A tryptic peptide from β -casein depresses protein synthesis and degradation and enhances ureogenesis in primary cultures of rat hepatocytes

Vol. 23, No. 4, pp. 483490, Printed in Great Britain. All rights reserved

fnr. J. Eiochem.

1991 Copyright

0

0020-71 IX/91 $3.00 + 0.00 1991 Pergamon Press plc

A TRYPTIC PEPTIDE FROM p-CASEIN DEPRESSES PROTEIN SYNTHESIS AND DEGRADATION AND ENHANCES UREOGENESIS IN PRIMARY CULTURES OF RAT HEPATOCYTES AKIO TAKENAKA, YUICHI OHISHI, TADASHI NCKXJCHI* and HIROSHI NAITO

Department

of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan [Tel. 03 812 2111 ext. 51181 (Received 9 July 1990)

Abstract-l.

A peptide which enhances ureogenesis in primary cultured hepatocytes of rats was isolated from a tryptic digest of bovine p-casein. 2. The structure of the peptide was Ala-Val-Pro-Tyr-Pro-Gh-Arg which is located from 177th to 183rd residues from N-terminal of bcasein. 3. The peptide also showed the activity to inhibit protein synthesis and protein degradation. 4. It also inhibited DNA synthesis of hepatocytes induced by insulin and/or epidermal growth factor.

INTRODUCTION Some tryptic peptides from casein show biological activities. Brantl (1979) discovered a morphine-like activity in a peptide derived from B-casein and named

/?-casomorphine. In 1979, Zioudrou found a peptide which shows an activity of exorphine (Zioudrou et al., 1979; Loukas et al., 1983). Yoshikawa et al. (1984) extended these studies and found several peptides of similar activity. Furthermore, they found some other peptides which show an antagonist activity of enkepharine (Yoshikawa et al., 1984). Maruyama and his coworkers (1982, 1985, 1987) found peptides which inhibit the activity of angiotensin-converting enzyme in a tryptic digest of /I-casein. These studies with others (Parker et al., 1984; Jolles et al., 1986) show that there are many biologically active peptides in partial digestion products of casein. In a series of attempts to obtain substances which regulate ureogenesis, protein synthesis and protein degradation in primary cultures of rat hepatocytes, we tried to find some peptides which affect the activity of the above-mentioned metabolic events. Here we report a tryptic peptide prepared from /I-casein which enhances ureogenesis and depresses protein synthesis and degradation in primary cultures of rat hepatocytes. MATERIALS AND METHODS Materials

Human epidermal growth factor (f-Met-EGF) was obtained from AMGen Biochemicals. Bovine serum albumin, porcine insulin, glucagon and dibutylated cyclic AMP (dbCAMP) were from Sigma. Trypsin was from P.L. Biochemical Inc. (Milwaukee). Williams’ E medium (WE) was obtained from Flow Laboratories. Phosphate buffered saline solution (PBS-) and Earle solution were from Nissui. PBS( + ) was prepared by dissolving MgCl, H,O (100mg/l) *To whom all correspondence

should be addressed.

and CaCl, (100 mg/l) in PBS(-). Arginine-free minimum essential medium (arginine-free MEM) was prepared by dissolving to Earle solution, the vitamin mixture, the non-essential amino acid mixture and essential amino acids the amounts of which were simulated to the essential amino acid mixture for MEM (Flow Laboratories) except arginine. The vitamin mixture and the non-essential amino acid mixture for MEM were obtained from Flow Laboratories. [3H]Thymidine and [i251]insulin(receptor grade) were purchased from New England Nuclear. [‘*‘I]EGF was prepared by iabelling 1 nmol of EGF with Nai2SI (1 mCi, for protein iodination, purchased from Amersham Japan, Tokyo, Japan) according to Greenwood et al. (1963). Preparation of a heptapeptide from tryptic hydrolysate qf /I -casein

Our preliminary studies elucidated that a peptide in tryptic hydrolysate of B-casein enhances ureogenesis in primary cultures of rat hepatocytes. We purified the activity by sequential employment of Sephadex G-25, CM-Sephadex C-25 and high performance liquid chromatography (HPLC) with octadecylsilica gel (data not shown). Amino acid analysis and identification of N-terminal amino acid elucidated that the peptide is the same as CEI, reported by Maruyama er al. (1985) as an inhibitor of angiotensin-converting enzyme. Therefore, we employed their method for the preparation of the heptapeptide. The method is as follows. /I-Casein was obtained from Meiji Seika Co. (Tokyo, Japan). A 2 g sample of this /?-casein was dissolved in 50 ml of 0.04 M sodium phosphate buffer @H 7.4). Bovine trypsin was dissolved in 0.01 M HCI at the concentration of 5 mg/250 ~1. These two solutions were mixed and kept at 37°C for 16 hr with continuous mixing. After the incubation, 2.26ml of 11 M HCl was added. The mixture was centrifuged at 8000g for 20 min. The supernatant was stored at - 20°C until it was used for peptide preparation. A 25 ml of the supematant was fractionated on LH-20 column (30 x 1050mm) which was preequilibrated with deionized water. The active peptide was obtained in the eluate of 4-90 ml. The eluate was lyophihzed and dissolved in 20 ml of deionized water. This sample was fractionated on SP-Sephadex C-25 column (H-form, 20 x 470 mm) which was equilibrated previously with 0.1 M ammonium formate (pH was adjusted with formic acid at 5.0). Peptides were 483

484

AKIO TAKENAKA et al.

eluted with linear gradient of ammonium formate from 0.1 to 0.5 M. The fractions indicated in Fig. 1 of Maruyama’s paper (Maruyama et al., 1985) contained solely a heptapeptide (CEI,) i.e., Ala-Val-Pro-Tyr-Pro-Gin-Arg, which is sited at from Ala (177) to Arg( 183) of bovine B-casein. The purity of the peptide was confirmed by HPLC (reverse phase chromatography employing an octadecylsilica gel column, see below), amino acid analysis and identification of N-terminal amino acid by dinitrophenylation (data not shown). This heptapeptide will be referred to as ureogenesis enhancing peptide (UEP) in the following sections of this paper. Synthetic UEP

UEP synthesized by a solid phase peptide synthesizer and purified by reverse phase HPLC was kindly donated by Dr Masaaki Yoshikawa, Department of Food Technology, Kyoto University, Kyoto, Japan. Hepatocytes

Hepatocytes of male Wistar strain rats of body wt ca 200 g were isolated as described previously (Takahashi et al., 1985). The cells were incubated in three kinds of Corning culture dishes, i.e. 24 wells, 35 and 60 mm. The volume of culture media was 250 ~1 for 24 wells, 1 ml for 35 mm dish and 2 ml for 60 mm dish. These cultured hepatocytes have been shown to maintain many functions of liver in vivo (Tanaka et al., 1978), although the metabolic activity may be more or less modified by isolation and culture procedure. Determination of the rate of ureogenesis

The isolated cells were cultured for 2 hr in WE with 10% calf serum. insulin (lo-* M) and dexamethasone (1O-6 M). Coming 24 well dishes wereemployed. After 2 hr incubation in WE with serum, the cells were maintained in serum-free WE fortified with insulin (lo-*M) and dexamethasone (10m6M) for 24 hr. The medium was changed to WE without insulin and dexamethasone and the cells were incubated in this medium For 18 hr. After this incubation, the medium was changed to arginine-free MEM (AFM). The cells were incubated in this medium for 15 min. The medium was renewed and the cells were incubated For further 15 min. The cells were then maintained for 6 hr in refreshed arginine-Free MEM in which the various agents were dissolved. After 6 hr, 200 ~1 of the medium was taken and urea in this portion was determined according to Geyer and Dabich (1971). The difference between 0 time and 6 hr was taken as the rate of urea synthesis. Protein synthesis

The isolated cells were inoculated to Coming culture dishes (35 mm) at a density of 1.0 x lo6 cells/35 mm dish. The cells were incubated in WE with 10% calf serum, insulin (10-s M) and dexamethasone (10m6M) for 24 hr. After 24 hr, the medium was changed to WE (without insulin, dexamethasone and serum), and Further incubated For 18 hr. After this incubation. the cells were washed with PBS( +) for 2 times and were transferred to three lines of the medium; WE, WE with 30 nM glucagon and WE with 30 nM insulin. Each line had two treatments; control (without anything) and with 107 nM (lOOpg/ml) of UEP. Then, 0.1 PCi of (‘Hlvaline (tracer amount dissolved in 100~1 of WE) was added and incubated for 6 hr. After 6 hr, the medium was discarded and the cells were washed with PBS( -). The washed cells were collected with a rubber policeman in 2 ml of 2% sulphosalicylic acid (SSA). The cells were disrupted with ultrasonic wave. The broken cells were centrifuged at 3000g for 20 min. The supematant was discarded and the radioactivity incorporated into the precipitate was assumed to be the rate of protein synthesis. Protein degradation

The isolated cells were inoculated at the density of 2.0 x lo6 cells/60 mm dish (Corning culture dish). The cells

were incubated For 24 hr in WE supplemented with 10% calf serum, insulin (lo-* M) and dexamethasone (10e6 M). After 24 hr, the cells were washed three times with WE. Then, the cells were incubated for further 18 hr in WE containing 2 y Ci of rH]valine. After this labelling period, the cells were washed twice with WE at an interval of 30min. Then, the cells were transferred into three lines of the medium; WE, WE with 30 nM ghtcagon and WE with 30 nM insulin. Each line was subdivided into two treatments; WE and WE with 86 nM UEP. The cells were incubated in these media for 6 hr. After 6 hr, 2 ml of 4% sulphosalicyclic acid was added to the medium and the cells were harvested with a rubber policeman. The remaining small amount of the cells were again collected with I ml of 2% sulphosalicylic acid. The collected cells were sonicated and centrifuged at 2000g for 20 min. The supematant was saved for the determination of the radioactivity and the precipitate was dissolved in I ml of 0.5 N NaOH. The supematant and the dissolved precipitate were mixed with NT scintillator and the radioactivity was determined (Takahashi et al., 1985). The radioactivity in the supematant divided by that in the combined supernatant and precipitate (%) was supposed to be the rate of protein degradation. Effect of UEP on [‘Hlthymidine incorporation into hepatocyte DNA

The cells were prepared as described at the section of Protein Synthesis. After the 18 hr incubation, the cells were washed by PBS (+) two times. Then, the hepatocytes were incubated with ghicagon, insulin, EGF, db-CAMP, glucagon with insulin orEGF_with insulin for 24 hr in the-presence or absence of UEP. Then. PHlthvmidine solution (0.2 uCi of [3H]thymidine dissolved’in l&l iI of WE) was added. The cells were incubated further For 2 hr. Thymidine incorporation during this 2 hr was measured as follows. After the incubation, the cells were washed 2 times with PBS( +) and dissolved in 0.5 ml of 0.5 N NaOH by keeping the dishes at 37°C overnight. An equal volume (0.5 ml) of 20% trichloroacetic acid (TCA) was added, and the mixture was transferred onto a’ glass filter (4 = 25 mm, Whatman GFIC). , , The orecioitate was washed with 10% TCA. The radioactivity trapped on the filter was measured employing a toluene based scintillation cocktail (200 mg of PGPOP and 4 g of PPO were dissolved in 11. of toluene). 1

L

Effect of UEP on binding of insulin and EGF to hepatocytes and degradation of these hormones by hepatocytes

The cells were prepared as described in the section of Protein Synthesis‘Afier washing with PBS( +) two times, the cells were keot in WE with or without UEP (68 uM For insulin and 85 pzM for EGF). 12JI-labelled insulin or EGF dissolved in 20~1 of WE was added to the culture dish. Then, the cells were incubated for 2,4 or 6 hr for insulin and 1, 2, 3 or 6 hr for EGF. After the incubation period, the medium was mixed with 1ml of 10% TCA solution and 100~1 of bovine serum albumin solution (dissolved at 10 &ml _, of deionized water). The mixture was centriFuged at 2000g for 20 min. The radioactivity in the precipitate was oresumed to be undenraded EGF or insulin. The radioaciivity in the supernatait was supposed to be degraded EGF or insulin. The cells on the dish were washed 5 times with PBS (-). To the dishes, 1 ml of 0.2 M acetic acid was added. Then, the dishes were kept on ice for 7min. The acetic acid solution was removed and saved For the determination of receptor-bound radioactivity. After that, the cells were harvested with 2 ml of 10% TCA solution. The harvested cells were sonicated and centrifuged at 2000g for 20min. The radioactivity in the supematant (degradation product in the cells) and the precipitate (undegraded EGF or insulin in the cells) were measured as described above.

Enhanced ureogenesis in cultured hepatocytes Rate

485 (% of ccntrco

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o

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Thr Ser GIU Gill Gb AIB Cl1 VSI CYS

0

6

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Time (mln)

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Fig. 1. Reversed phase HPLC of purified UEP. UEP was purified as described in Materials and Methods. Approximately 10 pg of UEP was applied to ODS TSK gel column (4.6 x 150mm). The column was eluted with 20% acetonitrile-80% water containing 0.1% TFA. The flow rate was 1 ml/min.

Phe OWl TOP LYS His Aw ASII

HPLCofUEP

Pr0

A column of octadecylsilica gel (TSK gel, 4.6 x 150 mm, Toyosoda, Tokyo) was employed. Approximately 10 pg of purified UEP was applied to the column which had been equilibrated with 20% acetonitrile-80% water containing 0.1% of trifluoroacetic acid (TFA). The peptide was eluted with the same solution. The flow rate was 1 ml/mitt.

RESULTS

Figure 1 shows the HPLC pattern of UEP prepared in the present experiments. The results show that the UEP preparation is practically homogeneous chromatographically. Figures 2(A) and (B) show that UEP enhances the ureogenesis in primary cultures of rat hepatocytes. UEP stimulated ureogenesis either in the presence or in the absence of glucagon in the medium. However, the enhancing activity was more prominent in the presence of glucagon. This activity of UEP was also observed in the presence of db-CAMP. These results

Fig. 3. Effect of UEP and amino acids on ureogenesis in the primary cultured hepatocytes of rats. The hepatocytes were cultured without (right) or with 30pM UEP (left) in the presence of 10nM glucagon (marked bars) or without hormones (blank bars). The medium contained only one amino acid which is indicated at the right side of the figure, except Earle (contained no amino acid) and AFM (contained all amino acids except arginine). Data are expressed as the rate of urea formation taking that in the control (AFM medium without glucagon) as 100%. Each value is the mean of duplicate experiments.

suggest that UEP modifies the activity of glucagon or db-CAMP. The results shown in this figure is a typical and usual observation among at least 100 repeats. Although basic ureogenesis deviated more or less among cell preparations, the enhancing effect of UEP was always observed. The effect of glucagon on ureogenesis also deviated more or less according to the preparation. Therefore, if the results were

Concantratlcn 01 VEP (pl)

Fig. 2. Effect of UEP on ureogenesis in the primary cultured hepatocytes of rats. The hepatocytes were cultured for 6 hr in the arginine free medium with various concentrations of natural UEP (A and B) in the presence of 10 nM glucagon (O), 10 PM db-CAMP (A), and 7 nM insulin (0). or without any hormones (a). After the incubation, 200 ~1 of the medium was collected and the concentration of urea was determined as described in Materials and Methods. The figures are the typical and usual observations of at least 100 repeats. The results were reproducible. (C) The results of synthetic UEP. Each value is the mean of duplicate experiments in one lot of hepatocytes. Symbols represent the same treatments as those in (A) and (B).

AKIOTAICENAKA et al.

486

.8 .

Fig, 4. Effect of bestatin on the action of UEP to enhance ureogenesis in the primary cultured hepatocytes of rats. The hepatocytes were cultured for 6 hr without ((),a) or with 0.1 m&ml bestatin (A, A) in the presence of 10 nM glucagon {O, A) or not (e, A). Each value is the mean of duplicate experiments.

expressed enhanced

as how many folds the ureogenesis was by giucagon and UEP, the deviation of the

results was expanded. However, the effect of UEP on ureogenesis was always reproducible and it was more prominent in the presence of glucagon or CAMP than in their absence. Figure 2(C) demonstrates the effect of synthetic UEP on the ureogenesis of cultured hepatocytes. This result is one of the typical observations of some 10 repeats. The results were reproducible. The effect of UEP was observed at the concentration > 10e6 M. The amount of urea synthesized was almost 10 times of the arginine added to the medium as UEP. Therefore, it is concluded that the arginine residue in UEP is not the direct substrate of ureogenesis quantitatively. The possibility that other amino acids was used as the substrate was excluded from the results of Fig. 3. Figure 3 depicts the activity of UEP to enhance ureogenesis in various media. The effect of UEP was observed in all of the media employed in the present experiments.

Fig. 5. Time-dependent urea synthesis by the primary cultured hepatocytes of rats in the presence or absence of UEP and hormones. The hepatocytes were cultured in the arginine free medium containing 3 FM UEP (0, A, Uj or not (a, A, a). Glucagon (IO nM, 0, l ) or insulin (7 nM [3,8) was added to the medium. The cultured medium was collected at the indicated time and the concentration of urea was determined. Values are the means f SEM for six determinations. +P < 0.05 and **P < 0.01 show statistically significant differences between the cells with and without UEP. Figure 4 shows the effect of bestatin, a microbial aminopeptidase inhibitor, on the activity of UEP. When added at the concentration of 0.1 mg/ml to the medium, bestatin little affected the rate of protein degradation of cultured hepatocytes. However, it induced an accumulation of small peptides in the

medium by inhibiting the step to degrade small peptides into free amino acids (Kato et al., 1989). As it is seen in Fig. 4, bestatin inhibited ureogenesis either in the presence or absence of glucagon in the medium. Particularly, the effect of bestatin was noticeable in the presence of UEP and glucagon. Under this condition, ureogenesis is enhanced as described above. These results strongly suggest that

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Fig. 6. Effect of UEP on the [‘Hlvaline incorporation in the primary cultured hepatocytes of rats in WE (A) or Earle solution [B). The hepatocytes were cultured for 6 hr with 100 &ml UEP (marked bars) or without UEP (blank bars) in the prcsexxx of indicated hormones and [3H]valine (0.1 ~~j/rnl~. t3H~V~ine incorporated into the 2% SSA insoluble fraction in the control c&s was estimated as 100%. Values an the means + SEM for three dishes of hepatocytes. *P c 0.05 and **P < 0.01 show statisti~l~y s~~i~~nt differences between the cells with and without UEP.

487

Enhanced ureogenesis in cultured hepatocytes B

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30

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(30nt.9

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Fig. 7. Effect of UEP on protein degradation in the primary cultured hepatocytes of rats in WE (A) or Earle solution (B). The hepatocytes were cultured with [‘Hlvaline (1 @i/ml) for 24 hr before the

exneriment. Then the cells were incubated for 6 hr with 100ualml UEP (marked bars) or without UEP (blank bars) in the presence of the indicated hormones. ProteG degradation was calculated as % of the radioactivity released from 2% SSA insoluble fraction of cells to the 2% SSA soluble fraction. Values are the means + SEM for three dishes of hepatocytes. *P < 0.05 and **P < 0.01 show statistically significant differences between the cells with and without UEP.

the nitrogen from amino acids released for ureogenesis, because the effect of UEP is depressed when the supply of amino acids is inhibited and is enhanced when the supply is increased. Figure 5 shows the time course of the effect of UEP to elevate the ureogenesis. When WE was changed to arginine-free MEM, the cultured hepatocytes began ureogenesis after a short lag period. Glucagon and UEP seemed to shorten this lag period. This effect of glucagon and UEP also can be explained by the assumption that the nitrogen for ureogenesis is supplied mainly by the amino acids released by degradation of endogeneous proteins. Figure 6 shows the effect of UEP on protein synthesis and Fig. 7, protein degradation of hepatocytes. UEP inhibited protein synthesis and degradation partially either in the presence or absence of

C

G

I

E

A

G+I

glucagon or insulin. The effect of UEP on protein synthesis and degradation was more prominent in Earle medium than in WE. The reason of this result will be discussed later in this article. Figure 8 shows the effect of UEP on thymidine incorporation into hepatocytes. UEP strongly inhibited the thymidine incorporation which was induced by insulin or EGF. This effect of UEP was_dependent on the concentration of UEP (Fig. 9). UEP also inhibited the proliferation of hepatocytes in the presence of insulin or EGF (data not shown). The relationship of the activity to enhance ureogenesis and inhibit thymidine incorporation is not elucidated yet. There is enough possibility that the two activities of UEP is not directly related. Figures 10 and 11 show the effect of UEP on the binding of insulin and EGF to hepatocytes and the

E+I

Fig. 8. Effect of UEP and hormones on the DNA synthesis in the primary cultured hepatocytes of rats. The hepatocytes were cultured for 24 hr with indicated hormones and with 100 pg/ml UEP (marked bars) or without UEP (blank bars). DNA synthesis was measured as the incorporation of [‘Hlthymidine (0.2 tiCi/dish) into trichloroacetic acid-orecipitable material d.uring the 2 hr of incubation. The results were expressed as the rate of the incorporation in the control cells (without hormones and UEP) as 100%. Values are the means f SEM for three or four dishes of hepatocytes. Abbreviations are: C, control; G, glucagon (30nM); I, insulin (30 nM); E, EGF (17 nM); A, db-cAMP (10 PM). **P < 0.01 shows statistically significant differences between the cells with and without UEP.

Fig. 9. Effect of graded levels of UEP on the DNA synthesis in the primary cultured hepatocytes of rats. The hep&cytes were cultured for 24 hr with 17 nM EGF (0). 30 nM insulin (a), or without hormones (0) in the presence of indicated levels of UEP in the medium. The rate of DNA synthesis was measured as described in Materials and Methods. Values are the means f SEM for three determinations. .-I.

Amo TAKWAKAet al.

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10. Effect of UEP on [1251]insulinbinding with its receptor (A) and degradation (B) in the primary cultured hepatocytes of rats. The hepatocytes were incubated with 68 PM UEP (0) or without UEP (e). (A) At the indicated time, the cells were sampled and the bound insulin was detached from the cells by acetate treatment (see Materials and Methods for details). (B) The degradation was measured as the radioactivity appeared in the 10% TCA soluble fraction. Non-snecific binding and degradation (0) was measured in the presence of excksive cokentration,i unlabeled insulin (30 nM). Values are the means f SEM for three dishes of the cultured hepatocytes. degradation of these growth factors by hepatocytes. The effect of UEP on the binding of insulin to hepatocytes and the degradation of insulin by hepatocytes is not obvious. However, UEP increased the binding of EGF to hepatocytes and partially inhibited the degradation of EGF by hepatocytes. This suggests that UEP affects the cycling of EGF receptor between cell surface and the inside of the cells. Figure 12 shows the displacement curve of the binding of insulin or EGF with the cell surface receptors and the effect of UEP on it. The figure also shows the Scatchard analysis of the results. UEP did not affect the binding constant both in the case of insulin and EGF.

affected the activity of such hormones as insulin or glucagon, it little affected the binding of insulin or EGF to their cell surface receptors, although UEP more or less increased the number of bound hormones. Considering these effects of UEP on the action of hormones to cells, the mechanism of the effect of UEP may be explained as follows. UEP depresses both protein synthesis and degradation. However, its effect is more extensive in protein synthesis. As a result, amino acids released after degradation of endogenous proteins will not be reutilized efficiently and increased amount of endogenous amino acids will be metabolized by the cells thereby enhancing ureogenesis. The effect of UEP was more prominent in the presence of glucagon or dbcAMP in the medium. These substances are known to enhance protein degradation in hepatocytes, and consequently are suggested to produce more endogenous amino acids. The effect of bestatin, an aminopeptidase inhibitor which inhibits the step to degrade endogenous small peptides into free amino acids, can also be explained by the above hypothesis. Bestatin decreases the production of endogenous free amino acids by inhibiting the step to produce free amino acids from small peptides. The possible action of UEP to enhance the activity of urea cycle directly was excluded by the observation that UEP did not enhance ureogenesis from arginine or ammonium salts. Our previous observations employing the same culture system showed that the rate of protein syn-

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DISCUSSION

The peptide isolated in the present investigation, showed interesting properties. At first, it enhanced the glucagon-induced ureogenesis. This was also observed in db-cAMP-induced ureogenesis. On the contrary, the peptide inhibited insulin- or EGF-induced DNA synthesis in primary cultures of rat hepatocytes. The peptide inhibited protein synthesis in the culture cells. The latter effects were more prominent in the amino acid-deprived medium than in the ammo acid-supplemented medium. However, this effect was not affected by insulin or ghtcagon. The peptide also inhibited protein degradation. Although UEP

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Fig. 11. Effect of UEP on [‘251]EGF binding with receptor (A) and degradation (B) in the primary cultured hepatocytes of rats. The hepatocytes were incubated with 85 pM UEP (0) or without UEP (@). (A) At the indicated time, the cells were sampled and the bound EGF was detached from the cells by acetate treatment. (B) The degradation was measured as radioactivity appeared in the 10% TCA soluble fraction. Non-specific binding and degradation (0) was measured in the presence of excessive concentration of unlabelled EGF (17 nM). Each value is the mean of duplicate experiments.

489

Enhanced ureogenesis in cultured hepatoeytes

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Fig. 12. Specific [‘2SI]insulin binding (A) and [izSI]EGF binding (C) and Scatchard plot analysis for [L2SI]insulin(B) and [iZSI]EGF (D) in the primary cultured hepatocytes of rats. The cells were incubated at 4°C for 12 hr with 10pM UEP (0) or without UEP (0) in the presence of graded levels of unlabelled insulin or EGF.

thesis is not affected by the valine concentration in the medium at least in the range of valine concentration from that in WE medium to 100 times of it (Takenaka et al., 1989). This means that the effect of UEP is not that of the defect of measurement of the protein synthesis rate. It is difficult to exclude the possibility that the effect of UEP on protein degradation is the result of increase in reutilization of endogenous amino acids. However, previous evidence suggest that the exchange of valine between intracellular and extracellular compartment proceeds relatively quickly (Kato et al., 1989). Furthermore, the present medium contained enough amount of cold valine. The effect of glucagon on protein degradation was not so prominent in the present experiments. As we reported previously (Kato et al., 1989), the effect of glucagon on protein degradation is more prominent in long-lived proteins than in short-lived proteins. In the present experiments, the cells were labelled for 18 hr, washed for 1 hr and the degradation was observed during the following 6 hr. This time interval is enough long to exclude the contribution of the degradation of “short-lived proteins” but not enough to observe the effect of prominent effect of glucagon. In other words, the protein degradation must be enhanced more prominently by glucagon (the effect of glucagon on longer-lived proteins) but the resulting product must be cold valine. Therefore, if we presume that the effect of UEP on the protein degradation is the same in short-, intermediate- and long-lived proteins (the latter proteins are quantitatively largest), more amino acids will be available for ureogenesis than those practically presumed by the

rate of release of [3H]valine. These considerations will favour the above assumption that the ureogenesis enhancing effect of UEP is presumed to be due mainly to increased availability of endogenous amino acids for ureogenesis. The mechanism of UEP to inhibit insulin- or EGF-induced DNA synthesis remains to be elucidated. UEP increased the number of bound EGF on the cell surface receptors through partial inhibition of internalization. However, we tentatively suppose that these effects will not explain the strict inhibition of DNA synthesis. Although we do not have any positive evidence, we assume that some reaction(s) in the signal transduction system, which transfers the signal of insulin or EGF to cell nucleus, is blocked by UEP. This may be due to a general property of protease inhibitors as we suggested in our previous paper (Takahashi et al., 1985,1989), because UEP is known to be an inhibitor of angiotensin~nve~ing enzyme (Maruyama et al., 1985). However, detailed studies are needed for elucidating the effect of UEP on DNA synthesis. At present, we presume that the effect of UEP on hepatocytes is a cytostatic effect of this peptide on the cells. This may be due, at least in part, to the toxic effect of this peptide on hepatocytes, because relatively high concentration (around lo-’ M) is required to observe the ureogenesis-enhancing effect. Even if this assumption is the case, the effect of UEP is specific to UEP because other fractions of tryptic peptides of /I-casein did not show such a prominent effect, if any, on ureogenesis of hepatocytes as UEP. Acknowledgement-The

authors thank Dr Masaaki Yoshikawa, Department of Food Technology, Kyoto University, very much for giving us synthetic UEP. REFERENCES

Brantl V., Teschemacher H., Henschen A. and Lottspeich F. (1979) Novel opioid peptides derived from casein (fl-casomorphins). I. Isolation from bovine casein peptone. Hoppe-Seyfer’s Z. Physiof. Chem. 360, 121I-1216. Geyer J. W. and Dabich D. (1971) Rapid method for determination of arginase activity in tissue homogenates. Anafyt. Biochem. 39,412-417.

Greenwood F. C., Hunter W. M. and Glover J. S. (1963) The preparation of ‘3iI-labelled human growth hormone of high specific radioactivity. Biochem. 2. 89, 114-123. Jolles P.. Levv-Toledano S.. Fiat A.-M.. Soria C.. Gillessen D., Thomaidis A., Dunn F. W. and Caen J.‘P. (1986) Analogy between fibrinogen and casein. Effect of an undecapeptide isolated from K-casein on platelet function. Eur. J. Biochem. 158, 379-382. Kato H., Takahashi S.-I., Takenaka A., Funabiki R., Noguchi T. and Naito H. (1989) Degradation of endogenous proteins and internalized asialofetuin in primary cultured hepatocytes of rats. Inr. J. B&hem. 21,483-495. Loukas S., Varoucha D., Zioudrou C., Streaty R. A. and Klee W. A. (1983) Opioid activities and structures of a-casein-derived exorphins. Bioclrenrisrry 22,4567-4573. Maruvama S. and Suzuki H. (1982) A nentide inhibitor of an$otensin I converting enzyme in the’ tryptic hydrolysate of casein. Agric. Biof. Gem. 46, 1393-1394. Maruyama S., Nakagomi K., Tomizuka N. and Suzuki H. (1985) Angiotensin I-converting enzyme inhibitor derived from an enzymatic hydrolysate of casein. II. Isolation and bradykinin-potentiating activity on the uterus and the ileum of rats. Agrfc. Bfof. Chem. 49, 1405-1409.

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