Molecular and Cellular Endocrinology.

@Elsevier/North-Holland

9 (1977) 205 Scientific Publishers, Ltd.

2 I4

EFFECT OF GLUCOCORTICOIDS ON AMINO ACID TRANSPORT ISOLATED RAT HEPATOCYTES *

IN

Alphonse LE CAM ** and Pierre FREYCHET Institut National de la Sante’et de la Recherche Mkdicale, Groupe de Recherchrs sur les Hormones Polypeptidiques et la Physiopathologie Endocrinienne (INSERM I/ 1451, Facultk de MPdecine (Pasteur). Chemin de Vallombrose, 06034 Nice C&dex. France

Received 27 June 1977; accepted 29 July 1977

The effects of glucocorticoids on neutral amino acid transport were studied in freshly prepared suspensions of isolated hepatocytes from adult rat. Glucocorticoids stimulated the active transport of or-aminoisobutyric acid by increasing the influx. The onset of effect was preceded by a lag period of about 1 h. Cortisol (ED,, - 2 PM) was lo-20 times less potent than synof transport without affecting the Km. thetic analogs. Glucocorticoids increased the V,,, Actinomycin D and cycloheximide virtually abolished this effect. The glucocorticoid effect involved predominantly the A system of transport. The effect was additive with that of glucagon, insulin and catecholamines. Keywords.

amino acid transport; isolated hepatocytes;

glucocorticoids;

hormone action.

The liver is an important target for glucocorticoids. Hydrocortisone has been found to increase protein synthesis in the liver (Clark, 1953; Goodlad and Munro, 1959) and the induction of specific enzyme synthesis in the rat liver has been thoroughly investigated (Baxter and Tomkins, 1970; Feigelson and Feigelson, 1965; Goldstein et al., 1962). It is well established that glucocorticoid treatment in vivo enhances the amino acid concentration in plasma (Kaplan and Shimizu, 1963) and in liver (Clark, 1953; Kaplan and Shimizu, 1962). Although a stimulatory effect of hydrocortisone on oaminoisobutyric acid (AIB) uptake in the isolated perfused liver has been reported (Chambers et al., 1965) the data did not demonstrate that glucocorticoids influence directly the amino acid transport systems in the hepatocyte. It has recently been reported in primary cultures of adult rat hepatocytes that dexamethasone alone either decreases (Bonney and Maley, 1975) or is without effect on AIB trans-

* Thus work was supported by Grants 75.7.1366 and 77.7.0247 from the Delegation G&&ale a la Recherche Scientifique et Technique and by the Fondation pour la Recherche Medicale Francaise. ** To whom all correspondence should be addressed. 205

206

A. Le Cam and P. Freychet

port but exerts a permissive effect on the glucagon (Kletzien et al., 1975) and catecholamines (Pariza et al., 1977) induction of AIB transport. We have recently characterized the neutral amino acid transport systems that are operative in freshly isolated hepatocytes from the adult rat (Le Cam and Freychet, 1977a). Only the A system of transport was found to be sensitive to glucagon (Le Cam and Freychet, 1976) and to catecholamines (Le Cam and Freychet, 1977b). The present study has shown that glucocorticoids specifically stimulate the A system of transport through a mechanism dependent on protein synthesis. Furthermore, this effect parallels the glucocorticoid and anti-inflammatory potency.

MATERIALS

AND METHODS

Isolated hepatocytes were prepared from 6--8-week-old male Wistar rats following the procedure previously described (Le Cam et al., 1976). The animals were maintained on a 12 h light/l2 h dark cycle (the light period starting at 08.00 h) and had free access to a laboratory chow containing 21% protein, 4% fat and 51% carbohydrate, until the time of sacrifice (09.00-l 1 .OOh). Freshly prepared hepatocytes have been demonstrated to retain well-preserved ultrastructure (Le Cam et al., 1976) metabolic capabilities (Le Cam et al., 1976) and active transport systems for amino acids (Le Cam and Freychet, 1977a). The intracellular levels of ATP (15 -20 nmol/106 cells) and Kt (130-l 50 meq/liter of cell water) remained stable over 3-4 h incubation periods at 37°C. AI1 experiments were carried out at 37°C in Krebs-Ringer bicarbonate buffer containing 1% (w/v) bovine serum albumin (Fraction V), gentamycin (50 pg/ml) and gassed with 5% CO,-95% 02. Cells at about 1.5 X 106/ml were incubated with various agents and a mixture of labelled (0.05-0.1 pCi/tube) and unlabelled amino acid as indicated in the legends to the figures. The analytical procedure employed to measure transport and the mode of expression of the results have been previously described (Le Cam and Freychet, 1976, 1977a). Unless otherwise stated, the experiments designed to compare the effects of several compounds were performed with the same cell preparation. All experiments were repeated at least twice. Hydrocortisone, dexamethasone, prednisolone, triamcinolone, deoxycorticosterone, cycloheximide, L(-)epinephrine, a-aminoisobutyric acid and cycloleucine were purchased from Sigma. Actinomycin D was from Boehringer Mannheim Co. Glucagon and insulin were pork monocomponents from Novo. The ammo acid analog a-(methylamino)isobutyric acid (N-methyl-AIB) was a gift of H.N. Christensen (Ann Arbor, Mich., U.S.A.); a-amino [ 1-14C] isobutyric acid and 1-aminocyclopentane [ 1-14C Jcarboxylic acid (cycloleucine) were purchased from the Radiochemical Centre (Amersham, U.K.).

~~~cocorr~co~deffect on amino acid transport

207

RESULTS Effect of glucocorticoids on the time-course of AIB uptake

Cortisol and dexamethasone at 10 PM enhanced by about 40% both the initial velocity and the steady-state of transport of AIB in isolated hepatocytes (fig. I). This strongly suggests that the ~ucocorticoid effect is restricted to the influx of AIB. Direct evidence of specific action on influx was obtained by testing the effect of glucocorticoids on AIB efflux. Fig. 2 shows the fractional efflux of AIB from cells treated with dexamethasone or cortisol, and from control cells. The data fitted the same single straight line, indicating a first-order dependence of the efflux upon time and the lack of glucocorticoid effect on the efflux. dependence of st~mu~tion of AIB transport on the time of pree~po~re to glucoeorticoids No effect of glucocorticoids on AIB transport was seen within 1 h of ceil expo-

sure to the steroid; the maximal effect was achieved only after 2-3 h exposure (fig. 3). A lag period of about 1 h has also been observed for the effect of cortisol on

BASAL

, 0

60

,

, 120

TIME , min

Fig. 1. Effect of glucocorticoids on the time-course of AIB uptake. Suspensions of hepatocytes were preincubated for 3 h in the absence (basal) and presence of cortisol or dexamethasone. The uptake of 2 mM [ 14C]AIB was then measured at the times indicated. The distribution ratio was calculated by dividing the intracellular concentration of AIB by the extracellular concentration; intracellular water (-4 &lo6 cells) was determined in the same cell preparation. Each point is the mean t SEM of 4 determinations.

BASAL

e-e

10L,

Q-V

CORTISOL

A,-*,

DEXAMETHASONE.~~$I

,50pM

i 30

0

60

90

TIME ,min

Fig. 2. Effect of glucocorticoids on AIB efflux. Suspensions of hepatocytes were preincubated for 1 h in the absence or presence of glucocorticoids; 2 mM [ 14C]AIB was then added and uptake was allowed to proceed for 2 h. Cells were then sedimented by centrifugation and immediately resuspended (time 0 on figure) in AIB-free medium, with or without glucocorticoid at the same concentration as in the fist incubation period, The amount of AIB remaining in cells was determined at the times indicated on quadruplicate samples.

0

1 TIME

2 of

EXPOSURE,

3

hours

Fig. 3. ,Effects of duration of cell preexposure to glucocorticoid on AIB influx. Suspensions of ,hepatocytes were incubated in the absence or presence of glucocorticoid. At the times indic&ted transport was measured by incubating aliquot samples with 2 mM (I 4C]AIB for 15 min. Each value is the mean t SEM of 4 determinations.

Glucocorticoid

effect on amino acid transport

209

AIB uptake by the isolated perfused rat liver (Chambers et al., 1965). Such a time dependence of glucocorticoid effect is analogous to that observed for the catecholamine stimulation of AIB transport (Le Cam and Freychet, 1977b), but differs from that of glucagon; with the latter, the onset of effect was rapid (Le Cam and Freychet, 1976) although its full expression also required about 2 h (Freychet and Le Cam, 1977). Dose-response relationships for glucocorticoids The synthetic glucocorticoids tested were found to be more potent than the natural hormone in stimulating AIB transport in isolated hepatocytes (fig. 4). Thus, cortisol (EDso - 2 PM) was 1O-20 times less potent than prednisolone, dexamethasone or triamcinolone (ED,, - 0.1-0.2 PM). Because of some interexperimental variations it was not possible to delineate a precise order of potency for the synthetic glucocorticoids which were found, however, consistently more potent than cortisol. Of particular interest was the lack of effect of deoxycorticosterone (at l50 PM) on AIB transport (not illustrated). Effect of inhibitors of RNA and protein synthesis on basal and glucocorticoidstimulated AIB transport As previously observed (Le Cam and Freychet, 1976, 1977b; Freychet and Le

[ GLUCOCORTICOID] , -log M

Fig. 4. Dose responses of glucocorticoid stimulation of AIB influx. Hepatocytes were preincubated for 3 h in the presence of varying concentrations of glucocorticoid before the addition of 2 mM [ 14C]AIB. Amino acid influx was then measured after 15 min. Each point is the mean f SEM of 5 separate experiments for cortisol, and of 3 separate experiments for the other compounds; within a single experiment, each point was done in quadruplicate.

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A. Le Cam and P. Freychet

c

30-

wathout steroid

Dexamathasone 100

VM

Fig. 5. Effect of actinomycin D and cycloheximide on basal and glucocorticoid-stimulated AIB transport. Hepatocytes were incubated for 30 min in the absence or presence of actinomycin D or cycloheximide prior to the addition of glucocorticoid. Incubations were continued for 2.5 h; 2 mM [ r‘kT]AIB was then added and transport was measured after 15 min. Each bar is the mean f SEM of 6 determinations.

Cam, 1977), actinomycin D and cycloheximide treatment of cells decreased the basal rate of AIB transport (fig. 5). Actinomycin D at 1 pg/ml inhibited the glucocorticoid stimulation of AIB transport by 90-100%; under this condition, RNA synthesis and protein synthesis were inhibited by 95% and 4%, respectively. Cycloheximide at 0.1 mM (a concentration which inhibited 90% of protein synthesis in isolated hepatocytes) virtually abolished the glucocorticoid effect (fig. 5). Effect of dexamethasone on the kinetic parameters of AIB transport The nonsaturable, sodium-independent component of AIB transport was not component affected by dexamethasone (fig. 6). The VrnaXof the sodium-dependent of AIB transport (-120 nmol/106 cells - 15 min) was increased by dexamethasone (-230 nmol/106 cells . 1.5 min). In contrast, the apparent K, (-20 mM) was not altered (fig. 6). Effect of glucocorticoids on transport systems AIB is transported in isolated hepatocytes through the A and ASC systems (Le Cam and Freychet, 1977a). When AIB influx was measured in the presence of an excess of N-methyl-AIB, a condition under which the entry of AIB through the A

Glucocorticoid effect on amino acid transport

211

saturable C~polWlt

[Ale]

, mM

Fig. 6. Effect of dexamethasone on kinetic parameters of AIB transport. Hepatocytes were preincubated for 3 h in a sodium-containing medium, and in the absence or presence of dexamethasone. Cells were then sedimented by centrifugation, washed twice and resuspended in medium with or without dexamethasone. [ 14C]AIB was then added to give the final concentrations indicated, and transport was measured after 15 min. Sodium was replaced by choline when a sodium-free medium was required during transport &says. Closed symbols represent dexamethasone-treated cells; open symbols represent control cells. The upper and lower dotted lines represent the saturable components of transport calculated for dexamethasone-treated and control cells, respectively, by subtracting from the total the transport measured in the absence of sodium. Each point is the mean + SEM of 4 determinations.

system is largely inhibited, the stimulatory effect of glucocorticoids was no longer observed (fig. 7). This indicates that glucocorticoids specifically stimulate the A system of transport. The saturable transport of cycloleucine occurs through the L system for about 80% and through the A and ASC systems for about 20%. The contribution of the L mediation for the transport of cycloleucine can be evaluated by inhibiting the entry through the A and ASC systems by an excess of AIB, or by measuring transport in a sodium-free medium. As shown in fig. 7, dexamethasone and cortisol did not appear to significantly affect the L system of transport specifically measured under these two conditions. Combined

effects of glucocorticoids

with glucagon, insulin and epinephrine

No evidence for a permissive effect of dexamethasone was found when maximally stimulating concentrations of the steroid and glucagon were combined; the effects were additive (fig. 8). Similarly, dexamethasone and epinephrine on the one

212

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A. I,e Cam and P. Frey&et

10

I-

CYCLOLEUCINE

1

.‘.Z .:.;

i

.

:.:.: ii.

::::: ::::: ..i. ..:.

:::::

. . ... . .. .. :::::

*>:. :.::: :::.:

. ..‘. :::::

.y.

*‘. . ..i. . .

+N -Methyl t t.-

1OmM Na’

MEDIUM-I

1

cAlf33Oti

AIEI ,

t CHOLINE MEDIUM

c HOLINE TEDIUM

Na+ MEDIUM

Fig. 7. Effect of glucocorticoids on amino acid transport systems. Cells were incubated for 3 11 with or without cortisol or dexamethasone, in a sodium-containing medium. After washing twice, cells were resuspended in buffer wrth or without sodium and with or without glucocorticoid. AIB (0.25 mM) influs was then measured in the absence or presence of 10 mM N-methyiAIB. Cycloleucine influx was measured in sodium-free medium, or in sodium-containing medium in the presence of 30 mM AIB. Each value represents the mean t SEM of 6 determinations. Cycloleueine transport in gIucocorticoid-treated cells was not sjgnificant~y different from that observed in control cells.

DEXAMETHASONE GLUCAGON EPINEPHRINE INSULIN

DEX GLU GUJ

DEX EPI

EPI Di+X

10 pM : 0.1 plbl : 104 . 0.1 +l

DEX

INS

INS D;X

Fig. 8. Combined effects of dexamethasone and other hormones on AIB transport. Hepatocytes were incubated for 3 h in the absence or presence of each hormone alone, or with dexamethasone plus glucagon or epinephrine or insulin, at the concentrations indicated. 2 mM [ 14CIAIB was then added and transport was measured after 15 min. Transport in control cells and dexamethasone-treated ceBs is shown (two bars on the left) for each of the three groups. Each bar represents the mean of 2 separate experiments; triplicate determinations were done for each experiment.

Glucocorticoid

effect on amino acid transport

hand, and dexamethasone and insulin on the other hand, had essentially effects when used at maximally effective doses (fig. 8).

213

additive

DISCUSSION This study clearly demonstrates that glucocorticoids stimulate directly the active transport of a-aminoisobutyric acid in isolated hepatocytes from adult rat. Our results closely parallel those obtained in cultured human fibroblasts (Hollenberg, 1977). They contrast with those reported in cultured rat hepatoma cells in which dexamethasone was found to inhibit AIB uptake although the glucocorticoid did induce the synthesis of tyrosine aminotransferase (Gelehrter et al., 1975). Our results are also at variance with those obtained in primary cultures of adult rat hepatocytes where dexamethasone per se did not affect AIB transport but strongly enhanced glucagon (Kletzien et al., 1975) and catecholamine (Pariza et al., 1977) effects. In freshly isolated hepatocytes we did not find evidence for such a permissive action, since the combined effects of glucocorticoids and other hormones were essentially additive. The reasons for such a discrepancy are presently unknown, although the systems and the experimental conditions used differed largely. It is worth mentioning that in the isolated perfused liver, hydrocortisone alone also stimulated AIB uptake and this effect was found to be merely additive with that of insulin (Chambers et al., 1965). As in cultured fibroblasts (Hollenberg, 1977), the stimulatory effects of the corticosteroids tested corresponded closely to their established glucocorticoid and antiinflammatory properties. The lower potency of cortisol (compared to synthetic glucocorticoids) can be explained by the higher metabolic degradation of the natural compound (Glenn et al., 1957). The EDso value (-2 PM) of cortisol effect on AIB transport in isolated hepatocytes is in the range of glucocorticoid concentration in blood. This value is, however, about three orders of magnitude higher than the dissociation constant reported for the cortisol-receptor complex in the cytosol of rat liver (Koblinsky et al., 1972). As previously observed with glucagon (Le Cam and Freychet, 1976) and catecholamines (Le Cam and Freychet, 1977b), glucocorticoids enhance amino acid transport in isolated hepatocytes by stimulating predominantly system A. It has also been demonstrated in mesenchymal cells that only the A system of transport is subject to regulation by substrate (i.e. amino acids) and insulin (Guidotti et al., 1974). All of these observations stress the physiological importance of .the A mediation of transport. Three lines of evidence suggest that glucocorticoids enhance amino acid transport in isolated hepatocytes by stimulating the synthesis of new carrier proteins: (1) the inhibitory effect of actinomycin D and cycloheximide; (2) the lag period (about 1 h) observed before the onset of glucocorticoid action; and (3) the increase in v,,,, of transport. All of these observations are consistent with an increase in

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the number of carriers through new protein synthesis, although one cannot rule out other types of effect. It is noteworthy that the stimulatory effect of cortisol on AIB transport by the isolated perfused rat liver was not inhibited by pretreatment of the animal with actinomycin D, although a lag period was also observed in this system (Chambers et al., 1965). This discrepancy may result from the use of different systems and different experimental conditions. In conclusion, glucocorticoids per se stimulate amino acid transport in freshly isolated hepatocytes. This effect is not of the permissive type and is additive with that of glucagon, insulin and catecholamines. As observed with these hormones, the glucocorticoid action involves new protein synthesis and affects predominantly the A system of transport. Because of the additive character of these hormonal actions, it is, however, likely that different mechanisms are implicated.

ACKNOWLEDGEMENTS We are indebted to G. Le Cam and G. Visciano for skillful technical and to J. Ciaramella for secretarial assistance.

assistance

REFE~NCES Baxter, J.D. and Tomkins, G.M. (1970) Proc. Natl. Acad. Sci. I.J.S.A. 65,709. Bonney, J.R. and Maley, F. (1975) In: Gene Expression and Carcinogenesis in Cultured Liver, Eds.: L.E. Gershenson and E.B. Thompson (Academic Press, New York) pp, 24-45. Chambers, J.W., Georg, R.H. and Bass, A.D. (1965) Mol. Pharmacol. 1,66. Clark, I. (1953) J. Biol. Chem. 200,69. Feigelson, M. and Feigelson, P. (1965) Adv. Enzyme Regul. 3,111. Freychet, P. and Le Cam, A. (1977) Hepatotrophic Factors, Proceedings of the Ciba Foundation (Excerpta Medica, Amsterdam) in press. Gelehrter, T.D., Risser, W.L. and Reichberg, S.B. (1975) In: Gene Expression and Carcinogenesis in Cultured Liver, Eds.: L.E. Gershenson and E.B. Thompson (Academic Press, New York) pp. 190-203. Glenn, E.M., Stafford, R.O., Lyster, S.C. and Bowman, B.J. (1957) Endocrinology 61, 128. Goldstein, L., Stella, E.J. and Knox, W.E. (1962) J. Biol. Chem. 237, 1723. Goodlad, G.A.J. and Munr0,H.N. (1959) Biochem. J. 73,343, Guidotti, G.G., Gazzola, CC. and Franchi-Gazzola, R. (1974) In: Advances in Cytopharmacology, Eds.: B. Ceccarelli and J. Meldolesi (Raven Press, New York) Vol. 2, pp. 1.5-162. Hollenberg, M. (1977) Mol. Pharmacol. 13,150. Kaplan, S.A. and Shimizu, C&N. (1962) Am. J. Physiol. 202,695. Kaplan, %A. and Shimizu, C.S.N. (1963) Endocrinology 72, 267. Kletzien, R.F., Pariza, M.W., Becker, J.E. and Potter, V.R. (1975) Nature (London) 256,46. Koblinsky, M., Beato, M., Kalimi, M. and Feigelson, P. (1972) J. Biol. Chem. 247,7897. Le Cam, A. and Freychet, P. (1976) Biochem. Biophys. Res. Commun. 72,893. Le Cam, A. and Freychet, P. (1977a) J. Biol. Chem. 252,148. Le Cam, A. and Freychet, P. (1977b) Endocrinology, in press. Le Cam, A., Guillouzo, A. and Freychet, P. (1976) Exp. Ceil Res. 98, 382. Pariza, M.W., Butcher, F.R., Becker, J.E. and Potter, V.R. (1977) Proc. Nat]. Acad. Sci. U.S.A. 74,234.

Effect of glucocorticoids on amino acid transport in isolated rat hepatocytes.

Molecular and Cellular Endocrinology. @Elsevier/North-Holland 9 (1977) 205 Scientific Publishers, Ltd. 2 I4 EFFECT OF GLUCOCORTICOIDS ON AMINO ACI...
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