EXPERIMENTAL

CELL

RESEARCH

201,

514-516

(1992)

Effects of Glucagon and an Analogue of CAMP on Tyrosine Aminotransferase in Isolated Chick Embryo Hepatocytes I. 0. Department

of Biochemistry,

ONOAGBE’ University

AND

A. J. DICKSON

of Manchester,

Manchester

Ml3

SPL,

MATERIALS

Hepatocytes were isolated from 17-day-old chick embryos by the use of collagenase. Glucagon and dibutyryl CAMP (bt,cAMP), individually or in combination, stimulated tyrosine aminotransferase (TAT) activity and synthesis in the isolated hepatocytes; maximal stimulation occurred 4 h after exposure of hepatocytes to the inducers. The stimulatory effects produced by glucagon and b&CAMP were abolished by treatment of hepatocytes with cordycepin or cycloheximide. The effects of the hormone and the cyclic nucleotide were not additive. The induction of the enzyme by glucagon suggests a physiological role for the hormone in the expression of TAT activity during chick embryonic development. 0 1992 Academic Press, Inc.

Tyrosine aminotransferase (TAT) (E.C. 2.6.1.5) catalyzes the first and rate-limiting reaction in the degradation of tyrosine in the liver [l]. In rodents hepatic TAT is stimulated by several factors including glucocorticosteroids and pancreatic hormones. Hormonal regulation of the enzyme in rat liver has been extensively investigated [ 21. Although there are published reports of TAT induction by glucocorticoids in chick embryo and adult chicken livers [3-51, very little is known about the stimulation of this enzyme by other inducers such as glucagon and CAMP in chick embryos. Glucagon appears in the plasma of embryos before the sixth day of development [6, 71 and the receptors for the hormone are functional at this early stage of embryonic life [8]. The presence of functional receptors for glucagon in embryo liver would suggest that the hormone might be an important physiological regulator of TAT during embryonic development. This paper examines the effects of glucagon and dibutyryl CAMP on TAT catalytic activity and synthesis in isolated chick embryo hepatocytes.

0014.4827/92 Copyright All rights

$5.00 0 1992 by Academic Press, of reproduction in any form

be adP.M.B.

514 Inc. reserved.

AND

Kingdom

METHODS

Fertilized white leghorn eggs Sources of animals and materials. (Gallus domesticus, Thornber strain) were obtained from the University of Manchester Medical School and used after 17 days of incubation (3 days prior to hatching). Glucagon, cordycepin, cycloheximide, collagenase, ATP, and firefly lantern extract (FLE-50) were obtained from Sigma Chemical Co. Ltd., (Poole, Dorset, IJK). Dibutyryl CAMP (bt,cAMP) was from Boehringer Corp. (Lewes, Sussex, UK). L-[4,5-3H]Leucine and L-[side chain-2,3-3H]tyrosine were purchased from Amersham International (Amersham, Bucks, UK). Eagle’s minimum essential medium (MEM) was obtained from Flow Laboratories (Irvine, Ayrshire, Scotland, UK). The stock MEM was diluted IO-fold and supplemented with 24.0 mM sodium hydrogen carbonate and 2.0 mM glutamine just before use. Bovine serum albumin (BSA) was purchased from International Enzymes Ltd. (Windsor, Berks, UK). It was defatted and dialyzed before use [9]. All other chemicals (Analar grade) were obtained from standard suppliers. Krebs-Henseleit bicarbonate buffer (KHB), without Ca”, was prepared just prior to use [lo]. A specific anti-TAT antiserum was raised against purified rat liver TAT by the method described earlier [ll]. Hepatocyte isolation. Embryos were eased out through the blunt end of the egg shell and decapitated immediately. Livers (6-8 g wet wt) from 16 to 20 embryos were pooled and minced in an iced beaker. The minced tissue was rinsed with 10 ml of gassed KHB (without Ca”) containing 1 mg collagenase/ml into a loo-ml flask. The flask was flushed for 2 min with a mixture of 95% 0, and 5% CO,, stoppered, and incubated at 37°C with shaking (100 cycles/min) for 15-20 min. At the end of the digestion period, the suspension was filtered through nylon mesh (pore size, 150 pm) into a polypropylene beaker. The retained material was dispersed in 10 ml of KHB and filtered for a second time. The filtrate was centrifuged at 125g for 3 min and the supernatant was removed by aspiration. The pellet was washed by successive centrifugations (75g, 3 min; 5Og, 2 min; 25g, 2 min). After each centrifugation step the supernatant was aspirated and the pellet was resuspended in 20 ml of KHH (without Ca*+). The pellet obtained in the final centrifugation step was resuspended in MEM (final resuspension medium) and supplemented with 1% (w/v) BSA and 0.2 mM Ca2+ (final concentrations). For measurement of protein or TAT synthesis, the hepatocytes were resuspended in supplemented MEM containing 0.4 mM L-[4,5-3H]leucine (final specific radioactivity, 25 Ci/mol). Hepatocyte incubation. Portions (0.5 ml) ofthe hepatocyte suspensions were added to 7.ml polypropylene vials in the presence of inducers or inhibitors. Control incubations were without additions. All concentrations of effecters used in this study were those producing maximal effects on TAT (determined in preliminary experiments). The vials were stoppered, gassed for 2 min with a mixture of 95% 0, and 5% CO,, and incubated at 37°C with shaking (100 cycles mini) for the desired time. Analytical procedures. ATP content of deproteinized and neutralized hepatocyte extracts was analyzed by chemiluminescence 1121.

INTRODUCTION

1 To whom correspondence and reprint requests should dressed at Department of Biochemistry, University of Benin, 1154, Benin City, Nigeria.

United

GLUCAGON

TABLE

AND

CAMP

EFFECTS

(2.87 X 10e6 M) (2.88 x 1O-4 M) plus bt,cAMP

202.8 188.1 190.3

TAT

2

Effects of Cycloheximide and Cordycepin on the Induction of TAT by Glucagon or b&CAMP

activity

f 15.4 f 10.0 f 8.9

Note. Values are expressed as percentages of control TAT at 4 h of incubation and are means f SEM of six independent ments.

activity experi-

TAT activity of hepatocytes was determined by a radiometric assay [13]. First, the incubations were stopped by centrifugation (5Og, 2 min). The cell pellets were resuspended in 0.2 ml (per incubation) of phosphate buffer (50 mM potassium dihydrogen orthophosphate, pH 7.6, containing 3 mM P-oxoglutarate, 1 mMEDTA, 1 mA4 dithiothreitol, and 0.2 mM pyridoxal5’-phosphate). The hepatocyte suspensions were then lysed by three cycles of freezing and thawing, followed by centrifugation (12,OOOg, 10 min) to provide a supernatant for analysis of TAT activity. Synthesis of TAT was measured by incorporation of radiolabeled leucine into immunoprecipitable TAT. TAT was extracted from cells as described above and mixed with 300 mU of carrier TAT in the presence of sufficient anti-rat TAT antiserum to precipitate 450 mU TAT. Immunoprecipitates were washed extensively and, after solubilization with nuclear Chicago solubilizer, counted for radioactivity [14]. Total protein synthesis was measured as described previously [14]. Expression of results. Data are expressed as means + SEM for the number of experiments indicated unless otherwise stated. In some cases the results are presented as percentages of control TAT activity at 4 h of incubation. In this study control TAT activity at 4 h of incubation was in the range of 0.4-0.5 U (g wet wt liver)-‘.

AND

515

AMINOTRANSFERASE

TABLE

% of control

RESULTS

TYROSINE

1

Effects of Glucagon and b&CAMP (Singly or Combined) on TAT Activity in Isolated Hepatocytes

Glucagon bt,cAMP Glucagon

ON

DISCUSSION

% of control Glucagon (2.87 X 10-a M) bt,cAMP (2.88 x 10m4 M) Cycloheximide (20 pg/ml) Cordycepin (20 @g/ml) Glucagon plus cycloheximide b&CAMP plus cycloheximide Glucagon plus cordycepin bt,cAMP plus cordycepin Note. Values are percentages bation and are means k SEM

TAT

activity

197.3 xi 7.1 189.6

88.3 81.0 78.9 79.7 83.4 80.2

i- 6.2

i i i -t -t k

2.4 4.3 2.5 4.1 3.9 1.8

of control TAT activity at 4 h of incuof four independent experiments.

physiological range [15]. Thus, the isolated chick embryo hepatocyte preparations are viable. Treatment of the cells with glucagon and bt,cAMP, singly or in combination, resulted in an approximately twofold increase in TAT activity; the effects of the hormone and the cyclic nucleotide were not additive (Table 1). TAT activity was maximally stimulated in 4 h following exposure of the hepatocytes to glucagon or b&CAMP (Fig. 1). Addition of cycloheximide or cordycepin (individually) to the glucagon- or b&CAMP-treated hepatocyte suspensions prevented the stimulation of TAT activity produced by either inducer (Table 2). This result suggests that continued synthesis of protein and mRNA is necessary for the induction of TAT activity by glucagon and the analogue of CAMP. The inhibitors (individually) also decreased basal TAT activity by about 20% relative to controls (Table 2). Inhibition of basal TAT

Intracellular values of ATP (6-7 nmol/mg dry it) obtained at all stages of incubation (l-6 h) are within the zoot

Time(h)

FIG. 1. Time course of TAT stimulation by glucagon or b&CAMP. Isolated hepatocytes were incubated in the presence of 2.87 X 10-s M glucagon (0) or 2.88 X lo-’ Mb&CAMP (0). Results from a single representative experiment are shown. Values are percentages of control TAT activity at each time of incubation and each point is the mean f SD of triplicate determinations.

Time(h)

FIG. ^~~

2.

Effects of glucagon or b&CAMP on incorporation of into TAT. Isolated hepatocytes were incubated with 2.87 glucagon (0) or 2.88 X 10e4 M bt,cAMP (0) or under control (A). At the end of indicated incubation periods, hepatocytes were analyzed for radioactivity incorporated into TAT. Results are the means +- SEM of three independent experiments. [“Hlleucine X 10-a M conditions

516

ONOAGBE

AND

600

v

* 12

3 G Time(h)

5

6

FIG. 3. Effects of glucagon or b&CAMP on incorporation of [3H]leucine into total proteins. Hepatocyte incubations were performed under control conditions (m) or in the presence of 2.87 X 1Om6 M glucagon (A) and 2.88 x 10F4 Mb&CAMP (A). At the end of indicated incubation times, cell suspensions (50 ~1) were analyzed for radioactivity incorporated into total proteins. Values are the means f SEM of four separate experiments. Error bars were less than 7% of mean values and are omitted for clarity.

activity (about 50%) by cordycepin in rat hepatome cells [16, 171 and in isolated adult rat hepatocytes [18] has been demonstrated. The mechanism of the inhibition of basal enzyme activity by cordycepin or cycloheximide is not known. It would appear that their effects are due either to a constant degradation of TAT while its synthesis is blocked or to a generalized toxicity on the hepatocyte. To investigate the relationship between TAT catalytic activity and synthesis, the isolated hepatocytes were incubated in MEM containing L-[4,5-3H]leucine in the presence of glucagon or bt,cAMP. Incorporation of radiolabeled leucine into TAT and subsequent immunoprecipitation showed TAT synthesis to be low under control conditions; either inducer produced about a threefold increase in TAT synthesis (Fig. 2). Glucagon or bt,cAMP selectively increased TAT synthesis as incorporation of radioactive leucine into total proteins was inhibited by each inducer (Fig. 3). Cordycepin abolished the stimulated increase in TAT synthesis by glucagon or bt,cAMP (results not shown). The increase in TAT synthesis produced by glucagon and bt,cAMP would appear to account for the twofold induction of TAT catalytic activity by the inducers. The mimicking of the effects of glucagon on TAT activity and synthesis by bt,cAMP is consistent with CAMP acting as the intracellular messenger of the hormone. It has, indeed, been demonstrated that exposure of isolated chick embryo hepatocytes to glucagon results in a rapid increase in CAMP concentrations [19]. It appears likely that glucagon has a physiological role to Received

February

25,1992

DICKSON

play in the expression of TAT activity in chick development. Glucagon can increase hepatic TAT activity after injection to chicks in uiuo [4] and it has been reported that plasma concentrations of glucagon increase at hatching [6]. We have shown previously that there is a transient threefold increase in TAT activity soon after hatching [ 201.In adult and fetal rats the glucagon-stimulated increase in TAT activity and synthesis is due to a corresponding selective increase in the concentration of TAT mRNA relative to total mRNA content [21, 221. This is an indication that glucagon specifically enhances the transcription of TAT mRNA. The inhibition of glucagon-induced increase in TAT catalytic activity and synthesis by cordycepin in the isolated hepatocytes suggests that the hormone may exert its action at the level of transcription in chick embryos. REFERENCES 1.

Dickson,

A. J., Marston,

F. A. O., and Pogson,

C. I. (1981)

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Lett. 127, 28. 2. 3.

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Thommes, R. C., and Firling, C. E. (1964) Gen. Comp. Endocrinol. 4, 1. Chen, R. F. (1967) J. Biol. Chem. 242, 173. Krebs, H. A., and Henseleit, K. (1923) Hoppe-Sey1erS.Z Physiol. Chem. 210,33.

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Rinaudo, M. T., Ponzetto, C., and Curto, M. (1976) Int. J. Biothem. 7, 239. Butcher, F. R., Bushnell, D. E., Becker, J. E., and Potter, V. R. (1972) Exp. Cell Res. 74, 115. Dethlefsen, L. A. (1975) J. Cell. Physiol. 86, 155. Marston, F. A., Dickson, A. J., and Pogson, C. I. (1981) Mol. Cell. Biochem. 34,59. Onoagbe, I. 0. (1985) PhD thesis, University of Manchester, England. Onoagbe, I. O., and Dickson, A. J. (1986) Ann. N. Y. Acad. Sci. 478,300. Perry, S. T., Rothrock, R., Isham, K. R., Lee, K., and Kenney, F. T. (1983) J. Cell. Biochem. 21,47. Hashimoto, S., Schmid, W., and Schutz, G. (1984) Proc. N&l. Acad. Sci. USA 81. 6637.

Effects of glucagon and an analogue of cAMP on tyrosine aminotransferase in isolated chick embryo hepatocytes.

Hepatocytes were isolated from 17-day-old chick embryos by the use of collagenase. Glucagon and dibutyryl cAMP (bt2cAMP), individually or in combinati...
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