Decreased liver cytosol glucocorticoid receptors in protein-deficient rats

Life Sciences, Printed in the

Vol. 28, U.S.A.

pp.

Pergamon

2719-2725

II‘J PKOTEIN-DEFICIENT RATS

l~tiCKEASk,ll LIVEK CY'I'OSOL GLUCOCUK~LCOlD KaCZPWKS D.R. Departments of Pharmacology 3655 Drlummond St., Montreal, (Received

Varma

and

S.

& Therapeut its, Quebec, Canada, in

final

form

Press

Mulay and Medicine, H3G lY6. April

2,

McGill

University,

1981)

-Summary

A low protein diet (5% as compared to a control 21% protein decrease in the concentraad libitum) caused a significant diet, ~_____ ticIn of liver cytoplasmic glucocorticoid receptors; the equilibrium The ,m~,,uld not be attributed to elevated plasma corticosterone levels since a comparable increase in plasma corticosteri)~rr’ <)E caloriedeficient rats (21% protein diet in restricted quantity) did not decrease glucocorticoid recepf.fJr,; 41111 the difference in receptor levels of control and protein deficient animals persisted following adrenalectomy. These results suggest that glucocorticoids might in the presence of protein not exert their usual biologic effects mal.ntltrition.

It is generally agreed that most biological actions of glucocorticoids are interactions (1) and alteraexerted through highly specific steroid-receptor t lens in glucocorticoid receptors can rrslrlt in changes in their effects Age-related changes in the responsiveness to glucocorticoids have been (2,3). attributed to alterations in glucocort icoid receptors (3) ; similar changes during other situations are not unlikely. We recently reported that dietary protein deficiency in rats was associated with a decrease in the anti-inflammatory and ulcerogenic effects of dexamethasone, which could not be attributed to any pharmacokinetic changes (4). Tnese results suggested that protein deficiency might induce alterations in glucocorticoid receptors. The experiments described below were done in order to test sucn a possibility using livers of protein-deficient animals as a model system. Materials

and Methods

Sprague-Dawley male rats weighing between lOO-1251: and approximately 5 weeks old at the beginning of these studies were placed individually in suspended wire-b:,t to~ns,J cngrs. Animals had free access to water and were either fed ad libitum isocaloric diets (Teklad Test Diets, Madison, Wisconsin) containing 21X (control) or 5% (low) protein, or were fed a restricted quantity (log/rat/day) of a 21% protein diet (pair-fed controls) (4,5). To minimize the influence of endogenous corticosteroids on receptor assays, some animals were adrenalectomized under ether anesthesia and maintained on 1% saline instead of 0024-3205/81/242719-07$02.00/O Copyright (c) 1981 Pergamon

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water for 3 days prior to receptor assay; controls were sham operated (6). For collection of blood (cardiac puncture) and tissues, animals ‘were anesthetized with a mixture of carbon dioxide and oxygen (50%:50%). plasma corticosterone was measured by radioimmunoassay (ant ibodies purchased from Endocrine Sciences, California). Proteins were measured according to the method of Lowry et al (7)and DNA was assayed by the diphenylamintd ,o~‘:‘I 1~1 (3). Preparation of liver cytosol and assay of cytoplasmic glucocort icoicl receptors was done as described by Giannopoulos (9); for this purpose c3H)dexamethasone (40 Ci/mmol, New England Nuclear, Mass.) was incubated with liver cytosol for 3 hours at 4OC in the absence and presence of a 100-fold excess nonlabeled dexa:nethnsone (Sigma Chemicals, St. Louis I Hiss.). Unbound steroid was separated witn dextran coated charcoal and specific binding was estimated as the difference between the total binding and the binding in the presence of excess nonlabeled dexamethasone. Linear sucrose density gradient (IO-30%) stlldies using (‘3il)-hovin,? s~=rum alhlllnin as a marker, competition studies with SeVer:sl st-roids (see results) and binding measurements at 37°C were done in order to ascertain the specificity of binding to receptors and exclude, as far 4s possible, any binding to albumin or other non-receptor macromolecules (9). Uata were analysed according to the technique of Scatchard (10). Differences between two means were compared according to Student’s t test, and were treated as significant at p< 0.05.

Results A decrease resulted in a plasma proteins, a significant influence of cant decrease tort icosterone

in dietary protein to 52 as compared to a control value of 21% significant reduction in body weight gain, liver weight, total plasma albumin and globulins, and in liver proteins; there was elevati,,, i:l pl.151~ ~:orf icoster.,ne levels (table I). The main food restriction (pair-fed controls) was a moderate but signifiin body weight dain and liver weight, and an increase in plasma levsls (Table 1-j.

Some Consequences

of

Dietary

Protein

or

Calorie

Deficiency

Dietary

protein

Parameters

21% -_ ad lib _--

5% __--ad lib

Initial body weight (g) Final body weigI?t (g:) Food consumed (g/day/rat) Plasma protei,‘; <::/,lL) Plasma albumin (g/dl) Plasma globulins (g/dl) [Liver weight (g:) Liver pr:‘teins (:ng/g) I.iver IUNA (mg/g) Plasma corticosterone (ng:/ml)

112 334 17 7.n 2.9 3.6 13.2 165 5.2 32

116 128 10.6 5.7 2.3 3.3 6.1 128 7.9 154

+2 i 7.7 +0.6 ?I). 12 io.02 +O.l 20.04 +4 +0.1 i8

in vale (4

+2 2 1.6* + 1.h -i +0.13” +0.08” iO.1” iO.25” _ + 11” _ 2 1.2 -t +ZY t

Rats

weeks) 21% (pair 114 254 10 7.5 2.8 3.6 9.9 168 7.1 162

i2 24.9 io 20.27 20.08 kO.06 +0.43 ?5 fO.2 +32

* Denotes of at least 8 expzrilnents. Each value is the mean + S.E.i+. significant difference from respective values on the left as well as -i_ denotes diEEerrnce only from the In the same row; on the right values on the left (p ~0.05).

fed)

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The binding of (3H)-dexamethasone to liver cytosol of control and proteindeficient animals as a function of its concentration and Scatchard plots are the binding pattern of labeled shown in Fig. 1; specific binding was saturable; dexamethasone to liver cytosol of pair-fed controls was similar.

-0

[q-oEX:HETHAs13r4E BO”LJ bmoles/ml

I 0

1

25 SUBSTRATE

CYTOSOL)

I

I

50 CONCENTRATION

FIG.

75 (nM)

1.

Specific binding of (3H)-dexamethasone to liver cytosol from male rats fed ad lib a 212 (control) or a 5% (low) protein diet for 4 weeks. Aliquots of 0.3 ml cytosol cant aining approximately 5 mg protein were incubated for 3 hours at 4°C with increasing concentrations of (3H)-dexamethasone in the absence and presence of a lOOfold excess of nonlabeled dexamethasone. The quantity of specifically bound labeled steroid was determined by charcoal assay. Inset Each point is the mean+ Scatchard (10) plot of the data. -2 S.E.M. of at least 8 separate experiments.

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The specific binding of (3H)-dexamethasone at 4°C corresponded to 6-8 S and it was negligible at 37°C. The ability of various steroids to compete for c3H)-dexamethasone binding sites in the cytosol of both control and proteindeficient animals were very similar and correlated well with their glucocorticoid potency (Table II).

TABLE II Effect

of Nonlabeled Competing Steroids on the to Liver Cytosol of Control and Protein

Binding of Deficient

(3H)-Dexamethasone Male Rats.

Control Group Protein Deficient Group ___-_ X Bound (3H)-Dexamethasone % Bound (3H)-dexamethasone _-Mean ?S.E.M. Mean &S.E.M. -None 100 100 Dexamethasone 11 +1.0 16 ?r3.0 Yu-fluoroprednisolone 11 +1.2 13 22.2 Fludrocortisone 14 &I .Y 14 tl .o Triamcinolone acetonide 14 k2.2 17 22.0 bu-Methylprednisolone 15 k2.5 19 +l.l Cortisol 23 +3.0 19 +2.5 ll-Dehydrodexamethasone 24 k2.1 19 k1.7 Corticosterone 33 i5.6 27 f2.6 Aldosterone 58 +4.2 5Y k6.1 Progesterone 89 +2.6 81 k4.2 Estradiol-17 D 71 +4.0 82 k4.2 ll-Deoxycortisol 88 k1.1 86 i-2.3 Cortisone 82 +8.0 87 i5.1 Pregnenolone 93 22.9 YO 22.0 Testosterone 92 k4.6 93 i2.0 ___--_----__ -----_-------Competing

Steroids

(3~)-Dexam~~thasone (25 nM) was incubated at 4°C for 3 lhours in the prcsenc? t>f 2.2 UM (IO~-f,>ld excess) of nonlabeled colnpeting sterJids with liver cytosols from rats fed __ad lib a 21% (control) or a Bound c3H)-dexa5% (protein deficient) protein diet for 4 weeks. methasone was determined following treatment with dextran coated nonspecific binding. charcoal; correct ions made for the 110 were None of the values in Each value is derived from 4-7 experiments. the control group are significantly different from the corresponding values in the protein deficient group.

The influence of a low protein diet on the specific binding OE c3H)-dexamethasone to liver cytosol was obvious within one week and reached a imaximum in when rats maintained on a 5% protein diet for 3 weeks were fed a two weeks; labeled dexn.nethasone tg> cytosol control diet (21% protein), the binding of returned to control levels in approximately one week (Figure 2). decrease in the A low protein diet ‘was associated with a significant specitic binding of (3H)-dexamethasone to liver cytosol; on the other hand, approximately equal to the restriction of a control protein diet (10 g/rat/day, food consumed by protein deficient animals) did not change the specific binding (Table III). Bilateral adrenalectomv increased the binding of [3H]-dexameti,asane to liver cytosol of both control and protein deficient rats; however the

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Vol. 28, No. 24, 1981

between the two groups of animals with respect to the binding drf ference The equilibrium dissociation constant (Kd) did maintained (Table III). deficient d~ri,n~l.<. differ brt,q<,:jl L’IIL ;~:)ntr~,l aild protein

Dietary

protein

LI

21% (control)

q

5%

q

5% for 3 weeks followed

0

7

14

28

21

by 21%

35

49

DAYS

‘Cine-cnllrse \)f changes in I iver ,cytoplasmic glucocorticoid receptors F~>I.I.owing drctary protei 1 k>ri v:lt i(>ll and repletion in male rats. DLrts Nere fed ad libitum and animals were sacrificed on the days of 4-13 separate experiL~.li~‘~f +d. Each value is the mean +S.E.M. ,,r’il.L5 .nll.i ,das derived by Scatchard aolalysis (10) of the data obtained following krclrbatic,n of (3H)-dexamethasone at 4°C for 3 hours with liver cytosol in the absence and presence of lOO-fold nonlabeled dexamethasone. * Indicates difference (p < 0.05) from the values corresponding to the same experimental duration.

2723

was not

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TABLE III Influence

of

Dietary

Protein on the Binding Liver Cytosol of Male

to

Dietary protein for 4 weeks -----------_21X .-__ ad lib 5i ad lib 21Xx-fed

----

(controls) (10

g/day)

Nonadrenalectomized Capacity (pmoles/m& ~~~--_-_-~~_~~~~ DNA) (2, ------5.79 1.26 4.76

10.38 +0.18* +0.70 _--_--~-~

of (3H)-Dexamethasone Rats Adrenalectomized _--_____-_--_______ Capacity (pmoles/mg DNA) __

43 +3.5 7.06 30 k7.1 3.94 54 +3 7” ------_---.-

Kd (nM)

kO.26 i_ 26 k5.4 cO.48jkt 18 +3.1 -----.__-_---__

Binding studies (n = b-13 each group) were done at 4°C in the, presence and absence of loo-fold excess nonlabeled dexamethasone; capacity and Kd were estimated by Scatchard (10) analysis. “Indicates significant difference (p < 0.05) fro.11 the control values in the top row and tindicates significant difference (p <0.05) from the corresponding value in non-adrenalectomized animals s 4 “62 d il
Discussion General very similar corticosterone been reported

effects of a low to those reported levels following by others (11).

protein diet in earlier (4,5); a protein or

rats found in tllese also, the elevation calorie deficiency

studies are of plasma in rats has

All the characteristics of the binding of (3H)-dexamethasone to cytoplasmic macromolecules found in these studies (saturable specific binding at 4°C; negligible binding at 37°C; binding peak corresponding to 6-8 S; reasonable correlation between the ability of various steroids to compete for dexa.neiiiasone binding sites and their glucocorticoid potency) are similar to those reported for binding with cytoplasmic glucocorticoid receptors (6,9,12,13). The results of this study show that a decrease in dietary protein is associated with a significant reduction in the concentration of liver cytoplasmic Since no such decrease was found in pair-fed conglucocorticoid receptors. trols, it can be assumed that these changes are the result of a protein rather The present findings extend our preliminary observathan calorie deficiency. tion (14) and are consistent with the reported reduction in the binding of The exact mechanism by cortisol to the lens of protein-deficient monkeys (15). leads to a reduction in glucocorticoid receptors is which a low protein diet It is unlikely to be the result of elevated not clear from these studies. plasma corticosterone levels since a comparable increase in the plasma corticosterone of pair-fed controls was not associated with any reduction in these cytoplasmic glucocorticoid Moreover, differences between liver receptors. animals persisted following receptors levels of control and protein deficient adrenalectomy (Table III). It may be argued that protein deficient animals changes such as an increase receptor-steroid degradation; certain the present study, example, protein deficiency

the observed decrease in glucocorticoid receptors in is more apparent than real and might result from inact ivat ing enzymes or in some inhibitory factor, although this cannot be ruled out on the basis of For observations point against such a possibility. in nuclear also associated with a decrease is

t +

Vol.

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24,1981

Glucocorticoid

Receptors

glucocorticoid receptors and in the tryosine of dexamethasone (unpublished observations). mechanism for the decrease in the specific protein deficient animals are in progress.

in Malnutrition

2725

aminotransferase inducing eEfect Studies designed to ascertain tha-! binding of (3H)-dexamethasone in

The studies on the time-course of these changes show that the onset of the influence of dietary protein deficiency on glucocorticoid receptors in rats is (approximately 1 week) and the process is reversible upon relatively quick repletion of dietary protein (Figure 2). Glucocorticoids are used in the treatment of diverse clinical conditions throughout tihe world including developing countries in many of which proteincaloric malnutrition is widespread. Assuming that an alteration in these receptors can lead to changes in glucocorticoid effects (2,3), it would appear that tnese valuable agents might not exert their expected effects in a fair proportion of patients requiring glucocorticoid therapy in developing countries; perhaps a correction of nutritional status prior to the institution of therapy may prove fruitful.

Acknowledgements This work was supported by grants Canada. We sincerely thank Ms. Christine skilled technical assistance.

from the Schwenter

Medical and Ms.

Research Joan Lee

Council, for their

References 1. 2. 3. 4. 5. 6. 7. 8. Y. 10 11 12 13 14 15

R.J.B. KING and W.I.P. MAINWARING, Steroid-Cell Interaction, p. 305 University Park Press, Baltimore (1974) A.W. HARRIS and J.D. BAXTER, Glucocorticoid Hormone Action Eds. J.D. _____I Baxter & G.G. Rousseau, p. 423, Springer-Verlag, New York (1979). G.S. KOTH, Mech. Ageing Dev. 9 497-514, (1979) D.R. VARMA and S. MULAY, J. Pharmacol, Exp. Ther. 214 197-202 (1980) U.K. VARMA, J. Pharmacol. Exp. Ther. -211 388-344 (1979) P.L. BALLARD, J.D. BAXTER, S.J. HIGGCNS, G.G. ROUSSEAU and G.M. TOMKINS, Endocrinology 94 998-1002 (1974) O.H. LOWKY, NT ROSEBKOUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. lY3 265-275 (1951) K. BUK'I'ON, Biochem. J. 62 315-323 (1956) G. GIANNOPOULOS, Endocr%ology 94 450-458 (1974) G. SCATCHARD, Ann. N.Y. Acad. Sz. 51 660-672 (1949) W.A. COWAKD, R.G. WHITEHEAD and P.GTLUNN, Hr. J. Nutri. 38 115-126 (1977) E.H. EPSTEIN, Jr. and N.H. MUNDEKLOH, Endocrinologv 108 703-711 (1981) R.G. MACDON4LD and .J.A. CIDLOWSKI, J. Steroid Biochem. 10 21-29 (1979) S. MULAY, C.A. BROWNE, D.R. VAKMA and S. SOLOMON, Federation Proc. 39, 261-263 (1980) S. ONO, H. HIRANO, H. UEMURA, H. TAKAHASHI, K. OBARA, T. HATAKEYAMA and H. WAKO, J. Nutri. Sci. Vitaminol. 122 249-254 (1979)