Chinese Journal of Oceanologyand Limnology Vol.19, No.2, P.135-140, 2001

UP-REGULATION OF HEPATIC RECEPTOR FOR GROWTH HORMONE IN THE FLOUNDER (PARALICHTHYSOLIVACEUS)AFTER ORAL ADMINISTRATION WITH EXOGENOUS GH" LIU Z o n g - z h u ( ~ ] ~ j ~ ) + ' ' , WANG J i n - b a o ( t ~ ) ' , XU Y o n g - 9h ("r~ , ~ 3 ~ ) t WANG Yong(~E ~ ) + , ZHANG P e i - j u n ( ~ ) * ( ffnstitute of Oceanology, ChineseAcademy of Sciences, Qingdao 266071, Gh/na) ( #Dep. Anita. Sci. LaiyangAgriculture College, Laiyang 265200) Received July 6, 1999; revision accepted Sept. 5, 2(100

Abstract The iodination efficiency of salmon GH(sGH) was 38.82 %, using a modification of the chlommine-Tmethod. The specific activity of the 1~I-sGH was about 40 ttCi/gg protein. The results of binding assay showed a single class of high affinity and low-capacity binding site in flounder liver. Long-term administration with exogenous GH can induce the up-regulation of hepatic GH receptor in total binding capacity though there was no significant difference of association constant among any groups. Considering that there was no significant difference in capacity of free binding sites of livers from control and experimental fish, this result also indicated that the liver from experimental fish, compared to that from control fish, had more occupied binding sites.

Key words: iodinatiun, GH, hepatic receptor, up-regulation INTRODUCTION It is well established that the mammalian liver is a major target organ for growth hormone ( G H ) . As for nonmammalian vertebrates, specific GH-binding sites are present in several tissues and are predominantly concentrated in the liver ( Hirano, 1991 ; Yao et a l . , 1991 ), furthermore, GH appears to be involved in regulating its own receptors by down- and up-regulation(Gray et a l . , 1992; Moil et al. , 1 9 9 2 ) . In our other experiment, we observed that long-term administration with exogenous GH improved the growth rate of flounder and elevated IGF-I concentration in plasma of experimental fish (Liu et a l . , 1999 ). The present study was undertaken to find out whether there was any difference of hepatic GH receptor capacity and binding affinity between control and experimental fish. MATERIALS AND METHODS

Collection of samples Young flounders ( 2 1 0 . 6 + 8 . 7 g ) were cultured with normal diet in 4 large cement pools (64 rnz , 4300 fish/pool, 2 for experiment and 2 for control). Recombinant yeast containing salmon GH was mixed into the diet of the experimental fish at a ratio of 1:1000 (wet weight). After 5 months' feeding, 12 fish were randomly sampled from both experimental and control groups. The livers of each fish were removed and stored at - 70~ for further study.

* Contribution No.247 of Experimental Marine Biology laboratory and 3798 of the Institute of Oceanology, Chinese Academy of Sciences. Project 96-C01-05-04 supported by National "Ninth Five Year Plan Key Problem".

136

CHIN. J. OCEANOL. LIMNOL., 19(2), 2001

Vo[.19

Iodination of sGH Purified salmon GH was supplied by Dr. C. L. Hew of Toronto University. The GH was iodinated using the chloramine-T method with some modification (Greenwood et a l . , 1963). Five hundred microcuries N a l ' I in 2.2 t.d of lmmol/L NaOH,pH 8-11 ([MS-30,Amersham) ,and 100 ttg chloramine-T (5 mg/ml in 500 mmol/L PB, pH 9.0) were added to 5 t~g sGH in 10 gl of 500 mmol/L PB, pH 9 . 0 . The above reagents were immediately mixed well by vortexing, and the reaction was terminated after 40 seconds by addition of 20 pl of sodium metabisulfite (12 mg/ml in 500 mmol/L PB, pH 9 . 0 ) . Unreacted iodide was separated from the labeled hormone by gel filtration on a Sephadex G-50 column (0.5 x 30 cm). The iodination mixture was eluted with 500 mmol/L PB (pH 9 . 0 ) at 400 pl/min, and 1200 pl fractions were collected in separate tubes and the radioactivity in each tube was counted in a gamma counter. Receptor preparation Frozen livers were minced and then homogenized for 3 minutes in 5 Vol(w/v) of 40 mmol/L Tris-HCl buffer (pH 7 . 4 ) , containing 10 mmol/L CaCI~, 1 mmol/L phenylmethyl sulfonyl fluoride (PMSF) and 5 nmol/L thioethylenglykol using a glass homogenizer. The tissues were then homogenized with an ultrasonic homogenizer for 5 min. The homogenizer were kept in an ice bath during all procedures. The homogenates were subsequently centrifuged at 600 g for 45 min. at 4 ~C. The supernatant was collected and centrifuged at 10000 g for 30 rain at 4 ~C. The resultant pellets were washed twice with 2 Vol of Tris buffer and resuspended at Ig starting tissue weight per milliliter of buffer. Half of the suspension was treated with 4 mol/L MgC12 to remove bound GH from the receptor as described by Sakamoto and Hirano( 1991 ) . An aliquot was taken to determine protein content by Bradford assay using bovine serum albumin (BSA) as a standard. The receptor preparation was then stored at - 70 ~C. Just before use, the frozen suspensions were thawed and diluted with the extraction buffer to appropriate concentrations and homogenized with ultrasonic homogenizer for 10 s to disperse the receptor thoroughly. Binding experiments AH solutions were prepared with 40 mmol/L Tris buffer containing 10 mmol/L CaC12 and 0 . 5 % BSA. Specific binding was estimated by incubation of 100 ttl receptor preparation with 20000 to 900000 CPM of 1~ I-sGH in 50/.d buffer in the presence or absence of an excess amount of unlabeled sGH (200 - 500 times that of labeled hormone). The total volume of the reaction mixture was 200 td. Plastic sample tubes used for incubation were placed at (8 - 15~ ) for 24 h, with 5 min shaking in every 6 h. Incubation was terminated by adding lml ice-cold Tris-HC1 buffer followed by centrifugation at 4000 r/rain for 30 min at 4~ The radioactivity of the resultant pellet was counted in an automatic gamma counter. Statistical analysis Equilibrium association constant ( K a ) and binding capacity ( N ) of GH receptors were calculated as described by Klotz (1982). Values were expressed as means • S. E . , with statistical difference compared using Student' s t-test. RESULTS

No.2

IAU et al. : UP-REGULATION OF HEPATIC GH RECEPTOR IN FLOUNDER

137

Iodinafion of sGH As shown in Fig. 1, iodinated sGH was eluted first, followed by a smaller free 1~ I peak. The total radioactivity of labeled sGH was 38.82% of that added to the reaction mixture (230190/ 92975, 10 s count), thus the specific radioactivity of labeled sGH in this experiment was about 40 ttCi/ttg protein. Considering our previous experience (decline fractions of the peak usually had high bioactivity). We took mixed fractions of No. 17 and No. 18 tubes, and diluted them with 50% BSA for further use in the binding experiment.

45000 40000 -~ 35000 30000 25000 ~ 20000 15000 :~ 10000 ,.r 5000 0 -5000 0 Fig.1

5

I0

15

20

25

30

35

Number of tube Separation of

12SI-labelled sGH from [lz~I]iodide on Sephadex G-50

Equilibrium association constants and concentration of sGH free binding sites in the flounder Hver The specific binding of labeled sGH to liver membranes was saturable with increasing amount of label. Scatchard plot analysis revealed a single class of high-afflnity binding site (Fig. 2 ) . The equilibrium constant of association and the capacity for specific binding in the livers from control and experimental fish were not significantly different.

25.00

5.00

~, 20.00

4.00

15.oo

3.00 x

=~ lO.OO ;~

~

2.00 1.00

5.00

0.00 0.00 0.000 0.500 1.000 1.500 2.000 2.500 3.00 3.500 Free(nmol/L) Fig.2

5.000

35.000 9 45.000 15.000 25.000 Binding (tmol/L)

Saturationcurve (a) and Scatchard plot of free specific binding ( b )

138

CHIN. J, OCEANOL. LIMNOL., 1 9 ( 2 ) , 2001

Vol.19

Fait~'lrium association eomlaias and eommaration of sGH total birding sites in the flounder liver As shown in Fig. 3, liver membranes treated with MgC12 were saturable at a much higher amount of labeled sGH. The Scatchard plot analysis also revealed a single class of high affinity binding sites. Although the equilibrium constant of association for specific binding showed no significant difference between control and experimental fish, the capacity of total binding site of experimental fish increased significantly compared with that of control fish (Table 1 ).

50.00

I0.00, 9.00} b 8.00|

40.00

6.oo t ",,,,

60.00

7.oo1~

30.00

5ool ~. 4.00~

20.00

"~ 3.06t " ~

10.00 0.0s 0.00

, 0.500 1.000 1.500 2.000 2.500 3,000 Free (nmol/L) Fig.3

35oo :

o

"x.

2o

-,,,.

4o 6o

,oo

8o

120

140

Binding (fmol/L)

Saturation curve (a) and Scatchard plot of total specific binding (b)

Table 1 Equilibrium association constant (/Ca ) and bindin~ capacity ( N ) of GH receptors in the liver h e m different fish (Values are means • S . E . ) liver n free 6 total 6 occupied exp. fi~e 6 total 6 occupied a) P < 0.05; b) P < 0.01 compared with control (Student's t-teat). con.

Ka (liter/nmol)

N (fmol/mg protein)

1.125+0.102

30.2+2.4

1.340 + 0.213

56.9 + 3.6

1.149+0.113

28.7+ 1.8

1.560 + 0.207

78,6 + 4.9 a) 49.9 + 1.4 b)

26.7 + 1.2

Effects of exogenous GH on equilibrium association constants and capacity of GH binding site in the flounder liver

There was no significant difference of equilibrium association constant among any groups, but the capacity of total binding sites of the experiment group was higher than that of the control, consequently, the occupied binding sites of the liver from experimental fish increased significantly over that from control fish(Table 1). DISCUSSION

There are many methods available for labeling GH with radioactive iodide and many factors affecting the efficiency of iodination. Bohon et al. (1986) developed a procedure for iodination of lower vertebrates GH with [lZSI] iodide, but the labeled hormone they got had much lower specific activity (2 - 5 ttCi/ttg). In our experiment, we mainly followed the procedure of Bohon except that we enlarged the amount of NanSI from 200 ktCi to 500 ttCi and prolonged the reaction time from 20 s to 40 s. With these modification, we got l~I-salmon GH with much higher specific activity (40 ttCi/ttg) 9

No.2

LIU et al. : UP-REGULATIONOF HEPATIC GH RECEPTOR IN FLOUNDER

139

The basic properties of hepatic receptors for GH of lower vertebrates had been studied using homologous GH as radioligand (Fryer, 1979; Hirano, 1991). In the present study, by using salmon GH as radioligand, we observed similar GH receptor binding activity in the liver of flounder, which characterized by high affinity ( Ka = 1 - 2 liters/nmol) and low capacity ( N = 1 - 100 fmol/mg protein). Hirano (1991) showed that recombinant salmon GH competed equally with t25I-eGH for the receptor sites in the eel liver. Our result indicated that the hepatic GH receptor of eel and flounder may have similar affinity to both salmon GH and ingenuous GH. Exposing membranes to 4 mol/L MgCI2 can remove the bound hormone from their receptors so that the datum of total binding sites can be obtained (Maiter et al., 1988). In our experiment, both free and total binding sites were tested. The results showed no significant difference in free binding sites between two groups; on the other hand, the total binding sites of experimental fish increased significantly compared with that of control fish, indicating that more GH had occupied hepatic receptors in experimental fish. Our result is in accord with the Mori et al. experiment (1992), in which they found that total binding sites increased gradually to almost twice that of the controls 5 days after intramuscular injection of GH, indicating that GH plays an important role in induction and maintenance of its own receptors in the fish liver. The mechanism of receptor regulation by homologous ligands probably depends on the ability of peptide hormone receptors to behave as mobile entities within the plasma membrane. It is generally accepted that in mammals, after the occupancy of the receptor with circulating GH, the GH-receptor complex is internalized, processed, and transported among intracellular organelles (Picard and Postel-vinary, 1984). Internalization processes seemed to stimulate intracellular synthesis of new receptors (Maiter et a l . , 1988). Our results also suggested that the flounder liver, as a major target organ of GH, responded to changes in ambient GH mainly by regulating the number not affinity of its surface receptor. The involvement of other hormonal factors and of nutritional factors in this up-regnlation remains to be examined. Duan et al. (1993) showed that injection of coho salmon GH resulted in a dose-dependent increase in hepatic IGF-I mRNA levels. In this study, significant occupancy of hepatic GH receptor was observed in experimental fish. Considering the elevation of IGF-I concentration in plasma of experimental fish observed in our experiment (Liu et a l . , 2000), it is safe to suggest that GH promotes growth mainly by binding to liver receptors and stimulating production of IGF-I in liver. Kawauchi et al. (1986) reported that growth rates increased during the post - treatment period in rainbow trout injected with natural chum salmon GH. Whether this post-effect of growth enhancement after termination of exogenous GH treatment is mainly due to the up-regnlation of hepatic GH receptors, or due to other endocrine changes ( e . g. thyroid gland, gonads, pancreas, etc. ) deserves further study (Higgs et a l . , 1976). References

Bolton, J. P., Takahashi, A., Kawauchi, H. et al., 1986. Developmentand validation of a salmon growth hormone radioimmunoassay. Gen. Comp. Endocrinol. 6 2 : 230- 238. Duan, C., l)uguay, S. J., Pliskaya, E. M., 1993. Insulin-like growthfactor I (IGF-I) mRNA expressionin coho salmon, Oncorhynchus kisutch: Tissue distribution and effects of growth hormone/prolacfinfamilyproteins. Fish Physiolo. Biochem. 1 - 6:371 - 379. Fryer, J. N., 1979. A radioreceptor assay for purified teleost growth hormone. Gen. Comp. Endocrinol. 39:123 - 130. Gray, E. S., Kelley, K. M., Law, S. et al., 1992. Regulationof hepatic growthhormone receptors in coho salmon ( Oncorhynchus kisutch). Generaland Comparative Endocr/no/ogy 8 8 : 2 4 3 - 252. Higgs, D. A., Donaldson, E. M., McBride, J. R. et al., 1976. Influenceof bovine growth hormone and L-thyroxine on growth,muscle compositionand histological structure of the gonads, thyroid,pancreas and pituitary of

140

CHIN. J. OCEANOL. LIMNOL., 19(2), 2001

Vo1.19

eoho salmon ( Oneorhynehus kisutch ). J. Fish Res. Boad Can. 33:1585 - 1603. Hirano, T. 1991. Hepatic receptors for homologous growth hormone in the eel. C,en. Corap. Endirinol. 81:383 390. Kawanehi, H . , Mosiyama, S . , Yaanda, A. et el., 1986. Isolation and charaeteilzation of chum salmon growth hormone. Arch. Biochem. Biophys. 244- 5 4 2 - 552. Klotz, I. M., 1982. Number of receptor sites from Scatehard graphs: fact and fantasies. Sc/ence 217. 1247 - 1254. Liu, Z. Z . , Xu, Y. L . , Xu, D. W. et a l . , 1999. Effects of exogenous growth hormone on muscle composition of flounder ( Paralichys olivaceus ). Marine Sciences 5. 51 - 53. Liu, Z. Z . , Xu, D. W . , Wang Y . , 2000. Endocrine changes of flounder (Paralichthys olivaceus) after oral administration with exogenous growth hormone. Chin. J. Oceanol. Limnol. 18(4) : 315 - 319. Malter, D . , Underwood, L. E . , Maes, M. et al., 1988. Acute down-regulation of the sematogenic receptors in rat liver by a single injection of growth hormone. Endocrinology 122. 1291 - 1296. Moil, I . , Sakaraoto, T., Hirano, T. 1992. Growth hormone(GH)-dependent GH receptors in the Japanese eel, Anguilla japonica : Effects of hypophysectomy and GH injection. C,en. Comp. Endocrinol. 85 : 385 - 391. Pieard, F . , Postel-vinary, M. C . , 1984. Hypephyseetomy and growth hormone receptors in liver membranes of male rats. Endocrino/ogy 114: 1328- 1333. Sakamoto, T . , Hirano, T., 1991. Growth hormone receptors in the liver and osmoregulatory organs of rainbow trout: characterization and dynamics during adaptation to seawater, J , Endocrinol. 130: 4 2 5 - 443. Yao, K . , Niu, P. D . , Le Gac, F. et al., 1991. Presence of GH specific binding sites in rainbow trout ( Oneorhynchus mykiss ) tissues: Characterization of the hepatic receptor. Gen. Comp. Endocrinol. 81 : 72 - 82.