Early effects of cortisone on nucleic acid and protein metabolism of rat liver

BIOCHIMICA ET BIOPHYSICA ACTA

EARLY

EFFECTS

OF CORTISONE

AND PROTEIN

METABOLISM

495

ON N U C L E I C A C I D OF RAT LIVER

MURIEL FEIGELSON', PAUL R. GROSS'* AND PHILIP FEIGELSON

Department o/ Biology, New York University and Departments o/ Biochemistry and Medicine, College o/ Physicians and Surgeons, Columbia University, New York, N.Y. (U.S.A.) {Received July 28th, 1961)

SUMMARY

Parenteral administration of a single low dose of cortisone acetate has been shown to exert anabolic influences on nucleic acids and proteins of both normal and regenerating rat livers. [3~P]Orthophosphate and [2-14C]glycine incorporation in vivo into RNA of all subcellular constituents of normal and regenerating livers was markedly stimulated. Maximal response was attained 4 h after hormone injection. Since increased RNA turnover has been shown to be associated with increases in the absolute quantity of RNA in the liver, it is concluded that net synthesis of RNA is an early response to cortisone. In normal liver, the stimulatory influence of cortisone on RNA metabolism was more marked when [2-1~C]glycine served as precursor than when radioactive orthophosphate was employed, suggesting hormonally-induced enhancement of purine nucleotide biosynthesis, as well as stimulated nucleotide polymerization into RNA. A cortisone-induced stimulation in incorporation of [l~C]glycine into the proteins of each subcellular fraction has been demonstrated in both normal and regenerating liver, which is of similar time course, although of lesser magnitude than the stimulation of this precursor into RNA of the same fraction. Cortisone administration was shown to result in a transient stimulation and a subsequent depression in the incorporation of precursors into the DNA of regenerating liver.

INTRODUCTION

Cortisone is known to rapidly induce a marked augmentation in the levels of several hepatic enzymes1,~. In view of the possible role of RNA in this process, it seemed important to investigate the effects of this hormone on liver RNA and total protein metabolism. It is well known that adrenal steroids exert a catabolic influence on protein and RNA metabolism in the mammalian organism as a whole3-s. Depressions in the rates of synthesis of these macromolecules in a variety of individual organs, including liver, have been reported ~-I~. Under different experimental conditions, however, anabolic responses in liver4,8,15-I~ have been observed. This study represents an attempt to elucidate the initial modifications in liver " Part of this work is derived from a doctoral dissertation, presented by M. F. to the faculty of New York University in partial fulfilment of the requirements for the degree of Doctor of Philosophy. "" Present address: Department of Biology, Brown University 12, R. I. (U.S.A.).

Biochim. Biophys. Acta, 55 (1962) 495-504

496

M. FEIGELSON, P. R. GROSS, P. FEIGELSON

metabolism as measured b y the incorporation of radioactively labeled precursors into liver RNA, DNA, and protein of various subcellular constituents, following a single low dose of cortisone. The absolute amounts of these substances have been quantitated to complement the isotopic tracer experiments. The influence of the proliferative state of the target organ on its response to hormonal stimulation was assessed by comparing the metabolic behavior of mitotically active regenerating liver with that of normal liver. METHODS

Male albino Sprague-Dawley rats weighing 230-330 g remained intact or, in experiments on regenerating liver, were subjected to 75 % partial hepatectomy 19. The rats always underwent surgery in the morning to minimize diurnal variations ~° and were allowed 5 % glucose during the first 24 post-operative hours. Rats were sacrificed 46-47 h after partial hepatectomy and at various intervals after a single intraperitoneal injection of 0.5 or IO.O mg of cortisone acetate (Merck Chemical Co.) per IOO g body weight, administered respectively as a 0.2 or 2.5 % suspension in 0.85 % NaC1. Zero-time control animals remained uninjected. Food was withdrawn 20 h prior to sacrifice to minimize biosynthetic fluctuations due to variations in dietary intake. In labeled precursor incorporation experiments, precisely two hours preceding sacrifice, each rat received 4 °/~C 32Pt alone or combined with IO/~C [2-14C]glycine/Ioo g body weight. Thus, incorporation i n vivo of 32p and 14C into nucleic acids, and 14C into proteins was obtained at various 2-h periods following a single dose of cortisone acetate. Approximately 5 g of normal or regenerating livers were promptly removed from decapitated rats and homogenized for 2 rain in two volumes of cold 0.25 M sucrose buffered with 3" lO-4 M KHCO v Inorganic phosphate was isolated 2~ from an aliquot of each homogenate. The remainder of each homogenate was fractionated b y differential centrifugation: nuclei were sedimented at 650 x g for IO rain and were separated from contaminant whole cells and debris b y resuspension in 2.2 M sucrose and further centrifugation 22 at 35 ooo x g; mitochondria were sedimented at 9000 x g for IO rain and washed in 0.25 M sucrose; microsomes were sedimented at lO5 ooo × g for 60 min; a RNA fraction was obtained from the final high-speed supernatant b y precipitation at p H 5.0 with acetic acid. Examination b y phase microscopy revealed no detectable contaminating particles in the mitochondrial, microsomal, and supernatant fractions; a few contaminating whole cells and cellular debris particles were present in nuclear preparations. RNA and DNA were isolated from appropriate subcellular fractions and the incorporation of 32p I and [2-1*C]glycine was determined in a manner analogous to that previously described 2~. DNA samples were submitted to a total of three t o four overnight alkaline hydrolyses in I N N a O H at 3 °0 followed b y alcoholic HC1 precipitations until constant specific activities were achieved, thereby insuring removal of all contaminant RNA. DNA and RNA samples thus isolated were quantitated b y ultraviolet spectrophotometry24; the isolated inorganic phosphate was estimated by the method of FISKE AND SUBARROW. The inorganic phosphate, RNA, and DNA samples were plated infinitely thin and the radiation emitted b y 3zp was determined with a 5 % probably counting error in a thin-window Geiger counter 2~. In doubly labeled samples, *zP was counted in the presence of 14C by interposing an aluminum Biochim. Biophvs. A a a , ~

(I96~) 495-504

CORTISONE EFFECTS ON LIVER METABOLISM

497

shield between the planchet and the Geiger tube. The weak beta particles of 14C were thereby completely absorbed, whereas 79 % of the zzp radiation penetrated; appropriate correction for loss of asp radiation through tile aluminum shield was made. To eliminate the influence of alterations in the inorganic phosphate pool on isotopic :incorporation, the 32p specific activities of RNA and DNA of each subcellular fraction have been expressed relative to the specific activities of the inorganic phosphate pool isolated from the same homogenate, z4C radiation in the presence of 3zp was quantitated b y counting unshielded planchets with a windowless gas Geiger tube twice, allowing elapse of two or more weeks to permit significant 3~p decay. The radiation emitted by I4C was determined, by deducting the counts due to 3zp as computed by the amount of radioactive decay occurring during the allotted interval, and has been expressed as specific activity. Incorporation of [14C]glycine into the total proteins, purified from each subcellular fraction, was determined in gel suspension using a liquid scintillation counter, as previously described 23. RNA and DNA were isolated quantitatively from unfractionated sucrose homogenates, and estimated using orcinol and diphenylamine reagents, respectively, according to a modification of the method of SCHNEIDER25. The residues remaining after quantitative hot trichloroacetic acid extraction of nucleic acids were washed with 95 % ethanol, solubilized in I N NaOH, and their protein content estimated b y a Biuret method 2e. RESULTS

The influence on normal liver nucleic acid metabolism of administration of a single dose of cortisone acetate within the physiological range 27 is depicted in Fig. I. 3~Pi incorporation into RNA and DNA of each subcellular fraction during a series of 2-h incorporation periods following a single intraperitoneal injection of 0. 5 mg cortisone acetate/Ioo g of body weight is presented as per cent of control values. Relative specific activities of the RNA of all subcellular fractions were considerably increased,

2.of

:-2_--: sN

-:~,

220

R ] NA

2O0

/~,~..'..i

8"U

~

t20

/.. ~

......

DNA

' ' ~ ...............

OC tOO HOURS POST C O R T I S O N E

Fig. I. Effects of cortisone on 3zPi incorporation into the nucleic acids of subcellular components of rat liver. Cortisone dose, o. 5 mg/ioo g body weight. Relative specific activity 32p = Specific activity 3~p of nucleic acid × IOO, where specific activity = counts/min/~ug P. Specific activity of 82Pt Biochim. Biophys. Acta, 55 (1962) 495-504

498

M. FEIGELSON, P. R. GROSS, P. FEIGELSON

exhibiting similar temporal patterns of response. Cortisone elicited greatest stimulation of s,p incorporation into microsomal RNA, which attained a m a x i m u m relative specific activity of 24 ° °/o that of control animals; m a x i m u m orthophosphate incorporation into the other principal RNA fractions was approximately double that of controls. Fig. I also demonstrates a transient but reproducible 4 ° °/o elevation in the incorporation of 32pi into the DNA of normal liver. No effects of cortisone on the specific activities of liver inorganic phosphate pools were observed. However, in order to eliminate the possibility that cortisone exerted its effects indirectly through alterations in organic phosphate metabolism lather than directly on RNA biosynthetic mechanisms, the incorporation of [2-14C]glycine, a purine precursor, into the liver nucleic acids of the same cortisone-treated rats was also measured. The incorporation of [2-14C]glycine into the nucleic acids of various subcellular constituents, illustrated in Fig. 2, revealed an anabolic response to corti-

I-

~

o_> 400

.J )t-

w

I I I

350

I

Mc-I

~

~,

N-0NA

\

v-

o

o. ~ 150 v

I00 ~ I

I I I r 2 3 4 5 6 HOURS POST CORTISONE

Fig. 2. Effects ot cortisone on E2-14C~glycine incorporation into the nucleic acids of subcellular c o m p o n e n t s of r a t liver. Cortisone dose, o. 5 m g ] i o o g b o d y weight. Specific activity 14C = c o u n t s / m i n //~g nucleic acid P.

sone treatment of even greater magnitude than observed with z2pl. Increases in [14C]glycine incorporation were most pronounced in nuclear RNA, where m a x i m u m specific activities at 4 h after steroid injection were more than 45o % that of the controls; cytoplasmic RNA specific activities were over three times that of control values. A more marked and protracted elevation in the rate of [14C~glycine incorporation into DNA was observed following cortisone injection than when inorganic phosphate was employed as precursor. The cortisone-induced enhancement of [14Clglycine incorporation into nucleic acid relative to the augmentation in 3*P1 incorporation, represented in Fig. 3, followed a temporal pattern similar to the stimulation of incorporation of either isotope (Figs. I and 2). This effect was most evident in the nuclear fraction at four hours post-cortisone injection, at which time the increased incorporation of [l*C]glycine was almost twice that of a~Pl. I t is thus evident that cortisone stimulates purine biosynthesis as well as nucleotide polymerization. To compare the responses to hormone administration of rapidly proliferating tissue with those of mitotically quiescent cells, the effects of cortisone on nucleic Biochim. Bio~hvs. Acta, 55 (1062) 495-504 `

499

CORTISONE EFFECTS ON LIVER METABOLISM

=--=.4

X-...~ S ~-RNA O--ON J e,-eN-DNA

1.8 13' N ~'~ 1.6

I I ! !

>- 1.5 __. >

< 0 < 1.3 IE L2

7.,

I.I tO

O

I I

I 2

I 3 4

I

I

I

5

6

HOURS POST CORTISONE

F i g . 3. E f f e c t s of c o r t i s o n e on t h e r a t i o of i n c o r p o r a t i o n of [2-ziC]glycine i n c o r p o r a t i o n of 32Pt i n t o n u c l e i c a c i d s of s u b c e l l u l a r c o m p o n e n t s of r a t liver. T h e o r d i n a t e d e n o t e s Specific a c t i v i t y xiC, as p e r c e n t of u n t r e a t e d c o n t r o l R e l a t i v e specific a c t i v i t y 8=p, as p e r c e n t of u n t r e a t e d c o n t r o l "

acid metabolism in regenerating rat liver were studied 46 h after partial hepatectomy. As shown in Figs. 4 and 5, the increases in 32Pt and [2-ziClglycine incorporation into cytoplasmic and nuclear RNA of regenerating liver following cortisone administration were of similar absolute magnitude and time course, as observed in nonproliferating liver. Metabolic acceleration accompanying regeneration is reflected in the elevated basal specific activities of RNA of all cell fractions. This acceleration

! "t ~ A L ma

~

2

4

6

8

IO

2

4

6

8

I0

HOURS POST CORTISONE

2

4

6

8

I0

Fig . 4. E f f e c t s of c o r t i s o n e on p r e c u r s o r i n c o r p o r a t i o n i n t o c y t o p l a s m i c R N A of n o r m a l a n d regen e r a t i n g liver. O - O , r e l a t i v e specific a c t i v i t y 3zp; x - x , specific a c t i v i t y t i c × 2; - - , normal l i v e r ; . . . . . , r e g e n e r a t i n g liver.

Biochim. Biophys. Acta, 55 (1962) 4 9 5 - 5 0 4

500

M. FEIGELSON, P. R. GROSS, P. FEIGELSON NUCLEAR 3.0 P-8 2.4~ )I-~2.0

~

\\

1.6 0

20,

°

6

1.2 n-

2

4

H6OUR~ PO~ST GO2RTIS~)NE e

8

,0

Fig. 5. Effects of cortisone on p r e c u r s o r i n c o r p o r a t i o n into nuclear D N A and R N A of n o r m a l and r e g e n e r a t i n g liver. 0 - 0 , relative specific a c t i v i t y s2p; x - x , specific a c t i v i t y 14C × I for DNA a n d specific a c t i v i t y ~4C x 2 for R N A ; , n o r m a l liver; . . . . . , regenerating liver.

is most evident when E14Clglycine is used as precursor, signifying potentiated purine biosynthesis as well as increased RNA formation during regeneration. Whereas the effect of cortisone on RNA metabolism in regenerating liver was similar to that in normal liver, the action of this hormone on DNA biosynthesis was strikingly different in each of these tissues. Fig. 5 illustrates the characteristic responses of resting and proliferating liver DNA to a single low cortisone dose, which m a y be further contrasted to the reponses of nuclear RNA in these two types of cells. The low basal rates of incorporation of radioactive orthophosphate and glycine into the DNA of mitotically inactive liver cells are evident; fifty- and twenty-fold increases, respectively, occurring in the basal incorporation rates of these two precursors into the DNA of regenerating livers reflect the accelerated mitotic activity of these livers. Whereas a slight transient increase in DNA synthesis occurred in normal liver following cortisone administration, regenerating liver DNA responded to steroid injection with a similar elevation which was followed b y a profound depression in its specific activity. Thus at 8 h following cortisone, only 30 to 40 ~/o of the basal DNA synthetic rate was retained. This cortisone-induced inhibition of DNA synthesis in regenerating liver is in sharp contrast to the exclusively anabolic effects of cortisone on RNA metabolism. Table I depicts the effects of cortisone injection on the metabolic incorporation of [2J4Clglycine into the total proteins of each subcellular fraction of normal adult and regenerating livers. Increased precursor incorporation into the proteins of all normal liver cell constituents was observed, although enhancement of protein turnover was considerably less striking than in the case of RNA. Maximal stimulation occurred at 4 h after cortisone injection in all cell constituents with greatest stimulation observed in the mitochondrial fraction. The basal incorporation rates of E14C]glycine into the protein of all regenerating liver subcellular fractions were clearly more rapid than those of normal adult liver. Cortisone elicited anabolic responses in the proteins of all subcellular compartments of regenerating liver which were similar in absolute magnitude to those in intact livers. In order to determine whether the increased precursor incorporation into RNA Biochim. Biophys. Acta, 55 (1962) 495-5o4

CORTISONE EFFECTS ON LIVER METABOLISM

501

TABLE I EFFECT OF CORTISONE

ON [2-14C]GLYCINE I N C O R P O R A T I O N INTO P R O T E I N O F S U B C E L L U L A R F R A C T I O N S O F NORMAL AND R E G E N E R A T I N G LIVER

N, nuclei; M e , microsomes; Mt, mitochondria; S, soluble cell fraction.

Liver

Normal

Regenerating

Hours post cortisone*

N

O 2 3 4

352 4IO 456 480

6

427

o 2 3 4 6 8

826 Io64 959 919 lO99 IOOI

Speci]ic activity (Counts/rain/rag protein) Mc S

452

508 5 I8 507 516 694 788 612

Mt

280 28I 334 376 338

195 239 200 296 226

677

414 524 368

720 667 752 745 807

779

679 578

477

45 ° 317

* Cortisone dosage: 0. 5 mg cortisone acetate/Ioo g body weight.

and proteins following cortisone administration was associated with net synthesis of these compounds, estimation of the absolute quantities of nucleic acids and proteins was undertaken. Larger cortisone doses (IO.Omg/Ioo g body weight) were used in these experiments since it was anticipated that changes in the quantities of metabolic end-products would be small compared with alterations in labeled precursor turnovers. As seen in Table II, increasing the dosage of cortisone twenty-fold augmented T A B L E II E F F E C T O F C O R T I S O N E O N 82Pt I N C O R P O R A T I O N INTO T H E N U C L E I C ACIDS O F S U B C E L L U L A R F R A C T I O N S OF

NORMAL L I V E R

N, nuclei; Mc, microsomes; Mr, mitochondria; S, soluble cell fraction. Relative specific activity** Hours post cortisone*

o o 2. 5 5.o 7.5 IO.O

RNA

DNA

N

Mc

S

Mt

N

6.8 7.2 8.2 15.2 14. 9 I 1.7

0.39 0.44 o.91 1.32 1.57 0.96

0.93 1.22 2.14 2.67 3.3 ° 2.31

o.51 0.53 1.31 x.49 1-51 0.95

0.0535 0.0459 0.0530 o.o526 0-0425 0.0467

" Cortisone dosage : IO mg cortisone acetate]ioo g body weight. ** Relative specific activity as defined in Fig. I. a n d p r o l o n g e d t h e anabolic effect on R N A . Q u a n t i t a t i v e e s t i m a t i o n of liver D N A , R N A , a n d p r o t e i n o f u n t r e a t e d r a t s e i g h t h o u r s a f t e r i n j e c t i o n of c o r t i s o n e , is p r e s e n t e d i n T a b l e I I I . A / t h o u g h t h e c o n c e n t r a t i o n s of R N A a n d p r o t e i n a c t u a l l y fell f o l l o w i n g c o r t i s o n e a d m i n i s t r a t i o n , t h e m a g n i t u d e of l i v e r h y p e r t r o p h y , a s e v i d e n c e d Biochim. Biophys. Aaa,

55 (1962) 495-504

502

M. F E I G E L S O N ,

P. R. GROSS,

TABLE

P. F E I G E L S O N

III

THE EFFECTS OF CORTISONE ON NORMAL LIVER R N A , D N A , AND PROTEIN LEVELS Rats were sacrificed 8 h after administration of io mg cortisone acetate/ioo g body weight; c o n t r o l a n i m a l s w e r e u n i n j e c t e d . R e s u l t s a r e r e p o r t e d a s m e a n s -}- s t a n d a r d e r r o r .

Liver weight #er ;too g body weight Cortisone (z) --

Wet (a) 3.05

(+0.06) +

3.57

Dry (3) 0.857

(+0.020) 0.997

(~:o.o3) (~o.oo4) Difference *

+0.52*

+o.14o*

DNA mg purine deoxyribose per g liver (4) 1.°9°

.too ~. body We_oht

RNA

g liver

(5)

(6)

3-38

2.48

(4-0.070) (:5o.15) (±0.06) 0.974

3.48

Protein

mg purine ribose per

2-37

too g body weight (7) 7.59

(±o.17) 8.45

mg per g z-oo g liver body weight (8) 171

(~4) 153

(9) 523

(zhl8} 553

(4-0.062) (4-0.23) (2-o.o5) (io.o6)

(zh3) (d- 7)

--o.116

--18"

+o.io

--o.n

+0.86*

+3 °

P
by increases in wet and dry liver weights after hormone treatment, exceeded these depressions. Thus, when computed as quantities per total liver (corrected for body weight) significant increases in liver RNA and suggestions of increased liver protein were observed at 8 h following cortisone (Table III, columns 7 and 9)- Total liver DNA (column 5), however, remained essentially unaltered. Increases in precursor incorporation into RNA and proteins following cortisone administration, therefore, reflect increased net synthesis of these macromolecules. DISCUSSION In both resting and regenerating rat liver, stimulated RNA and protein turnover is an early response to a single dose of cortisone. The increases in ~zP1and [14C]glycine incorporation into RNA of all cell fractions may be due to acceleration of purine and pyrimidine nucleotide synthesis and/or increased nucleotide condensation into RNA. The observed elevations in liver ribonucleic acids support the interpretation that increased nucleotide polymerization is taking place. The greater elevations in incorporation into RNA of the purine precursor [2-14C]glycine than of I32P] orthophosphate indicate a cortisone-induced acceleration of de novo purine biosynthsis as well. It seems quite possible that these processes are closelycoupled. A single inj ection of a small quantity of cortisone, therefore, suffices to achieve the anabolic responses to cortisone observed after repeated treatment with low doses of hol-mone~,s,15--17. The reported catabolic effects in liver observed in chronic experiments using repeated relatively high hormone doses1°-Izmay represent secondary effects, perhaps reflecting precursor depletion or attrition of metabolic processes due to prior anabolic overstimulation. The mechanisms underlying cortisone-induced acceleration of hepatic protein and RNA metabolism remain conjectural. Cortisonemay stimulate RNA and protein anabolism directly, in an unknown manner, or indirectly, through increases in the concentrations of amino acid or nucleotide precursors derived from the breakdown Biochim.

Biophys.

Asta,

55 (1962) 4 9 5 - 5 o 4

CORTISONE EFFECTS ON LIVER METABOLISM

503

of extrahepatic tissue and transported to the livere, 28. These extrahepatic breakdown products may, alternatively, increase energy reservoirs within the liver xS, thereby implementing macromolecule synthesis. These effects of cortisone m a y be further enhanced b y facilitated amino acid transfer from blood into liver cells2L Accelerated x4C amino acid incorporation into liver proteins in vivo following cortisone treatment, as herein reported, provides support for the finding that liver microsomes of cortisone-treated intact animals demonstrate enhanced capacity for incorporation of 14C amino acids in vitro 3°. The glucocorticoid stimulation in protein synthesis observed in vivo and in vitro m a y be the resultant of marked accelerated synthesis of but a few protein species. Among these responsive proteins m a y be the enzymes t r y p t o p h a n pyrrolase31m, xanthine oxidase 33, glucose 6-phosphatase ~, transaminases 3~,3e, and othersLSL The influences of cortisone on various tissues illustrate an interesting aspect of target organ specificity. The net effect of cortisone administration on the organism as a whole is clearly catabolic, as evidenced b y a decrease in total body weight, total carcass protein, and in a negative nitrogen balance following cortisone 3-6. Tissuespecific catabolic influences of adrenal glucocorticoids have been reported for eosinophils 38 and for lymphoid 8,13,~4 and muscle 18 tissue. Under the influence of cortisone, however, hepatic tissue hypertrophies with concomitant increases in the rates of nucleic acid and protein synthesis. Thus, the concept of target organ specificity must include not only the fact of organ response to a hormone, but also the nature and direction of the response. Whereas a single low dose of cortisone similarly influenced RNA and protein metabolism in livers of intact and partially hepatectomized rats, it exerted characteristic effects on DNA synthesis in these livers. In normal liver, where the basal DNA synthetic rate is low, a transient increase at three hours post-cortisone administration was followed b y a return to control values. Furthermore, total quantities of DNA in normal liver were not depressed after cortisone. In regenerating liver, however, cortisone treatment results in suppression in DNA synthetic rate, as observed in this study, which presumably underlies the reported depression in D N A levelsg,SL The demonstrated cortisone-induced inhibition of DNA replication is consistent with the reported anti-mitotic effect of this steroid on the livers of partially hepatectomized animals 9,3". I t seems possible that the rapid basal rate of DNA synthesis, characteristic of regenerating liver further stimulated b y cortisone administration, m a y result in depletion of deoxyribonucleotide pools, with consequent suppression of DNA synthesis. ACKNOWLEDGEMENTS This investigation was supported in part b y grants from the United States Public Health Service (CY 2332 and A-23o2). This study was carried out during the tenure b y M. F. of a United States Public Health Service Pre-doctoral Fellowship. P. F. is an Established Investigator, Health Research Council, City of Net" York. REFERENCES

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Biochim. Biophys. Acta, 55 (I962) 495-504