Regulation of protein synthesis by estradiol 17β, dexamethasone and insulin in primary cultured Xenopus hepatocytes

Experimental

Cell Research 148 (1983) 423-436

Regulation of Protein Synthesis by Estradiol 17(3, Dexamethasone and Insulin in Primary Cultured Xenopus Hepatocytes AKIRA ‘Cell and Developmenial ‘Department

KAWAHARA,’ KAZUKI MINORU AMANO’ Biology Lahoralo?, of Zoology, Fuculty Hiroshima

SATO’ and

Fuculty qf‘lntegrutrd of Scienc,e, Hiroshima 730. Jupun

Arts and Sciences und University.

Changes in the protein synthesis of Xenopus hepatocytes caused by insulin. estradiol17j3 (estradiol) and dexamethasone were studied by using a primary culture in serum-free medium. All of these hormones stimulated the synthesis of secretory and intracellular proteins. Dexamethasone induced or stimulated the synthesis of many proteins (though limited in number). whereas estradiol induced or stimulated relatively few proteins. including the yolk precursor protein vitellogenin. The majority of these proteins differed in molecular weight and/or isoelectric point. When hepatocytes were treated with both steroids, most of the proteins were synthesized at the rates expected from the single treatment of the respective steroids. Thus, each steroid selectively stimulated the synthesis of its specific proteins. However, exceptional proteins were observed. whose syntheses were stimulated only by double treatment. In contrast. insulin seemed to cause an overall increase in individual secretory protein synthesis.

Steroid hormones regulate the expression of restricted sets of genes in a tissuespecific manner and there are two opposite directions of regulation: repression and stimulation of gene expression ]I]. Numerous reports have indicated that steroid hormones regulate the gene expression by a steroid-receptor complex interacting directly with highly specific sites on the chromosome [2]. Furthermore, it has been demonstrated that there are specific sequences of DNA around inducible genes responsible for steroid induction [3]. However, it is not known whether or not the repression of specific genes by a steroid is also due to the direct interaction of a steroid-receptor complex with the genes. The induction of the yolk precursor protein vitellogenin in oviparous vertebrates by estrogen is one of the steroid-regulated events, providing a unique and excellent system for studying hormonal regulation of gene expression in non-proliferating and terminally differentiated cells of the liver [4]. In this system, non-specific-like responses have been observed, concomitantly with the induction of vitellogenin synthesis, i.e., an increase in the number of ribosomes, proliferation of the endoplasmic reticulum and a greatly enhanced overall RNA and protein syntheiss [5-l I]. However, there is no evidence that such non-specific-like responses are caused by the direct effect of estrogen on hepatocytes. Furthermore, available data suggest that albumin synthesis is inhibited by estrogen injection in animals

424 Kawahara et al. 112-151. However, a quantitative measure seems insufficient for elucidating this decreases. On the other hand, the synthetic glucocorticoid, dexamethasone has been found to stimulate specific sets of protein synthesis including albumin synthesis 116, 171. It is very interesting that albumin genes may be regulated in opposite directions by two different classes of steroids. Recently, we developed a primary culture of Xenoplrs hepatocytes in an insulin-supplemented synthetic medium and reported that estrogen induces vitellogenin synthesis directly, accompanied by a decrease in albumin synthesis [IX]. In contrast, other culture systems revealed that the synthesis of albumin and other secretory proteins decreased with culture time, irrespective of the presence of estrogen [l&20]. These different results probably arise from the use of insulin in our culture medium. To resolve these problems, direct responses toward estradiol, dexamethasone and insulin were analysed by SDS-polyacrylamide gel electrophoresis (PAGE) and two-dimensional gel electrophoresis quantitatively. MATERIALS

AND METHODS

Primayy Cultrrre of Heputocytes Hepatocytes were obtained from adult male frogs by collagenase-perfusion and parenchymal cells were enriched by several low speed centrifugation to comprise a more than 95% of hepatocyte population. The final hepatocyte preparations were cultured in a tissue culture dish (Falcon plastic dish. 35 mm in diameter) at densities within the range of SX IO“-1 x IO” cells/cm in 60% Leibovitz’s L-15 medium (Gibco) supplemented with 0.5 mgiml glucose. 5 mu/ml insulin (bovine pancreas. Sigma), 100 U/ml penicillin and 100 ugiml streptomycin. All procedures for the primary culture of hepatocytes have been previously described in detail 1181. In addition. microtest plates (Falcon. 6 mm 0) were used to study the kinetics of [‘HI leucine incorporation into the hepatocyte protein. Each well of microtest plates received 2.4~10~ cells and 0.36 ml of culture medium. A half-volume of culture medium was replaced with fresh medium at 24 h intervals. Dexamethasone and estradiol-17/1 (Sigma) dissolved in propylene glycol were added to the culture medium one day after inoculation of the cells. Control cultures were administered an appropriate volume of propylene glycol.

Measurement of Protein Secretion Rate Hepatocytes were cultured in 3 ml of the medium. The medium was changed at 24 h intervals and the spent media from 3-5 dishes were pooled and centrifuged at I SO0 g for I5 min. whereafter the supernatant was made 107~’ with respect to trichloroacetic acid (TCA) and kept overnight in a refrigerator. The resulting precipitates were collected by centrifugation at I 500 g for 15 min. washed with 5 ml 5% TCA and once with ethyl ether. The final pellet was dissolved in 0.1 % sodium dodecylsulfate (SDS) - 0.01 N NaOH aqueous solution. The aliquots were used for protein determination by Lowrys’ method 1211, using bovine serum albumin as the standard.

Measurement of Cell Number and (3H]Leucint~ Incorporation into Protein Hepatocytes were labelled with 10 pCi/ml [‘Hlleucine (I.-[4. 5-3H]leucine, the Radiochemical Centre. Amersham, UK) after the culture medium was replaced with fresh medium. The final specific radioactivity in the culture medium was 17.6 Ciimole. The medium was stored at -20°C until use. The cells were washed with fresh medium and then homogenized in I % Triton X-100 - I mM phenylmethylsulfonylfluoride (PMSF) - 10 mM Tris-HCI (pH 7.6) - 0.7% NaCI. The nuclei in the homogenate were stained with 0.1 % gentian violet in a I % acetic acid solution. The number of nuclei was then Exp

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Dose-teiponse relationships on overall protein secretion. Dexamethasone (r
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counted on a hemocytometer. The number of nuclei determined in thi> way was expressed as the number of cells attached to the substratum of the dish. Radioactivities of 10% TCA-insoluble materials were measured on fiber glass filters (Whatman GFIC) in a toluene-DPO scintillator solution. as described elsewhere 1181. The radioactivity counting efficiency was within 2@25’ii.

Proteins secreted from hepatocytes for 24 h were analysed by SDS--7.5% polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [22]. Molecular weight standards (HMW and LMW. Rio-Rad) were used as markers of the electrophoresis. The gels were stained with 0.1 ‘;i Coomassie brilliant blue in 50% TCA aqueous solution and then destained in a 7R acetic acid solution. For quantitative measurement of the intensity of each band on the gels. the gels were scanned with a densitometer (FD-A 111. Fuji Riken) at 570 nm of light (slit. 0.5 mm x 3 mm). The peaks on the charts of the densitometric patterns were cut off and weighed. All weights were corrected for each band width. Hepatocytes were labelled with I.-]‘S]methionine (40 uCi/ml, 104 mCi/mmole) in a methionine-free culture medium and then homogenized in 2% Nonidet P-40-I mM PMSF - 90% urea. The homogenates were centrifuged at 8000 g for 4 min and applied on a two-dimensional gel electrophoresis according to O’Farrell [23]. The gels for the first dimension were 6 cm in length and two gels were applied in parallel on one SDS - 10% polyacrylamide gel for the second dimension. After electrophoresis. the gels were fixed with 10% TCA. treated with EN’HANCE (New England Nuclear) and dried on filter papers. The gels from an experimental group were placed in contact with the same X-ray film (Fuji X-ray film. Medical) for 2 weeks at -70°C and then developed.

RESULTS Primary culture of male Xcnopus laeuis hepatocytes was recently established in this laboratory [18], using a serum-free culture medium containing insulin which effectively prevents detachment of cells from the substratum of the dish. In the present study, insulin was added to the culture medium at 5 mu/ml which was sufficient to maintain cells without loss of cell for at least the duration of experimental period. This allowed a quantitative measurement of the rate of protein synthesis. As shown in fig. 1, estradiol was capable of stimulating

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Fig. 2. Dose-response relationships on overall protein synthesis. Hepatocytes were labelled with r3H]leucine for 24 h on day 5 of the hormone treatments and the incorporations into secreted and intracellular proteins were determined as described in Materials and Methods. The data were expressed as the mean of three determinations. Vertical range bars represent SDS. Incorporation into vitellogenin was determined by the direct immunoprecipitation method. as described elsewhere [ 181. Open and closed circles represent the percentages of protein secretion and vitellogenin production to the total protein synthesis, respectively. 0, Total: 0. secreted; III, vitellogenin.

secretory protein formation at IO-’ M and an increase in the amount of dose was accompanied by an increase in the formation of secretory protein. At IO-’ M, a near maximum response was obtained on day 6. A relatively lower concentration of dexamethasone (IO-’ M) was sufficient to secrete the maximum amount of secretory protein (fig. 1). This stimulation capability of estradiol was twice of dexamethasone at maximum responses. In addition, the response toward dexamethasone reached a plateau more rapidly than that toward estradiol. Labelling with 13H]1eucine on day 5 was carried out for 24 h in order to determine the extent to which the total protein synthesis was altered by estradiol or dexamethasone. The results obtained are shown in fig. 2. Total incorporations of 13H]1eucine(the sums of the radioactivities incorporated into secreted proteins and intracellular proteins) were stimulated by both steroids. The magnitudes of maximum stimulation (approx. 1.8-fold) relative to that of the steroid-free control were similar for estradiol (10e6 M) and dexamethasone (more than IO-” M), but estradiol mainly increased secretory protein formation, whereas dexamethasone enhanced both types of protein synthesis (secretory and intracellular protein synthesis). With increasing doses of estradiol, the percentage of radioactivity of the secreted proteins to the total radioactivity increased to 45 70. Dexamethasone Erp Cell Re\ 148 tlY83J

Hormonal control of hepatocyte protein synthesis

427

Est.+Dex.

0

Fig. 3. Stimulation of overall protein secretion by single or double treatment of estradiol and dexamethasone. Hepatocytes were treated with either none (control), estradiol (lo-“M), dexamethasone (IO-’ M) or dexamethasone (IO-* M) plus estradiol (IO-’ M). Protein amounts secreted from the hepatocytes were determined as described in Materials and Methods. 0123456

Days after Hormone Addition

treatment did not result in such an increase but the ratio remained constant. The incorporation of radioactive leucine into vitellogenin increased with the dose of estradiol, to comprise approx. 20% of total incorporation at IO-” M. Independent Actions of Dexamethasone and Estradiol on Formation of Secretory Proteins Experiments were carried out to determine whether or not two different steroid hormones act independently on secretory protein formation. Hepatocytes were treated with estradiol, dexamethasone and estradiol plus dexamethasone at concentrations required for the maximum stimulation of protein synthesis (see fig. 2). First, the overall secretion rate of protein was compared for the treatments described above. The secretion rate in the double hormone treatment increased as if the two steroids each had the ability to stimulate formation of secretory protein independently, as shown in fig. 3. The secretion rates in the double treatment were approximately equal to the control rate plus the sum of the additional rates which increased by a single treatment of estradiol or dexamethasane during the period of treatment. Thus, the two different steroids additively stimulated the overall secretion rate. The secreted proteins were prepared daily from the culture medium and resolved in SDS-PAGE (fig. 4). Three major proteins appeared in the control pattern. Two of these were albumins (alba and alb b) as reported previously [18] and the third had a molecular weight of 84000 (84K protein). Dexamethasone induced or stimulated at least six different size proteins (84K protein, alb a, Dl , D2, D3 and D4), whereas estradiol induced or stimulated vitellogenin mainly and at least one more protein (E). Thus, each steroid stimulated the formation of specific and different secretory proteins. When hepatocytes were treated with Exp Cell Res 148 (19831

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Fig. 4. SDS-PAGE patterns of secreted proteins In single or double treatment of dexamethasone and estradiol. Hepatocytes were treated with the indicated hormones at concentrations descrtbed in fig. 3. Protema secreted at 24 h intervals were electrophore\ed and stained with (a) Coomassie brilliant blue. (h) The Intensities of several protein bands (A, 84K protein; 0, alba; 0, alb b; 0, vrtellogenin: 0. 6: A, DI; V. D3) were determined by a densitometrlc assay as described in Material5 and Methods. The number at the top of each lane indicates the day of hormone treatment. The amounts of secreted proteins charged on SDS-PAGE were equivalent to those from 2.4x IO’ cells.

Hormonal control of hepatocyte protein synthesis 429

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Fi,c. 5. Insulin effects on protein synthesis. One day after culture in a hormone-free medium, insulin was added to the culture medium at the indicated concentrations. ((1) The protein amounts secreted from the hepatocytes were determined at 24 h intervals. (h) On day 5. [‘Hlleucine-lahelling of the hepatocytes was carried out for 24 h and incorporations were determined. The data were expressed as the mean of three determinations (0. total; q . secreted). Vertical range bars represent the SD. (c) Proteins secreted on day 5 were resolved by SDS-PAGE. The amounts of protein charged on SDS-PAGE were equivalent to those from 3x 10” cells. It should he noted that in the absence of or at low concentrations of insulin, hepatocytes became slightly detached from the substratum of the dish (about IO% of the inoculated cell number was lost during the tirst 6 days of culture). Left lane. MW markers: 200000; 116250: 92 SOO; 66200 and 4.5000 D.

430

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Fig. 6. Time course of [3H]leucine incorporation into hepatocyte protein in the single or double treatment of dexamethasone and estradiol. Hepatocytes cultured in the microtest plate were labelled with [3H]leutine for the indicated times and then the radioactivities incorporated into the intracellular (0) and secreted (0) protein fractions were determined in duplicate cultures (data at the 24 h labelling were expressed as the mean + SE of four determinations) as described in Materials and Methods. The lines marked with crosses represent the sum of intracellular and secreted radioactivities (total incorporation). (a) Control; (h) 1O-6 M estradiol; (c) 10-a M dexamethasane; (& 10m6 M estradiol plus IO-~* M dexamethasone. Labeling

Time

(hr)

both steroids, all these proteins appeared in the pattern of SDS-PAGE (fig. 4a). Their staining intensities seemed similar to those stimulated by single treatment of either estradiol or dexamethasone. This observation was corroborated by the densitometric measurement of each band on SDS-PAGE (fig. 4b). Previously, we found that estradiol reduces albumin secretion in the culture medium containing 10 mu/ml of insulin [18]. This was also observed in the present study but less clear than that in the presence of 10 mu/ml. Dexamethasone increased the secretion rate of alba by three-fold, but seemed not to affect that of albb. Such an asymmetric production of albumin did not occur in the culture treated with estradiol or insulin or both. However, it may be possible that the alba band on SDS-PAGE co-migrated with other protein(s) whose synthesis is markedly stimulated by dexamethasone. Similarly, changes in the SDS pattern may be due to an event similar to that described above. However, this was not the case since the results of protein separation by two-dimensional gel electrophoresis eliminated this possibility (data not shown).

Hormonal control of hepatocyte protein synthesis

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Insulin .!Tffeecton Heputocytr Protein Synthesis In the present study insulin was added to the culture medium to avoid loss of cells during the culture period. We attempted to determine insulin effects on hepatocyte protein synthesis. Insulin stimulated the overall secretion rate of protein (fig. 5 a) as well as the total protein synthesis (fig. 5 h) but there seemed to be no selective increase in the formation of individual secretory proteins (fig. 5 c). In addition, proportions of the radioactivity of the secreted protein to the total radioactivity did not change with the dose insulin (fig. 56). However, in an insulin-free medium, hepatocyte protein synthesis was directed to secrete in lesser amounts than in the case in which insulin was present. This decrease in protein secretion in a hormone-free culture medium has been reported previously 117. 19. 211. Absolute Rate of Protein Synthesis Altered by Different Hormonal Treatments [3H]leucine-labelling of hepatocytes was carried out on day 6 of the treatment. using a microtest plate for the culture. Fig. 6 illustrates the incorporation of [jH]leucine into the total, intracellular and secreted protein under different hormonal conditions (none, IO-” M estradiol, 10P8 M dexamethasone or 10eh M estradiol plus 10P8dexamethasone). Incorporation into the total acid-precipitable materials (intracellular plus secreted) linearly increased for at least 24 h in all treatments. This indicates that there is no significant turnover of newly synthesized protein during at least 24 h of labelling. Therefore, the radioactivity incorporated into the protein should be proportinal to the absolute amount of newly synthesized protein. All the figures shown in fig. 6 represent similar kinetics of [‘Hlleucine incorporation. As described in the previous report[l8] and by other authors 1201, the appearance of labelled protein in the medium was delayed 1-2 h from the start of labelling, whereas the line representing the incorporation into intracellular protein shows two different phases of accumulation. Apparently, this initial phase includes intracellular accumulation of secretory proteins before secretion. By comparing the slopes of the lines representing the total and secreted protein, it can be estimated that 45, 66, 40 and 60% of the total protein synthesis are directed toward secretion in the control, estradiol, dexamethasone, and estradiol plus dexamethasone treatments, respectively. Hormonal effects may possibly be accompanied by changes in the uptake and/or pool size of the amino acids, thus making it difficult to compare the rates of protein synthesis under different hormonal conditions. In the present study, the specific radioactivity of newly synthesized secretory protein could be directly measured and compared for different hormonal treatments. In dealing with this problem, cell number and radioactivities of the secreted and intracellular proteins were determined simultaneously, following the labelling with (‘Hlleucine for 24 h. This long period of labelling may minimize error due to variations in the initial 28--X38337

Err, Cdl Rrs 148 (1983)

432 Kawahara et al. Table 1. Absolute rates of protein synthesis with different hormonal treatment Control Specific radioactivity” of secreted protein (cpmiyg protein) Total incorporation” (cpm/lO’ cells) Rate of total protein synthesi? (pg/lO’ cells/day) Rate of intracellular protein synthesis’ (ng/lO’ cells/day) Rate of secretory protein synthesis’ (rig/l@’ cells/day)

4 190+657 40 500+ 1 750

9.67+1.51

IO-’ M Estradiol

2 85Ok280 57 700+3 630

20.24k2.23

IO-’ M Dexamethasone

3 2101121 70 8OOk2 490

10mh M Estradiol + IO ’ M Dexamethasone

3 460+260 I I5 60029 330

22.05kl.33

33.4lk3.68

13.41

5.32

6.88

13.23

4.35

13.36

8.82

20.0

Hepatocytes were treated with the indicated hormones and on day 6 of treatment were labelled with [3H]leucine for 24 h as described in Materials and Methods. ” Data expressed as the mean + SE of three determinations. ’ Calculated by the formula: rate of protein synthesis = total incorporation/specific radioactivity of secreted protein. ’ Calculated using the ratio of the slope representing the intracellular or secreted protein synthesis to that of the total synthesis (shown in fig. 7 and see text).

kinetics of the incorporation and overestimation of the amount of labelled protein due to the intracellular secretory protein pool present before the start of labelling. The results are shown in table I. The specific radioactivities obtained were similar for various treatments, indicating that the efficiency of using [3H]leucine for protein synthesis was not altered significantly by different hormone treatments. From these data, the absolute rates of intracellular protein synthesis could be calculated by the simple formula described in table 1 on the assumption that intracellular proteins synthesized for the labelling period of 24 h has the same specific radioactivity as secreted protein. The results indicate that there was an additive increase in the synthesis of secretory protein as well as intracellular protein in the double hormone treatment. This was also true in the case of Fig. 7. Fluorograms of the two-dimensional gel electrophoresis of newly synthesized intracellular proteins in single or double treatment of dexamethasone and estradiol. Hepatocytes were labelled with [35S]methionine for 8 h on day 6 of the hormone treatment. The radioactive intracellular proteins (equivalent to 0.51 x lo5 cells) were electrophoresed and fluorographed. as described in Materials and Methods. (A) None (control); (B) IO-(’ M estradiol; (C) IO-’ M dexamethasone: (D) 10mh M estradiol plus IO-’ M dexamethasone. Incorporations of [‘5S]methionine into intracellular proteins were 89000 cpm (control), 137000 cpm (estradiol), 220000 cpm (dexamethasone) and 256000 cpm (estradiol plus dexamethasone) per IO5 cells. (I?) Schematic diagram of (D), in which the closed and hatched spots represent two sets of protein whose synthesis appeared or were intensified by dexamethasone and estradiol, respectively. Symbols of bars and arrowheads are explained in text. Exp CellRes 148 (1983)

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434 Kuwahara et al. [“S]methionine-labelling (se caption to fig. 7). These results suggest that secretory as well as intracellular protein synthesis is independently controlled by each steroid. In addition, we confirmed again that estradiol mainly stimulated secretory protein synthesis, whereas dexamethasone enhances both types of protein synthesis (secretory and intracellular). Hormone-specific Stimulution of Intracellulur

Protein Synthesis

To extend the comparison further, newly synthesized intracellular proteins were resolved in two-dimensional gel electrophoresis on day 6 of the hormone treatment. The fluorograms of [35S]methionine-labelled proteins prepared from the same number of cells (fig. 7) revealed that dexamethasone induced or stimulated many intracellular proteins, whereas estrddiol induced or stimulated few proteins (compare fig. 7B and C with A and see E). The majority of proteins can be divided into three categories: those whose syntheses are induced or stimulated by either estradiol or dexamethasone; those whose syntheses are unchanged significantly; and those whose syntheses are reduced by either estradiol or dexamethasone (denoted by bars in fig. 7B, C). In addition, it was found that there were minor proteins whose syntheses were stimulated by both steroids (fig. 7 B, C, arrowheads). Within the limits of our analysis, the majority of the proteins described above seemed to be synthesized additively in hepatocytes treated with both steroids (compare fig. 70 with A-C and see E). However, there were exceptional proteins whose syntheses seemed to be stimulated only by the double treatment of both steroids (fig. 70, arrowheads). The results in this section also demonstrate that the increase in intracellular protein synthesis by the respective steroids is due to the appearance and stimulated synthesis of hormone-specific proteins. DISCUSSION All three different hormones used in this study had a stimulatory effect on the protein synthesis of hepatocytes. Both dexamethasone and estradiol stimulated selectively the synthesis of restricted sets of proteins. In contrast, insulin seemed to cause an overall increase in the synthesis of individual secretory proteins. A simple mechanism by which such a specific gene expression by the steroid hormone can be realized is that both steroids act directly on the specific target genes to induce or stimulate their message productions. Even if there is an increase in translational and/or transcriptional efficiency, such an increase must be specific to the message directed by each steroid hormone. This view is strengthened by the fact that in the double steroid-treated hepatocytes, there is an additive in protein synthesis, essentially quantitative and qualitative, although this model does not provide a simple explanation of the presence of exceptional proteins whose syntheses are stimulated only by the double treatment of two different steroids (see fig. 8). Clemens & Tata 161reported that estradiol treatExp Cdl REA 148 (1983)

Hormonal control of hepatocyte protein synthesis

435

ment in vivo produces an intrinsic activation of translation capacity in ribosomes, using the hepatocyte cell-free translation system directed with poly (U). However, it is not known as yet whether such an increase in translational efficiency is specific or preferential to estrogen-inducible messages. This increase may result from estrogen-mediated indirect action(s) on hepatocytes in vivo. Albumin synthesis was found to be controlled in various ways by insulin, dexamethasone and estradiol. Insulin stimulates the syntheses of two albumins in nearly an equal number of molecules, but dexamethasone stimulates the synthesis of only one of these. Such different stimulatory effects on two albumins by the most effective glucocorticoid, dexamethasone, may explain the asymmetric presence of these albumins in serum (see fig. 5). It has been reported that there are one or two albumin genes in Xenopus laeuis. The present data suggest the presence of two albumin genes whose expressions are controlled differently by dexamethasone, as described above. On the other hand, estradiol tends to reduce both albumin syntheses in the presence of insulin whcih stimulates their syntheses. It has recently been found that the level of albumin mRNA sequence is not altered by estrogen in vivo [24]. On the other hand, Stanchfield & Yager [20] suggest that insulin prevents a decrease in albumin synthesis in an insulin-free medium by increasing the initiation of the translation. Therefore, it is likely that the inhibition of albumin synthesis by estradiol is a result of translation initiation competition between albumin messages and estradiol-inducible mRN,4s. It has also recently been found that, using a culture of Xenopus liver pieces, the addition of thyroid hormone during the first 2 weeks of the culture period can sustain the synthesis of ‘glucocorticoid-inducible secretory proteins’ which decrease in the absence of thyroid hormones [25, 261. It has been ptoposed that thyroid hormones enhance ‘the endogeneous glucocorticoid activity’, assumed to be present in fully-perfused liver pieces in culture. However. ‘endogeneous glucocorticoid activity’ is considered to be lost in freshly isolated hepatocytes. since two albumins are not synthesized asymmetrically but formed in nearly an equal number of molecules in the absence of dexamethasone (see figs 4, 5). The decrease in the synthesis of ‘glucocorticoid-inducible proteins’ (such as alb a and 84K protein), as well as uninducible proteins (alb b) may be due to the absence of insulin or other unknown factor(s) unlike in vivo. Further research on this matter is also necessary. This work was supported by Grants-in-Aid Scientific Research from the Japanese Ministry

for the Encouragement of Education.

of Young

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Received February 23. 1983 Revised version received May 2. 1983