Growth hormone acts at a pretranslational level in hepatocyte cultures

Vol. 115, No. 3, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

September 30, 1983

Pages 882-887

GROWTH HORMONE ACTS AT A PRETRANSLATIONAL LEVEL IN HEPATOCYTE CULTURES S. Seelig, C.N. Mariash, D.J. Topliss, J.H. Oppenheimer Divisions of Endocrinology and Metabolism Departments of Pediatrics and Medicine University of Minnesota Minneapolis, Minnesota 55455 Received August II, 1983

We have examined the effects of ovine growth hormone and recombinant DNA synthesized human growth hormone on hepatocytes maintained in serum free cultures~ Both growth hormon~ preparations augmented or attenuated 3 specific mRNA sequences as revealed by two-dimensional gel electrophoresis of [35S] methionine labeled products synthesized in vitro in an mRNA dependent rabbit reticulocyte lysate system. The results clearly indicate that growth hormone, free of potential pituitary contaminants, acts directly on hepatocytes at a pretranslational level.

All previous studies on growth hormone regulation of hepatic mRNA sequences are subject to limitations in interpretation either because pituitary extracted growth hormone was used or the experiments were performed in vivo.

The use of pituitary extracted growth hormone complicates

interpretation, since these preparations frequently contain bioactivities unrelated to the growth hormone moiety

(1-3); whereas in vivo administration

of GH may alter hepatic mRNA sequences by an indirect rather than a direct action on hepatocytes.

To avoid these serious experimental limitations,

we have used recombinant DNA synthesized growth hormone in conjunction with hepatocyte cultures.

With this strategy we demonstrate for the first time

that growth hormone, per se, acts directly on hepatocytes to augment and attenuate the level of specific messenger RNA sequences. METHODS Hepatocyte suspensions were prepared from normal animals by a collagenase perfusion technique (4,5) and plated on collagen coated mesh according to the method of sirica et al., (6). The cells were permitted to attach to the collagen mesh for four hours in Williams Media E

Abbreviations:

Ovine growth hormone, oGH; Methionyl-human growth hormone, Met-hGH

0006-291X/83 $I .50 Copyright © 1983 by Academic Press, Inc. All rights o f reproduction in any form reserved.

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without linoleic acid w h i c h was supplemented with insulin (100mU/ml) and dexamethasone (10-8M) (7). F o l l o w i n g the attachment period, the a t t a c h m e n t media was removed and fresh media was added w h i c h contained the additional hormones to be tested. Media changes were p e r f o r m e d every 48 hours and the cells were m a i n t a i n e d in culture for a total of seven days. At the end of the experiment, the hepatocytes were released from the collagen m e s h by a 15 m l n u t e incubatlon at 37 C in m i n i m u m essential media (MEM) containing 70mg collagenase (Worthington) per i00 ml. The released cells were h a r v e s t e d by centrifugation at 50xg for five minutes and w a s h e d twice with MEM. •

.

.

o

,

The final cell p e l l e t was extracted for total RNA by a guanidine hydrochloride m e t h o d as d e s c r i b e d by M u n n i c h et al (8) w i t h minor modification. A p p r o x i m a t e l y 2-3xi07 hepatocytes were h o m o g e n i z e d in 2.5 ml of 20 mM sodium acetate, p H 5.5, containing 7M guanidine-HCl, 240 m M ammonium acetate, 1 mM dithiothreitol, i0 m M iodoacetate, 0.5% lauroyl sarcosine, and 0.125 mg/ml E. coli tRNA. The initial homogenate was centrifuged at i0,000 xg at 10°C and RNA was p r e c i p i t a t e d from the supernatant with 1/2 volume of ethanol. The r e m a i n d e r of the extraction was as d e s c r i b e d except no lauroyl sarcosine was added to the buffers. In vitro t r a n s l a t i o n of total RNA was p e r f o r m e d in the rabbit reticulocyte l~--ysate system in the presence of [35S] methionine as d e s c r i b e d (9). Analysis of the [35S] m e t h i o n i n e labeled proteins was p e r f o r m e d by two-dimensional gel electrophoresis (i0) and spot intensity was q u a n t i f i e d by video imaging (ii). Ovine growth hormone, (oGH: NIH-GH-SII) was provided Pituitary A g e n c y d i s t r i b u t i o n p r o g r a m and m e t h i o n y l - h u m a n (Met-hGH) was a generous gift of Genentech, Inc.

by the National growth hormone

RESULTS The chemical

purity

(12) was d e t e r m i n e d of GH induced

of the oGH used in our previous

by isoelectric

4 and a t t e n u a t e d

chemical

heterogeneity

previous

interpretation

focusing

4 hepatic

of the oGH raised

(Figure

i).

investigations This p r e p a r a t i o n

m R N A sequences. serious

that the observed

changes

The extensive

questions

about our

were related

to the

GH moiety. To avoid in culture, the question

this p r o b l e m we examined

rather

than p u r i f y

of whether

but it also a d d r e s s e d

the oGH.

the effect

of M e t - h G H

This p e r m i t t e d

the in vivo effects

the issue of w h e t h e r

on hepatocytes

us to address

were caused by growth hormone the changes

observed

were the

+ IEF

Figure i. Isoelectric focusing of approximately 25~g oGH (NIH-GH-SII) was performed as described (i0). The gels were fixed in 10% trichloroacetic acid for 18 hours and stained with Coemassie Brillant Blue R-250. The cathode is on the left; the anode on the right.

883

not only

VoI. 115, No. 3, 1983

consequence

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of a direct action of GH on the liver.

Hepatocytes

were maintained

for seven days in control medium with and without growth hormone. culture,

total RNA was extracted

rabbit reticulocyte

and used to direct protein synthesis

lysate system in the presence

of

labeled proteins were analyzed by two-dimensional Three spots

(3,16,20)

previously

and Met-hGH in the culture

gel eleetrophoresis

The other previously

(Spots 6,11,15,17,19)

to either growth hormone preparation

in the

[35S] methionine.

identified as GH responsive,

system.

GH responsive mRNA sequences

Following

The (Figure 2).

responded

identified

to oGH

in vivo

did not respond in vitro

or were not visible on the two-dimensional

gel pattern. Quantitation

of the visible

spots for various doses of oGH and for Met-

hGH indicated

a dose response relationship

approximately

i00 with oGH and the half maximal concentration

2.5~g/ml

for Spot 3 mRNA.

(10~g/ml)

Of particular

essentially mimics

(Table i).

interest

The fold induction was was approximately

is the finding that Met-hGH

the effects of oGH in vitro.

DISCUSSION Until recently only albumin and alpha 2U globulin mRNA sequences have been available synthesis

for the investigation

of growth hormone action.

rate of albumin and alpha 2U globulin parallel

their respective

mRNA sequences with GH treatment,

believed that GH exerts control of hepatocellular translational presumptions

level

(13,14).

This conclusion,

that GH is the only bioactive

Since the

the changes

in

it is currently function at a pre-

however,

substance

is based on the

in the injected material

and that the apparent effect of GH is the result of a direct action on liver.

Lewis and colleagues

insulinemic

activity in relatively

more purified preparations upon insulin intake

have demonstrated

(i).

impure preparations

(16), it is possible

and hyper-

of hGH but not in

Since alpha 2U globulin mRNA is dependent

(15), and urinary alpha 2U globulin

mRNA is secondarily mediated Motwani

a hyperglycemic

is dependent on dietary

that the in vivo effect of GH on alpha 2U globulin through alterations

et al have demonstrated

increased

884

in insulin or caloric

alpha 2U globulin

intake.

in the media

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IEF

Figure 2. Two-dimensional electrophoretograms of in vitro synthesized products of the rabbit reticulocyte lysate translation system when directed by total RNA from hepatocytes maintained in control media (panel A), in control media with 10~g/ml of oGH (panel B) and in control media with 10~g/ml of Met~hGH (panel C). Equal radioactivity was applied to each gel.

of imprint cultures dexamethasone

of h e p a t o c y t e s

and g r o w t h hormone;

m R N A level or the relative established

(17).

Although

w i t h a c o m b i n a t i o n of triiodothyronine, however,

neither

rate of synthesis

of alpha

this c i r c u m s t a n t i a l

885

the alpha

2U globulin

2U g l o b u l i n was

evidence

suggests

that growth

Vol. 115, No. 3, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS T~BLE 1

Effect of oGH and Met-hGH on the Level of Four mRNA Sequences in Hepatocyte Culture

Spot Number

#3

Control

#16

#20

#17

.0016

.216

.469

.277

(.0011-.0024)

(.143-.328)

(.310-.713)

(.182-.421)

.163

.553

,253

+oGH (~g/ml) .01

.003*

.i

.011"

.211

.512

.280

1

.044*

.I01"

.457

.257

i0

.154"

.073*

.985*

.274

+Met-hGH

.173"

.070*

.874*

.188

10~g/ml Hepatocytes were prepared as described in the text. Control medium was William's Medium E without linoleic acid with 100mU/ml of insulin, 10-8M dexamethasone and llmM glucose. Additional hormones were added to the control media as indicated. RNA was extracted6as described and the yield of RNA was approximately 30 to 50~g per 2x10 cells. Total [35S] methionine incorporated into protein in the reticulocyte lysate system did not depend on whether the RNA was obtained from control or growth hormone treated cells. Results are expressed as cpm incorporated into a spot x i00 divided by the total [35S] methionine incorporated into protein. Analysis of each experimental condition was performed by a single twodimensional gel and the spot intensity of that single gel represents the average mRNA level for 8 individual culture dishes. To determine whether the changes observed in spot intensity were the result of random variation, 35 spots which were not previously identified as growth hormone responsive were quantitated and a population variance determined. The range in parenthesis below the control values represents the two standard deviation range for the growth hormone responsive spots as derived from the population variance.

hormone

acts directly

on hepatocytes

not been unambiguously hormone,

level

It is o f i n t e r e s t cultures exist

established.

free of pituitary

pretranslational

fully mimic

effects

possibility

contaminants,

that neither

action

milieu

to s u p p o r t

886

at a

in h e p a t o c y t e

Several

the possibility

and metabolic

is i n s u f f i c i e n t

that growth

sequences.

are the result of an indirect

culture

this has

on hepatocytes

the oGH or Met-hGH

including

level,

establish

acts directly

the in vivo observations.

is the h o r m o n a l

of hepatocyte

Our observations

for s e v e r a l m R N A

for t h i s o b s e r v a t i o n ,

in vivo

at a pretranslational

possible

that

reasons

some of the

o n t h e liver.

Another

of our current method all growth

hormone

action.

Vol. 115, No. 3, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Finally, culture of hepatocytes may alter cellular function sufficiently so that all normal function of adult hepatocytes is not expressed. In the rat growth hormone levels range from i0 to 500ng/ml.

Concen-

trations of oGH in a range from i0 to 100ng/ml are sufficient to c a u s e a significant augmentation of spot 3 mRNA.

Since the observed in vitro

effects of oGH strongly suggests a physiological response to the hormone, this experimental approach should permit a more detailed evaluation of the interaction of growth hormone with other hormones and metabolites at the hepatocellular level. Acknowledgements: This work was supported by NIH Grant AM-19802 (JHO), Clinical Investigator Award AM-0080 (CNM), Minnesota Medical Foundation Research Grant (CNM), National Research Service Award AM-06478 (SS) and a Biomedical Research Support Grant from the Division of Research Resources, NIH, to the University of Minnesota Medical School (SS). We thank Nate Zilz and Robin Manthei for their excellent technical assistance and Ms. Sheryl Frankel for secretarial assistance. Ovine growth hormone was generously supplied by the National Pituitary Agency; Met-human growth hormone was generously provided by Genentech, Inc. REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17.

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