufficient to increase mRNA levels for the urea cycle enzymes and to determine whether or not their effects are additive or synergistic. Coordinate regulation. The present study has shown that long-term changes in mRNA levels for the urea cycle enzymes induced by diet are largely coordinate but that short-term responses of these mRNAs to hormones are not. These results suggest that the coordinated dietary responses involve differential responses of individual mRNAs to multiple regulatory signals, including indirect effects which are not evident at short times after exposure to hormones. This suggestion is supported by recent studies using primary cultures of rat hepatocytes which demonstrate that mRNAs for the various urea cycle enzymes

352

MORRIS

clearly differ in their responses to several specific hormones (V. L. Nebes and S. M. Morris, Jr., unpublished work). Thus, regulatory mechanisms for modulating enzyme concentration are not identical for each of the enzymes in the pathway. Many studies involving coordinate gene expression in animal cells have been concerned with developmental transitions (e.g. (76,7’7)) rather than modulation of expression in fully differentiated cells. Moreover, many of these same studies have examined expression of secreted proteins (e.g. (77)) rather than the enzymes of a metabolic pathway, where there may be greater constraints on stoichiometric relationships. One example of stringent constraints on stoichiometric relationships in eukaryotes is afforded by expression of ribosomal proteins. Although ribosomal proteins accumulate coordinately, distinct steps are independently regulated for different ribosomal proteins in yeast (78). Thus, longterm regulation of urea cycle enzyme levels is achieved primarily by coordinate regulation of corresponding mRNA levels, but mRNA levels for the yeast ribosomal proteins are not coordinately regulated. Future experiments will determine whether the coordinate regulation of mRNA levels for urea cycle enzymes is the result of coordinate or independent regulation at different pretranslational steps. ACKNOWLEDGMENTS We thank Drs. Michael Stallup, David Loose, and Donald Back for advice on in z&o transcription assays; Julian Huang for assistance in the diet studies; Dr. Richard Hanson for providing the clone pPCK10; and Drs. Alan Goodridge and Robert Glew for helpful comments on the manuscript. REFERENCES 1. LIGHTBODY, H. D., AND KLEINMAN, A. (1939) J. Bid Chem. 129,71-‘76. 2. SCHIMKE, R. T. (1962) J. BioL Chem. 237,459-468. 3. SCHIMKE, R. T. (1962) .I Biol. Chem 237, 19211924. 4. MANDELSTAM, J., AND YUDKIN, J. (1952) Biochem. J. 51,681-686. ASHIDA, K., AND HARPER, A. E. (1961) Proc SC. Exp. BioL Med. 107.151-156. FREEDLAND, R. A., AND SODIKOFF, C. H. (1962) Proc. Sot. Exp. Biol Mea! 109,394-396. FREEDLAND, R. (1964) Proc Sot Exp. Biol. Med 116.692-696.

ET AL. 8. CHRISTOWITZ,D., MAITHEYSE, F. J., AND BALINSKY, J. B. (1981) Enzyme 26.113-121. 9. DAS, T. K., AND WATERLOW, J. C. (1974) Brit. J Nutr. 32,353-373. 10. FOLIN, 0. (1905) Amer. J. Physiol 13,66-115. 11. MCLEAN, P., AND NOVEL.LO, F. (1965) B&hem. .I 94,410-422. 12. SCHWARTZ, A. L. (1972) Biochem. J. 126,89-99. 13. SNODGRASS,P. J., LIN, R. C., MIJLLER, W. A., AND AOKI, T. T. (1978) J. Biol Ch.em. 253,2748-2753. 14. GEBHARDT, R., AND MECKE, D. (1979) Eur. J B&hem 97.29-35. 15. BREBNOR, L., PHILLIPS, E., AND BALINSKY, J. B. (1981) Enzyme 26,265-270. 16. LIN, R. C., SNODGRASS,P. J., AND RABIER, D. (1982) J. Biol. Chem. 257,5061-5067. 17. SCHIMKE, R. T. (1963) J. Biol Chem. 238, 10121018. 18. MCLEAN, P., AND GURNEY, M. W. (1963) Biodwm. J. 87,96-104. 19. LAMERS, W. H., ZONNEVELD, D., AND CHARLES, R. (1984) Deu. Biol. 105,500-508. 20. SAHEKI, T., KATSUNUMA, T., AND SASE, M. (1977) J. Biochem (Tokyo) 82,551-558. 21. SNODGRASS, P. J., AND LIN, R. C. (1981) J. Nutr. 111,586-601. 22. NICOLETTI, M., GUERRI, C., AND GRISOLIA, S. (1977) Eur. J. Biochem. 75,583-592. 23. SCHIMKE, R. T. (1964) J. Biol. Chem 239, 38083817. 24. TSUDA, M., SHIKATA, Y., AND KATSUNUMA, T. (1979) J. B&hem (Tokyo) 85,699-704. 25. MORI, M., MIURA, S., TATIBANA, M., AND COHEN, P. P. (1981) J. Biol. Chem 256,4127-4132. 26. RYALL, J., RACHUBINSKI, R. A., NGUYEN, M., ROZEN, R., BROGLIE, K. E., AND SHORE, G. C. (1984) J. Biol. Chum. 259,9172-9176. 27. DE GROOT, C. J., VAN ZONNEVELD, A. J., MOOREN, P. G., ZONNEVELD, D., VAN DEN DOOL, A., VAN DEN BOGAERT, A. J. W., LAMERS, W. H., MOORMAN, A. F. M., AND CHARLES, R. (1984) Biochem. Biophys. Res. Ccnnmun 124,882-888. 28. DE GROOT, C. J., ZONNEVELD, D., DE LAAF, R. T. M., DINGEMANSE, M. A., MOOREN, P. G., MOORMAN, A. F. M., LAMERS, W. H., AND CHARLES, R. (1986) B&him. Biophys. Acta 866, 61-67. 29 RYALL, J. C., QUANTZ, M. A., AND SHORE, G. C. (1986) Eur. J. Biochem 156,453-458. 30, VOLPE, J. J., AND VALGELOS, P. R. (1976) Physiol. Rev. 56,339-417. 31. WAKIL, S. J., STOOPS,J. K., AND JOSHI, V. C. (1983) Annu. Rev. B&hem. 52,537-579. 32. GOODRIDGE, A. G. (1985) in Molecular Basis of Insulin Action (Czech, M. P., Ed.), pp. 369-383, Plenum, New York. 33. WILSON, S. B., BACK, D. W., MORRIS, JR., S. M., AND GOODRIDGE, A. G. (1986) J. Biol Chem 261, 15179-15182.

mRNA

LEVELS

FOR UREA

CYCLE

34. LALLEY, P. A., AND MCKUSICK, V. (1985) in Eighth International Workshop on Human Gene Mapping; Cytogenet. Cell Genet. 40,536-566. 35. ADCOCK, M. S., AND O’BRIEN, W. E. (1984) J. BioL Chem 259,X3471-13476. 36. DIZIKES, G. J., SPECTOR, E. B., AND CEDERBAUM, S. D. (1986) Somat.CeU Mol. Genet. 12,375-384. 37. YOO-WARREN, H., MONAHAN, J. E., SHORT, J., SHORT, H., BRUZEL, A., WYNSHAW-BORIS, A., MEISNER, H. M., SAMOLS, D., AND HANSON, R. W. (1983) Proc N&L Ad Sci USA 80,36563660. 38. Su, T.-S., BOCK, H.-G., O’BRIEN, W. E., AND BEAUDET, A. L. (1981) J. BioL Chem 256,11826-11831. 3!1. NUSSBAUM, R. L., BOGGS, B. A., BEAUDET, A. L., DOYLE, S., POTTER, J. L., AND O’BRIEN, W. E. (1986) Amer. J. Human Genet. 38,149-158. 40. O’BRIEN, W. E., MCINNES, R., KALUMUCK, K., AND ADCOCK, M. (1986) Proc. NatL Acad Sci. USA 83,7211-‘7215. 41. MORI, M., MORRIS, S. M., JR., AND COHEN, P. P. (1979) Proc N&L Acad Sci USA 76,3179-3183. 42. CHIRGWIN, J. M., PRZYBLA, A. E. , MACDONALD, R. J., AND RUTTER, W. J. (1979) Biochemistry l&5294-5299. 43. GOODRIDGE, A. G., JENIK, R. A., MCDEVIT, M. A., MORRIS, S. M., JR., AND WINBERRY, L. K. (1984) Arch. Biochem Biophys. 230,82-92. 44. CHURCH, G. M., AND GILBERT, W. (1984) Proc NatL Acad Sci. USA 81,1991-1995. 45. LEHRACH, H., DIAMOND, D., WOZNEY, J. M., AND BOEDTKER, H. (1977) Biochemistry 16, 47434751. 46. DERMAN, E., KRAUTER, K., WALLING, L., WEINBERGER, C., RAY, M., AND DARNELL, J. E. (1981) Cell 23, 731-739. 47. RIGBY, P. W. J., DIECKMAN, M., RHODES, C., AND BERG, P. (1977) J. Mol. BioL 113,237-251. 48. FEINBERG, A. P., AND VOGELSTEIN, B. (1983) Anal. B&hem. 132, 6-13. 49. AMASINO, R. M. (1986) Anal. Biochem. 152, 304307. 50. LAMERS, W. H., HANSON, R. W., AND MEISNER, H. M. (1982) Proc Nut1 Acud Sci USA 79,51375141. 51. MARZLUFF, W. R., AND HUANG, R. C. C. (1984) in Transcription and Translation. A Practical Approach (Hames, B. D., and Higgins, S. J., Eds.), pp. 89-129, IRL Press, Oxford. 52. WAGNER, F. K., KATZ, L., AND PENMAN, S. (1967) Biochem Biophys. Res. Commun. 28,152-159. 53. TURCOTTE, B., GUERTIN, M., CHEVRETTE, M., AND BE’LANGER, L. (1985) Nucleic Acids Res. 13, 2387-2398. 54. LIS, J. T., NECKAMEYER, W., DUBENSKY, R., AND COSTLOW,N. (1981) Gene 15,67-80. 55. SCHIMKE, R. T. (1962) Biochim. Biophys. Acta 62, 599-601.

ENZYMES

IN RAT

LIVER

353

56. SCHIMKE, R. T. (1964) J. BioL Ch,em. 239,136-145. 57. IRR, J. D., AND JACOBY, L. B. (1978) Somatic Cell Genet. 4,111-124. 58. HUDSON, L. D., ERBE, R. W., AND JACOBY, L. B. (1980) Proc. Nat1 Acad Sci. USA 77,4234-4238. 59. Su, T.-S., BEAUDET, A. L., AND O’BRIEN, W. E. (1981) Biochemistry 20,2956-2960. 60. BOYCE, F. M., ANDERSON, G. M., AND RUSK, C. D., AND FREYTAG, S. 0. (1986) Mel CelLBid 6,12441252. 61. JACKSON, M. J., O’BRIEN, W. E., AND BEAUDET, A. L. (1986) Mol. Cell Biol 6,2257-2261. 62. NOGUCHI, T., DIESTERHAFT, M., AND GRANNER, D. K. (1982) J. BioL Chem 257,2386-2390. 63. HASHIMOTO, S., SCHMID, W., AND SCHIJTZ, G. (1984) Proc. NatL Acad. Sti USA 81,6637-6641. 64. LEE, L.-L., ISHAM, K. R., STRINGFELLOW, L., ROTHROCK, R., AND KENNEY, F. T. (1985) J. BioL Chem. 260,X433-16438. 65. VAULONT, S., MUNNICH, A., MARIE, J., REACH, G., PICHARD, A. L., SIMON, M. P., BESMOND, C., BARBRY, P., AND KAHN, A. (1984) Biochem Bie phys. Res. Commun 125,135-141. 66. VAULONT, S., MUNNICH, A., DECAUX, J. F., AND KAHN, A. (1986) J. BioL Chem. 261,7621-7625. 67. PICHARD, A. L., MUNNICH, A., MEIENHOFER, M. C., VAULONT, S., SIMON, M. P., MARIE, J., DREYFUS, J. C., AND KAHN, A. (1985) Biochem. J. 226,637644. 68. JUNGMANN, R. A., KELLEY, D. C., MILES, M. F., AND MILKOWSKI, D. M. (1983) J. BioL Chem 258, 5312-5318. 69. NOGUCHI, T., INOUE, H., AND TANAKA, T. (1985)5. BioL Chem. 260,14393-14397. 70. BACK, D. W., WILSON, S. B., MORRIS, JR., S. M., AND GOODRIDGE, A. G. (1986) J. BioL Chem. 261, 12555-12561. 71. HAGGERTY, D. F., SPECTOR,E. B., LYNCH, M., KERN, R., FRANK, L. B., AND CEDERBAUM, S. D. (1982) J. BioL Chem. 257,2246-2253. 72. HAGGERTY, D. F., SPE(;TOR,E. B., LYNCH, M., KERN, R., FRANK, L. B., AND CEDERBAUM, S. D. (1983) MoL Cell. Biochem53/54,57-76. 73. MURAKAMI, A., KITAGAWA, Y., AND SUGIMOTO, E. (1983) Biochim. Biqphys. Acta 740,38-45. 74. KITAGAWA, Y., AND SUGIMOTO, E. (1985) Eur. J. Biochem. 150,249-254. 75. KITAGAWA, Y., RYALL, J., NGUYEN, M., AND SHORE, G. C. (1985) B&him. Biophys. Acta 825, 148153. 76. GREENGARD, 0. (1971) in Essays in Biochemistry (Campbell, P. N., and Dickens, F., Eds.), Vol. 7, pp. 159-205, Academic Press, New York. 77. HAN, J. H., RALL, L., AND RUTTER, W. J. (1986) Proc. NatL Acad Sci. USA 83.110-114. 78. WARNER, J. R., MITRA, G., SCHWINDER, W. F., STUDENY, M., AND FRIED, H. M. (1985) MoL Cell. BioL 5, 1512-1521.