The effect of bovine growth hormone on formation of RNA by rat liver slices

BIOCHIMICAET BIOPHYSICAACTA

419

BBA 95648

T H E E F F E C T OF B O V I N E G R O W T H HORMONE ON FORMATION OF RNA BY RAT L I V E R SLICES

C. D. JACKSON ANDB. H. SELLS Laboratory o[ Biochemistry, St. Jude Children's Research Hospital, and Department o] Biochemistry, University o/ Tennessee, Memphis, Tenn. (U.S.A.)

(Received January i6th, 1967)

SUMMARY I. Rat-liver slices incubated with [3H]orotic acid incorporated label into RNA at a linear rate for at least 4 h. Labelled orotic acid was incorporated almost exclusively into 45-55-S RNA during the first 2 h incubation. Ribosomal RNA (I8-S and 28-S RNA) as well as soluble RNA became labelled after 4-6 h incubation. 2. Actinomycin D (io/~g/ml) reduced the incorporation of labelled orotic acid into the 45-55-S RNA approx. 75 %. 3. Liver slices from hypophysectomized rats sacrificed 6 or 12 h after bovine growth hormone administration incorporated significantly more label during a I-h incubation than did slices from untreated animals. Addition of growth hormone directly to the incubation medium had no demonstrable effect on the incorporation of labelled orotic acid into the 45-55-S RNA by liver slices from hypophysectomized rats. 4. Liver slices from hypophysectomized rats sacrificed 12 h after growth hormone administration incorporated approximately five times more label into ribosomal RNA (18 S and 28 S) during a 6-h incubation than did slices from untreated hypophysectomized rats. Growth hormone added directly to the incubation medium did not increase labelled orotic acid incorporation into ribosomal RNA of liver slices.

INTRODUCTION Administration of growth hormone to hypophysectomized rats has long been known to stimulate liver growth I and to increase the concentration of liver RNA and protein s. KORNER3 has shown that 12 h following treatment with growth hormone hypophysectomized rats incorporated larger amounts of RNA precursors into liver RNA than the untreated controls. Results obtained in this laboratory 4have confirmed these data and shown that this increased incorporation can be demonstrated after even shorter periods of growth hormone treatment. Biochim. Biophys. Acta, 142 (1967) 419-429

42o

c.D.

J A C K S O N , B. H. S E L L S

Growth hormone has been reported to have no effect on protein synthesis when added to cell-free systems 5 nor when added in vitro on the incorporation of [14CiGTP by nuclear preparations 6. Since liver often is used in studies of growth hormone action, it was of interest to determine if growth hormone would be effective in vitro on the incorporation of labelled precursors into RNA by whole liver cells. Although considerable work has been published on the incorporation of various precursors into the RNA of rat-liver preparations iu vitro, little or no information is available concerning the nature of the RNA synthesized in rat-liver slices or cell suspensions under conditions in vitro. JACOB AND BHARGAVA7 compared the synthesis of RNA in rat-liver slices and in cell suspensions. These authors showed that both preparations readily incorporated several precursors into RNA, but did not characterize the types of RNA synthesized. Investigations by RAKE AND GRAHAM8 and PERRY9 using L cells and HeLa cells respectively, have demonstrated that the precursors of RNA are incorporated initially into heavy material with sedimentation coefficients of 45 and 55 S. Much of this material apparently is converted into ribosomal RNA. The present studies have been designed to examine the formation of RNA in rat-liver slices, to determine the quality of the RNA produced, and to examine the influence of growth hormone when administered in vivo and in vitro upon the synthesis of RNA in the slice preparation.

M A T E R I A L S AND M E T H O D S

Female, Sprague-Dawley rats (go-loo g) were purchased from Hormone Assay Laboratories, Inc., Chicago, Ill. The normal rats were fed Wayne Blox ad libitum. The hypophysectomized rats were maintained on a synthetic dieO ° for two weeks or longer before being used. Growth hormone (bovine N.I.H. B-9, 0.98 units/ rag) was dissolved in slightly-alkaline saline for intraperitoneal injection or was dissolved in Krebs-Ringer phosphate buffer, without Ca ~+ (ref. II) for incubating iu vitro. [5-aH]Orotic acid (5.0 mC/2o.4 rag) or [6-14Clorotic acid (I mC/34.8 rag) was obtained from New England Nuclear Corporation.

Preparation and incubation o~ rat-liver slices After various treatments, the rats were killed with a blow on the head and decapitated. The animals then were allowed to bleed lO-2O sec after which time the livers were removed rapidly and placed in saline at 0-4 ° . Individual lobes of the liver were sliced with a Stadie-Riggs slicer 12 (A. H. Thomas Co.), which had been modified to cut slices of approx. 1.5 mm thickness. The first and last slice of each lobe was discarded. Immediately after slicing, the slices were placed in a small volume of Krebs-Ringer phosphate buffer, without Ca ~+. The slices were divided into a number of samples, each of which contained approximately the same weight of tissue. Large slices were sectioned and distributed among two or more samples to further randomize the sample.s. The entire procedure was carried out at 3-5 ° and the slices were never left in the cold buffer longer than 15 rain before incubating. The samples were transferred to 5o-ml beakers containing 3 ml Krebs-Ringer phosphate buffer, Biochim. Biophys. .4cta, 14z (1967) 419-429

FORMATION OF

RNA

BY RAT LIVER SLICES

421

without Ca 2+, which previously had been equilibrated at 37 ° and with a gas phase of oxygen. All additions to the incubation medium were made prior to the equilibration period unless specifically stated otherwise. At the end of the incubation period tile slices were removed quickly with forceps, washed in 25 ml saline at 0-5 ° and immediately frozen on solid CO2.

Isolation o~ R N A RNA was isolated by a modification of the procedures described by RAKE AND GRAHAMs. Sodium dextran sulfate (IOO/zg/ml) instead of polyvinyl sulfate was added to the sodium dodecyl sulfate buffer. Bentonite (5 #g/ml) was present in both the sodium dodecyl sulfate and EDTA buffers to further inhibit ribonuclease activity. Bentonite was purified by the procedure described by FRAENKEL-CONRAT13. The two RNA extraction procedures gave results with liver tissue similar to those reported with L cells s. The cytoplasmic RNA was extracted by the phenolEDTA procedure while the DNA and rapidly labelled RNA remained in the insoluble interface. Both the DNA and rapidly labelled RNA then were extracted from the insoluble interface by the sodium dodecyl sulfate buffer. DNA was removed by shaking the solution with an equal volume of phenol at 65 ° and chilling rapidly at 4 °. The total RNA (both nuclear and cytoplasmic) was extracted by the phenol-sodium dodecyl sulfate procedure by omitting the phenol-EDTA procedure. Phenol was removed from the isolated material either by extracting with ether or by passing the preparation over a colunm of Sephadex G-25 in a solution containing IO 2 M Tris (pH 7.6), o.14 M LiC1, lO -a M Mg2+ (TLM buffer). The radioactivity of the isolated material was measured in a Packard liquid scintillation counter using the dioxane system ~4.

Sedimentation analysis o/ R N A Isolated RNA was fractionated on a linear sucrose density gradient 15 (either 5-20 o/ /o or 5-4 ° °/o) made up in TLM buffer s or acetate buffer (lO -2 M NaC1, o.I M sodium acetate, pH 5.1) by centrifuging at 0-5 ° in a Spinco SW-39 rotor at 39 ooo rev./min for 220 rain. At the end of the centrifugation the bottom of the tube was pierced with a 2o-gauge needle and approx. 30 fractions of IO drops each were collected. After adding 0.5 ml water, the absorbance at 260 m# was measured with a Zeiss spectrophotometer. Approx. 25/~g bovine serum albumin was added as a carrier and the acid-insoluble material was precipitated by adding an equal volume of Io o~, trichloroacetic acid. The precipitates were collected on Millipore filters (type HA 0.45/~ pore size), dried, and counted in IO ml dioxane scintillation system containing 0. 5 ml water.

RESULTS

Orotic acid incorporation into R N A in liver slices Preliminary experiments indicated that the incorporation of labelled orotic acid by liver slices was limited by the lack of oxygen and that the incorporation of Biochim. Biophys. Acta, 142 (1967) 419-429

422

C . D . JACKSON, B. H. SELLS

orotic acid could be increased 5-fold or more by incubating the slices in the presence of oxygen as the gas phase. The kinetics of the incorporation of labelled orotic acid were studied by determining the specific activity of the RNA extracted by the phenolsodium dodecyl sulfate method from slices after various periods of incubation. The results of a typical experiment are shown in Fig. I. Slices from normal rats incorporated labelled orotic acid at a linear rate for 3 h or more. Labelled orotic acid added to the medium after 12o min pre-incubation in the absence of label was incorporated into liver RNA at approximately the same rate as that present at the beginning of the incubation.

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Fig. I. I n c o r p o r a t i o n of [~H]orotic acid b y liver slices from a n o r m a l rat. Liver slices were inc u b a t e d in K r e b s - R i n g e r p h o s p h a t e buffer. Samples were t a k e n at the times s h o w n and the R N A was extracted b y the p h e n o l - s o d i u m dodecyl sulfate method. After filtration of R N A t h r o u g h S e p h a d e x G-25, the specific activity (counts/min per A260 m~ unit) was determined. [3HlOrotic acid (2 ffC/ml) was added at the beginning ( O - © ) or after i2o min incubation (Ax- - - ~ ) .

Incorporation o/ labelled orotic acid into 45- to 55-S RNA To determine the nature of the RNA synthesized by liver slices from normal rats during the early periods of incubation, RNA extracted by the phenol-sodium dodecyl sulfate method from slices incubated 30, 60 and 12o min in the presence of [14C]orotic acid was analyzed on 5-40% sucrose gradients. Equal amounts of RNA (estimated by absorbance at 260 raft) from each sample were placed on the gradients. The results are shown in Fig. 2. The absorbance profile of each was similar and only one is shown for reference. The RNA was not passed over a Sephadex column and thus a high absorbance in the 4-S region was observed, due to low-molecular-weight compounds. The sedimentation profile of the radioactivity illustrates that during the first 2 h labelled orotic acid was incorporated almost exclusively into RNA having a sedimentation coefficient greater than the 28-S ribosomal RNA. In agreement with Fig. i, the amount of [14C]orotic acid incorporated into the 45- to 55--S RNA increased with time.

Effect o/actinomycin D on the synthesis o/45- to 55-S RNA The effect of actinomycin D, an inhibitor of DNA-dependent RNA synthesis 16, upon the incorporation of labelled orotic acid was determined in the liver-slice system. Liver slices from hypophysectomized rats were incubated in the presence of [14C]orotic acid with or without actinomycin D (IO ffg/ml) for 80 min and the RNA was extracted by the phenol-sodium dodecyl sulfate method. After passing the RNA

Biochim.Biophys.Acla,I42

(1967) 419-429

FORMATION OF

RNA

BY RAT LIVER SLICES

423

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Fig. 2. I n c o r p o r a t i o n of [t+C]orotic acid into 45-55-S R N A b y liver slices f r o m a n o r m a l rat. Liver slices were i n c u b a t e d in K r e b s - R i n g e r p h o s p h a t e buffer containing [14C]orotic acid (o. 5/~C/ ml). The R N A w a s e x t r a c t e d b y the phenol s o d i u m dodecyl sulfate m e t h o d and equal a m o u n t s (estimated b y a b s o r b a n c e at 26o m#) were centrifuged on a linear (5-4 ° %) sucrose gradient. Only one a b s o r b a n c e profile ( - - ) is s h o w n for reference and the distribution of radioactivity insoluble in trichloroacetic acid after 3 ° min (- - -), 6o min (-- - -- ), and 12o min ( . . . ) incubation are s u p e r i m p o s e d for comparison.

over a column of Sephadex G-25, the specific activity was determined. The R N A from slices incubated with and without actinomycin D was 13 5 o o ± 3 o o counts/min per mg and 28 4oo&_-IOOO counts/min per rag, respectively. Thus an inhibition of approx. 50 % was observed. In another experiment slices were incubated in the presence of ~aH]orotic acid with or without actinomycin D (IO #g/ml). The extracted R N A was passed over a column of Sephadex G-25 and analyzed on a sucrose density gradient. As shown in Fig. 3, the incorporation of labelled orotic acid was inhibited approx. 75 07o by ac-

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Fig. 3. Effect of a c t i n o m y c i n D on i n c o r p o r a t i o n of [3Hlorotic acid into 45-55-S RNA. Liver slices f r o m h y p o p h y s e c t o m i z e d r a t s were divided into two g r o u p s and incubated in I { r e b s - R i n g e r p h o s p h a t e buffer containing p H ] o r o t i c acid (2/,C/ml). Actinomycin D (io/~g/ml) w a s added to the i n c u b a t i o n m e d i u m of (A) while no addition w a s m a d e to (B). After i h incubation, tile R N A w a s e x t r a c t e d b y the p h e n o ~ s o d i u m dodecyl sulfate m e t h o d and equal a m o u n t s of R N A (estimated b y a b s o r b a n c e at 26o m/~) were centrifuged on a linear (5-4 o %) sucrose gradient. The abs o r b a n c e at 26o m/~ ( ) a n d trichloroacetic acid-insoluble radioactivity ( - - - ) are shown.

Biochim. Biophys. Acta, 142 (1967) 419-429

424

C . D . JACKSON, B. H. SELLS

tinomycin D. These experiments indicate that orotic acid ]abelling resulted from incorporation into newly formed RNA.

Incorporation o~ labelled orolic acid into ribosomal and soluble RATA The following experiment was designed to determine if labelled orotic acid could be incorporated in vitro into the ribosomal and soluble RNA of normal rat livers. Slices from normal rats were incubated in the presence of [3H]orotic acid and samples were taken at the end of 4 h and 6 h incubation. The RNA was extracted by the phenol-EDTA method and analyzed on a sucrose density gradient. The results are shown in Fig. 4. The sedimentation profiles of the incorporated label indicate that

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Fig. 4. I n c o r p o r a t i o n of [3H]orotic acid into ribosomal and soluble n o r m a l r a t were incubated in K r e b s Ringer p h o s p h a t e buffer w i t h for 4 h (A) and 6 h (B). The R N A was e x t r a c t e d by the p h e n o l - E D T A a linear (5-20 %) sucrose gradient. The a b s o r b a n c e at 260 m/~ (--insoluble radioactivity ( - - - ) were distributed as shown.

RNA. Liver slices from a [3H]orotic acid (2/~C/ml) m e t h o d and centrifuged on ) and trichloroacetic acid-

the 4-S RNA was labelled after 4 h incubation but very little incorporation of labelled orotic acid into 28-S and I8-S RNA occurred. At the end of 6 h, however, a significant amount of label was found in both the 28-S and I8-S RNA as well as in the 4-S RNA.

E//ect o/growth hormone on synthesis o~ rapidly labelled RNA To determine what, if any, effect growth hormone would have on the synthesis of the 45- to 55-S RNA, two types of experiments were performed. First, growth hormone was administered by intraperitoneal injection to hypophysectomized rats and at various intervals the livers were removed, sliced, and incubated in the presence of [aHlorotic acid. Control animals were injected with saline. Secondly, growth hormone was added directly to the incubation medium and slices from hypophysectomized rats were incubated in the presence of EaH]orotic acid. Table I shows the results of an experiment in which hypophysectomized rats (3 or 4 in each group) were given growth hormone (2 mg per rat) for 40 rain, 6 h and I2 h before sacrificing, along with control animals. The livers were sliced and samples from each were incubated in the presence of [3H]orotic acid for I h. After the incubation the RNA was extracted by the phenol-sodium dodecyl sulfate method, Biochim. t~iophys..4cla, i42 (t967) 419-429

FORMATION OF

RNA

BY RAT LIVER SLICES

425

TABLE I EFFECT OF BOVINE GRO'~VTH HORMONE in vivo ON INCORPORATION OF [3H]OROTIC ACID BY LIVER SLICES

H y p o p h y s e c t o m i z e d r a t s were given bovine g r o w t h h o r m o n e (2 mg per rat) b y i n t r a p e r i t o n e a l injection at various t i m e s before sacrificing. The livers were sliced and incubated I h in K r e b s Ringer p h o s p h a t e buffer containing [aH]orotic acid (2/~C/ml). The R N A w a s extracted b y the p h e n o l - s o d i u m dodecyl sulfate m e t h o d , passed over S e p h a d e x G-25, and precipitated w i t h 3 vol. of 95 % ethanol. The R N A was dissolved in a small v o l u m e of distilled w a t e r and the specific a c t i v i t y was d e t e r m i n e d b y m e a s u r i n g the a b s o r b a n c e at 260 m # in a Zeiss s p e c t r o p h o t o m e t e r a n d the radioactivity of an aliquot in a P a c k a r d Tri-Carb liquid scintillation spectrometer.

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Time

Speci/ic activity o / R N A (Counts/rain per A260ma unit ±S.E.)

Saline Bovine g r o w t h h o r m o n e Bovine g r o w t h h o r m o n e Bovine g r o w t h h o r m o n e

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Number o/ animals

4 4 4 3

passed over a Sephadex G-25 column, and the specific activity was determined and expressed as counts/min per A260ms unit. No difference was found in the amount of label incorporated in liver slices from treated (growth hormone 40 min before sacrificing) or untreated animals. In some experiments 3o-4o min pretreatment with growth hormone caused a significant decrease in incorporation of labelled orotic acid as compared with animals treated with saline. This response to short periods of pretreatment with growth hormone was

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Fig. 5. Effect of bovine g r o w t h h o r m o n e added in vitro on incorporation of [aH]orotic acid b y liver slices. Liver slices f r o m h y p o p h y s e c t o m i z e d r a t s were divided into two g r o u p s and incubated in K r e b s - R i n g e r p h o s p h a t e b u f f e r containing [3H]orotic acid (2 ffC/ml) w i t h ( A - A ) or w i t h o u t ( O - - - O ) g r o w t h h o r m o n e (5 ° / z g / m l ) . S a m p l e s were t a k e n after 4 ° and 6o m i n incubations and the specific activity of the R N A was d e t e r m i n e d as in Table I. The range of values for duplicate i n c u b a t i o n s are indicated b y (&) for slices w i t h and ( O ) for slices w i t h o u t g r o w t h h o r m o n e .

Biochim. Biophys. Acta, x42 (1967) 419-429

42b

C. D. JACKSON, B. H. SELLS

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Fig. 6. E f f e c t of b o v i n e g r o w t h h o r m o n e in vitro a n d in vivo o n i n c o r p o r a t i o n of [ 3 H ] o r o t i c acid i n t o r i b o s o m a l a n d s o l u b l e R N A b y l i v e r slices. H y p o p h y s e c t o m i z e d r a t s w e r e d i v i d e d i n t o t w o g r o u p s . O n e g r o u p r e c e i v e d 2 m g g r o w t h h o r m o n e i n t r a p e r i t o n e a l l y 12 h b e f o r e s a c r i f i c i n g (in vivo g r o u p ) w h i l e t h e c o n t r o l g r o u p r e c e i v e d s a l i n e . A f t e r s a c r i f i c i n g , t h e c o n t r o l g r o u p w a s div i d e d i n t o t w o s u b g r o u p s a n d g r o w t h h o r m o n e (5 ° / ~ g / m l ) w a s a d d e d t o t h e i n c u b a t i o n m e d i u m of o n e s u b g r o u p (in vitro g r o u p ) . A f t e r 6 h i n c u b a t i o n t h e R N A w a s e x t r a c t e d b y t h e p h e n o l E D T A m e t h o d a n d e q u a l a m o u n t s of R N A ( e s t i m a t e d b y a b s o r b a n c e a t 260 raft) w e r e c e n t r i f u g e d on a linear (5-20 %) sucrose gradient. The absorbance (----) and trichloroacetic acid-ins o l u b l e r a d i o a c t i v i t y (- - -) of e a c h p r e p a r a t i o n a r e s h o w n for t h e c o n t r o l (A), g r o w t h h o r m o n e in vitro (B), a n d g r o w t h h o r m o n e in vivo (C).

Biochim. Biophys. Acta, 142 ( i 9 6 7 ) 419 429

FORMATION OF R N A BY RAT LIVER SLICES

427

found to vary from one group of animals to another and the cause has not been determined. Similar observations have been made b y V~7IDNELLAND TATA17. However, slices taken after treatment in vivo for 6 h or 12 h incorporated more labelled orotic acid than the controls. The second experiment was performed to determine the influence of growth hormone added in vitro on RNA synthesis. The liver of a hypophysectomized rat was sliced, and divided into several samples. To half of these samples was added growth hormone (5o/~g/ml). After various periods of incubation with E~H~orotic acid, slices were removed and the RNA was extracted by the phenol-sodium dodecyl sulfate method. The specific activities were determined as above and are shown in Fig. 5- It m a y be seen that growth hormone added in vitro had no significant effect on incorporation of labelled orotic acid.

E//ect o/growth hormone on ribosomal and soluble R N A To determine the effects of growth hormone on the incorporation of labelled orotic acid into ribosomal and soluble RNA in liver slices, the hypophysectomized rats were divided into two groups. One group received 2 mg growth hormone 12 h before sacrificing while the other group received saline. The slices from the rats receiving saline were divided into two subgroups. Growth hormone (50 #g/ml) was added to the incubation medium of one subgroup and the slices then were incubated in the presence of E3H~orotic acid for 6 h. At the end of the incubation RNA was extracted by the phenol-EDTA method and analyzed on a sucrose density gradient. The results are shown in Fig. 6. Addition of growth hormone in vitro had 11o significant effect on the incorporation of labelled orotic acid while growth hormone administered in vivo 12 h before sacrificing the animal produced a marked increase in the amount of label incorporated in the 28-S and I8-S RNA, as well as in the 4-S material.

DISCUSSION

These studies have revealed that rat-liver slices can effectively incorporate [l*Clorotic acid into RNA for at least 4 h. JACOB AND BHARGAVA 7, studying incorporation of various RNA precursors into liver slices have also demonstrated that Ii*Clorotic acid can be incorporated into the RNA but have not described the quality of RNA produced. Our studies indicate that the rat-liver slice incorporates orotic acid initially into RNA having sedimentation coefficients greater than 28 S (45 and 55 S). HIGASHI AND BUSCHTM reported that freezing and thawing liver preparations resulted in degradation of heavy RNA. In our studies we have been able to freeze and thaw the tissue without obtaining extensive degradation of the heavy RNA. The difference between our procedure and that of B u s c h appears to be the use of dextran sulfate which either effectively inhibits ribonuclease and/or allows a more complete extraction of the RNA from the nuclear region as reported by TAMAOK119. Labelling of this heavy material has been observed in HeLa cells and L cells s,9. This material, believed to be a precursor of 28-S and I8-S RNA, is located in the nucleus of the cell. During Biochim. Biophys. Acta, 142 (1967) 419-429

42g

C. D. JACKSON, B. H. SELLS

the first 2 h little or no radioactivity can be detected in the cytoplasm of the cell and the ribosomal RNA's at this time are not labelled. Rat liver-slice preparation incubated with I14C]orotic acid for 6 h results in the appearance of label in the 28-S and I8-S RNA peaks. The rat-liver slice preparation was developed to measure RNA metabolism and to establish a system in which hormone action might be determined in vitro. These studies have been only partially successful in the study of growth hormone action. When growth hormone is administered to the hypophysectomized rat, liver slices obtained from the injected animal are metabolically more active in incorporating precursors of RNA into the various types of RNA. The rate of incorporation into heavy material (45 and 55 S) is stimulated about 2-fold over that obtained with slices from those animals which have not been treated with the hormone. The incorporation of isotope into the ribosomal RNA's (28 and 18 S) in animals which have received growth hormone is also stimulated. Using nuclear preparations, WlD~ELL AND TATA17 have reported that the activity of the enzyme DNA-dependent RNA polymerase is increased, at least 1-2 h after administration of the hormone. Efforts to obtain stimulation of RNA labelling by addition of the hormone to the cell slices in vitro has met with no success. It would appear that either the hormone does not act directly on the liver tissue or it has need of some component in the plasma to carry it into the liver cell. On the other hand, growth hormone may act on another target organ to indirectly influence liver metabolism. Concerning the possibility that growth hormone may act upon another organ within the body, the current report of SAKURAI AND KIPNIS is pertinent 2°. They have observed that administration of growth hormone increased hepatic DNA-dependent RNA polymerase activity and have shown similarly that insulin when administered in vivo also increased the activity of the enzyme. Furthermore, when antiserum to insulin is administered along with insulin, the increased activity of the RNA polymerase was abolished. When growth hormone and anti-insulin serum were administered concurrently, the effect of growth hormone upon hepatic RNA polymerase levels also was abolished. These observations would lend support to the possibility that growth hormone acts on liver RNA metabolism indirectly by stimulating the pancreas to release insulin. DAUGHADAY21 has indicated the presence of a component in the serum of growth hormone-treated hypophysectomized rats which stimulates incorporation of tritiated thymidine into DNA of cartilage slices in vitro. The identity of this factor is not known, however, it was not found in the serum of untreate'~ hypophysectomized rats. We have observed that when growth hormone was administered to hypophysectomized animals there was extensive mobilization of fat which accumulated in the liver. Whether this condition influences the rate of RNA synthesis in these livers is yet to be determined. The present results with liver slices correlate well with previous measurements in vivo and indicate that administration of growth hormone to hypophysectomized rats modifies the metabolism of the liver cells resulting in increased labelling of the various types of ribonucleic acid. This investigation suggests that the growth hormone acts indirectly on liver metabolism or requires a second component(s) with which to act synergistically. Biochim. Biophys. Acts, 142 (1967) 419 429

FORMATION OF

RNA BY RAT LIVER SLICES

429

ACKNOWLEDGEMENTS

This study was supported by grants from the National Institutes of Health (AMo7375-o4), Damon Runyon Memorial Fund (DRG-743D), the National Science Foundation and the American Lebanese Syrian Associated Charities (ALSAC). One of the authors (C.D.J.) is a predoctoral trainee of Grant ITICA-5176-o 3. REFERENCES

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