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3 1293 00790 8522

I“ V

LIBRARY
Michigan State
University

\ I

 

 

 

 

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This is to certify that the

dissertation entitled

UTILIZATION OF SOLUTION HYBRIDIZATION TO EXAMINE
DIFFERENTIAL EFFECTS OF GROHTH HORMONE AND
RACTOPAMINE ON THE RELATIVE ABUNDANCE OF IGF-I
mRNA IN LIVER AND SKELETAL MUSCLE OF PIGS

presented by

ALAN LESLIE GRANT

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in Animal Science

,& 4/C
Major professor

6/477 a"

MS U it an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

PLACE lN RETURN BOX to remove this checkout from your record.
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MSU Is An Affirmative Action/Equal Opportunity Institution
emu-no.1

 

UTILIZATION OF SOLUTION HYBRIDIZATION TO EXAMINE
DIFFERENTIAL EFFECTS OF GROWTH HORMONE AND RACTOPAMINE ON
THE RELATIVE ABUNDANCE OF IGF-I mRNA IN LIVER

AND SKELETAL MUSCLE OF PIGS

BY
Alan Leslie Grant

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of
DOCTOR OF PHILOSOPHY
Department of Animal Science

1990

ma- 07X¢¢

ABSTRACT
UTILIZATION OF SOLUTION HYBRIDIZATION TO EXAMINE
DIFFERENTIAL EFFECTS OF GROWTH HORMONE AND RACTOPAMINE ON

THE RELATIVE ABUNDANCE OF IGF-I mRNA IN LIVER
AND SKELETAL MUSCLE OF PIGS

BY
Alan Leslie Grant

It was necessary to develop a sensitive solution
hybridization-nuclease protection assay to examine the
effects of growth hormone and ractopamine, a
phenethanolamine, on the low abundance of IGF-I mRNA in
liver and skeletal muscle of market weight pigs. An
antisense RNA probe was synthesized from a porcine IGF-I
cDNA and utilized in a solution hybridization-nuclease
protection assay to quantitate relative abundance of IGF-I
mRNA. Optimum hybridization and digestion conditions were
determined. Intrassay and interassay coefficients of
variations were 2.8 and 2.7%, respectively. A linear
hybridization response was obtained using from 50 to 150 ug
of liver RNA.

In Experiment 1, barrows (average initial body weight
83.7 kg) were administered 50 ug of recombinant porcine
growth hormone per kg of body weight daily i.m. for 24

days. Pigs were fed diets containing either 14% or 20%

crude protein. At the end of the experimental period, pigs
were slaughtered and tissues collected for RNA analyses.
Relative abundance of IGF-I mRNA was determined in liver
and longissimus dorsi (LD) muscle by a sensitive solution
hybridization-nuclease protection assay. Administration of
growth hormone increased abundance of liver IGF-I mRNA 2.7-
fold in pigs fed the 14% protein diet and 3-fO1d in pigs
fed the 20% protein diet relative to controls. Muscle IGF-
I mRNA levels in the treated pigs were only 77% and 84% of
levels in control pigs fed the 14% and 20% protein diets,
respectively.

In Experiment 2, barrows (average initial body weight
72 kg) were administered ractopamine at either 0 or 20 ppm
in 16% crude protein diets for 28 days. IGF-I mRNA
abundance was determined in liver and skeletal muscle as in
Experiment 1. Ractopamine had no effect on the relative
abundance of IGF-I mRNA in either tissue.

Results from these experiments indicate that abundance
of IGF-I mRNA in liver and skeletal muscle of pigs can be
monitored, is dependent on dietary protein, and is altered
following administration of growth hormone, but not

ractopamine.

ACKNOWLEDGMENTS

I would like to thank the Department of Animal Science
and College of Agriculture and Natural Resources for their
support during my degree. I thank Dr. W.G. Bergen for
serving as my major professor and for allowing me to pursue
a Ph.D. degree in his research program. Dr. Bergen’s
support, patience, understanding, and kindness is greatly
appreciated. I wish to express my appreciation to Dr. Bill
Helferich for serving on my graduate committee and for the
use of his laboratory facilities and equipment. Bill has
contributed much time to my research projects with guidance
and helpful suggestions. Working with Bill has been a
pleasure. I would like to thank Dr. D.B. Jump for serving
on my graduate committee and for his many helpful
suggestions. I wish to thank Dr. R.A. Merkel for serving
on my graduate committee. I am thankful for having the
opportunity to work with him in both research and teaching
programs. Dr. Merkel’s guidance and time has been greatly
appreciated.

There have been a number of faculty, staff, graduate
students and undergraduate students that have helped me

during my degree program. I wish to acknowledge their many

iv

contributions.

I wish to express my deepest gratitude to my wife,
Brenda, for her patience and understanding during my
studies. Brenda's help, support and encouragement have
been major contributions to my success. I also wish to

,thank her for typing this dissertation.

TABLE OF CONTENTS

Page
LIST OF TABLES.......................................viii
LIST OF FIGURES........................................ix
LIST OF APPENDICES...................................xvii
LITERATURE REVIEW.......................................l

Introduction............................. ..... .....1
Regulation of IGF-I Gene Expression................6
Effects of IGF-I on Muscle Growth..................9
Involvement of IGF-I in Mediating Growth

Hormone-Induced Muscle Growth in Pigs.........12
Involvement of IGF-I in Mediating Beta

Adrenergic Agonist-Induced Muscle Growth......17
Summary of the Literature Review..................20

CHAPTER 1: DEVELOPMENT OF A METHOD TO DETERMINE THE
RELATIVE ABUNDANCE OF IGF-I mRNA IN LIVER AND
SKELETALMUSCE OF PIGSOOOOOOOOOCOOI.........OOOCOOOOZZ

Abstract..........................................22
Introduction......................................23
Materials and Methods.............................25
Tissue Sample Collection and RNA Isolation......25
Northern Blot Analysis..............¢...........26
Synthesis of Antisense Probe....................27
Results and Discussion............................32
Summary...........................................50

CHAPTER 2: ADMINISTRATION OF GROWTH HORMONE TO PIGS
ALTERS THE RELATIVE ABUNDANCE OF IGF-I mRNA IN
LIVERAND SIGIIETALMUSCLEeeeoeoeeeeeoeeoeee000000000051

Abstract..........................;...............51
Introduction......................................53
Materials and Methods.............................55
Animals, Care and Treatments....................55

Tissue Sample Collection, RNA Isolation,
and Analysis..................................57

vi

Statistical Analysis............................60
Results and DiscuSSion. I O O O O I O O O O O O O O O I O I O I I O O O O I O 61
smary.eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee00.079

CHAPTER 9: EFFECTS OF RACTOPAMINE ON THE RELATIVE
ABUNDANCE OF IGF-I mRNA IN LIVER AND SKELETAL
MUSCIE OF PIGS...‘0.000.000.........OOOOOOOOOOOOOO0.080

Abstract..........................................80
Introduction......................................81
Materials and Methods.............................83
Animals, Care and Treatments....................83
Tissue Sample Collection, RNA Isolation,
and Analysis..................................85
Statistical Analysis............................86
Results and Discussion............................87
Summary...........................................97

CONCLUSIONS AND RECOMMENDATIONS........................98
APPENDICESOOOOO0.0.0....I.........OOOOOOOOOOOOOOO0.0.0103
Appendix A: Acid Guanidine Phenol Chloroform
RNA Extraction Pr°t°C°1eeeeeeeeeeeeeeeeeeeeee0.103
Appendix B: IGF-I Solution Hybridization-
Nuclease Protection Assay......................107

LITEMWRE CITED.........OOOOOOOOOOOOOO00.0.0......00.117

vii

Table

Table

Table

Table

Table

Table

Table

3.1.

LIST OF TABLES

Page

Composition and calculated analyses
of diets fed to crossbred barrows.............56

Effects of growth hormone for 24 days on
average daily gains, liver weights, and

carcass measurements of pigs fed 14% and

20% crude protein diets.......................62

Relative abundance of IGF-I mRNA in liver

and LD muscle from growth hormone-treated

(GH) and control (CO) pigs fed 14% (14) or

20% (20) crude protein diets ........ ..........67

Relative abundance of liver beta-tubulin and
beta-actin mRNA and LD muscle beta-tubulin

mRNA in growth hormone-treated (GR) and

control (C0) pigs fed 14% (14) or 20% (20)

crude protein diets...........................77

Composition and calculated analyses of diet
fed to barrOWSOOOO00......0.000.000.0000....0084

Effects of ractopamine for 28 days on
skeletal muscle and body weight gains of
crOSSbréd pigSOOOOOOOOOOOO......OOOOOOO....0.088

Relative abundance of IGF-I mRNA and beta-

tubulin mRNA in liver and LD muscle of
ractopamine-treated (R) and control (C) pigs..94

viii

LIST OF FIGURES

Page

Figure 1.1. Maps of the plasmid vector (top) and IGF-
I cDNA insert (bottom) that were used for
the synthesis of the IGF-I cDNA and RNA
probes. The cDNA was subcloned into the
EcoRI site of the plasmid vector, pGEM-I
(Promega, Madison, WI), and generously '
provided by Dr. Frank Simmen (Tavakkol et
a1.,
1988)....................... ...... . .......... 29

Figure 1.2- Autoradiograms from Northern blot
analysis of IGF-I mRNA in 40 ug of total
RNA from uterine tissue of a pregnant pig
(12 days of gestation), 23 ug of Poly(A)
RNA from liver of a market weight pig,
and 38.3 ug of Poly(A)+ RNA from liver of
a newborn pig. RNA was denatured and
electrophoretically separated in 1.2%
agarose gels containing 2.2 M
formaldehyde and then transferred to
Zetabind (Cuno). Blots were prehybridized
at 42°C for 2 hours in 50% formamide, 5X
SSC, 5X Denhardt's solution, 0.1% SDS, 1
mM EDTA, 50 mM sodium phosphate buffer
(pH 6.5), and 0.5 mg/ml tRNA. RNA was
hybridized with a porcine IGF-I cDNA
probe at 42°C for 16 hours. Composition
of the hybridization solution was
identical to the prehybridization
solution except that the hybridization
solution contained only 1X Denhardt’s and
contained two million cpm of probe/ml.
Following hybridization, blots were
washed with three cycles of 0.1x SSC and
0.1% SDS at 65° C. Uterine and liver blots
were exposed to film with two
intensifying screens at -80 C for 3 and 7
days, respectively...................... ..... 34

Figure 1.3. Agarcse gel electrophoresis of

ix

Figure 1.4.

Figure 1.5.

restriction enzyme digestion products.
Following digestion of pGEM-1/IGF-I
plasmid DNA with restriction enzymes,
products were electrophoresed in 1%
agarose, stained with ethidium bromide,
and visualized utilizing a uv light (302
nm). Lambda and PhiX174 cut DNA were used
as DNA size markers. Marker fragment
sizes are indicated in base pairs (bp).
The 580-bp IGF-I cDNA insert was also
electrophoresed........................ ...... 37

Autoradiogram from a solution
hybidization assay utilizing an IGF-I RNA
probe contaminated with endogenous
template DNA. A porcine IGF-I cDNA was
utilized a? a template to synthesize an
antisense 2P-labeled RNA probe. The
probe was hybridized with 0 to 1000 pg of
exogenous plasmid template DNA at 42 C
for 14 hours. Following hybridization,
133 units of nuclease 81 were added and
samples incubated for an additional 1
hour at 37°C. Protected RNA fragments
were electrophoretically separated in
1.2% agarose gel containing 2.2 M
formaldehyde and then subjected to
autoradiography. The probe was also
electrophoresed alone [Probe; 678
nucleotides (nt)in length] and following
digestion with nuclease 81 (S1)......... ..... 39

Autoradiogram from a solution
hybridization assay utilizing an IGF-I
RNA probe purified by gel

electrophoresis. Fifty and 100 ug of RNA
from liver (A) and LD muscle (C) of a fed
finishing pig and from liver of a 48 hour
fasted pig (B) was included in the assay.’
A porcine IGF-I cDNA was utilized as 3
template to synthesize an antisense 3 P-
labeled RNA probe. The probe was
hybridized to RNA at 42 C for 14 hours.
Following hybridization, 133 units of
nuclease 81 were added and samples
incubated for an additional 1 hour at
37°C. Protected RNA fragments were
electrophoretically separated in 5%
acrylamide/8 M Urea gels and then

-subjected to autoradiography. Transfer

RNA (tRNA) was included as a negative
control to account for nonspecific
background hybridization. The 81

Figure 1.6.

Figure 1.7.

Figure 1.8.

nuclease-protected RNA fragments are 580
bases in length. The IGF-I probe was also
included alone and is 678 bases long.. ....... 42

Autoradiogram from an IGF—I solution
hybridization assay in which four amounts
of nuclease 81 were used following
hybridization. Each hybridization
reaction contained 100 ug of finishing
pig liver RNA and 2 million cpm of IGF-I
RNA probe. A porcine IGF-I cDNA was
utilized agz a template to synthesize an
antisense 2P-labeled RNA probe. The
probe was hybridized to RNA at 42° C for
14 hours. Following hybridization, 266,
133, 26. 6 or 13. 3 units of nuclease 81
were added and samples incubated for an
additional 1 hour at 37° C. Protected RNA
fragments were electrophoretically
separated in 5% acrylamide/8 M Urea gels
and then subjected to autoradiography.
The 580-base pair (bp) protected RNA
fragment is shown.................... ........ 44

Autoradiogram from an IGF-I solution
hybridization assay in which
hybridization was conducted with 100 ug
of pig liver RNA at either 42 or 65 C. A
porcine IGF-I cDNA was utilized as a
template to synthesize an antisense 32P-
labeled RNA probe. The probe was
hybridized to RNA at 42 or 65°C for 14

hours. Following hybridization, 133

units of nuclease 81 were added and

samples incubated for an additional 1

hour at 37° C. Protected RNA fragments

were electrophoretically separated in 5%
acrylamide/8 M Urea gels and then

subjected to autoradiography. Transfer

RNA (tRNA) was included as a negative

control to account for nonspecific

background hybridization. The S1
nuclease-protected RNA fragments are 580

bases in length. The IGF-I probe was also
included alone and is 678 bases long.........44

Autoradiogram from an IGF-I solution
hybridization assay in which RNA from
liver (LIV) and LD muscle (LD) of a
control pig (C) and a growth hormone-
treated pig (GH) was utilized. Fifty, 75,
100, and 150 ug of C-LIV RNA was included
in the assay for examination of a dose-
response relationship. A porcine IGF-I

xi

Figure 1.9.

Figure 2.1.

cDNA was utilized as a template to
synthesize an antisense P-labeled RNA
probe. The probe was hybridized to RNA at
65°C for 14 hours. Following
hybridization, 133 units of nuclease 81
were added and samples incubated for an
additional 1 hour at 37 °C. Protected RNA
fragments were electrophoretically
separated in 5% acrylamide/8 M Urea gels
and then subjected to autoradiography.
Transfer RNA (tRNA) was included as a
negative control to account for‘
nonspecific background hybridization. The
$1 nuclease-protected RNA fragments are
580 bases in length. The IGF-I probe was
also included alone and is 678 bases
long.Transfer RNA (tRNA) was included as
a negative control to account for
nonspecific hybridization. The IGF-I RNA
probe was included alone in the gel and
is 678 nucleotides (nt) long. Protected
fragments are 580 base pairs (bp) in
length.................................. ..... 47

Dose-response relationship between
quantity of total RNA used and
hybridization signal obtained in the
solution hybridization-nuclease
protection assay. A porcine IGF-I cDNA
was utilized 33 a template to synthesize
an antisense P-labeled RNA probe. The
probe was hybridized to 50, 75, 100, and
150 ug of porcine liver RNA at 65° C for
14 hours. Following hybridization, 133
units of nuclease 81 were added and
samples incubated for an additional 1
hour at 37° C. Protected RNA fragments
were electrophoretically separated in 5%
acrylamide/8 M Urea gels and then
subjected to autoradiography. Transfer
RNA (tRNA) was included as a negative
control to account for nonspecific
background hybridization. The signals
obtained in the 580-base pair nuclease-
protected fragments were quantitated by
liquid scintillation analysis. Results
have been corrected for nonspecific
hybridization by subtracting the signal
obtained from hybridization to tRNA (y-
intercept=80; slope=7.7; r squared=.978).....49

Autoradiograms of IGF-I mRNA abundance in

liver and LD muscle from growth hormone-
treated (GH) and control (C0) pigs fed

xii

Figure 3.1.

Figure 3.2.

(pH 6.5), and 0.5 mg/ml tRNA. RNA was
hybridized with beta-tubulin or beta-
actin cDNA probes at 42 °C for 16 hours.
Composition of the hybridization solution
was identical to the prehybridization
solution except that the hybridization
solution contained only 1X Denhardt’s and
contained two million cpm of probe/ml.
Following hybridization, blots were
washed with three cycles of 0. 2X SSC and
0.1% SDS at 55° C and then subjected to
autoradiography. Visualization of 18S
and 288 RNA following methylene blue
staining of the Northern blots is also
shown. Each lane represents one pig (n=4
pigs/treatment)..............................76

Autoradiograms of IGF-I mRNA abundance in
liver and LD muscle from ractopamine-
treated (R) and control (C) pigs. Liver
and muscle RNA was isolated and analyzed
via a solution hybridization-nuclease
protection assay. A porcine IGF-I cDNA
was utilized 33 a template to synthesize
an antisense P-labeled RNA probe. The
probe was hybridized to 100 ug of RNA at
65°C for 14 hours. Following
hybridization, 133 units of nuclease 51
were added and samples incubated for an
additional 1 hour at 37° C. Protected RNA
fragments were electrophoretically
separated in 5% acrylamide/8 M Urea gels
and then subjected to autoradiography.
Transfer RNA (tRNA) was included as a
negative control to account for
nonspecific background hybridization. The
81 nuclease-protected RNA fragments are
580 bases in length. The IGF-I probe was
also included alone and is 678 bases
long. Each lane represents one pig (n26
pigs/treatment)..............................91

Autoradiograms from Northern blot
analyses of beta-tubulin mRNA abundance
in liver and LD muscle from ractopamine
(R) and control (C) pigs. Twelve ug of
muscle RNA and 20 ug of liver RNA were
denatured and electrophoretically
separated in 1.2% agarose gels containing
2.2 M formaldehyde and then transferred
to nitrocellulose. Blots were
prehybridized at 42°C for 2 hours in 50%
formamide, 5X SSC, 5x Denhardt’s
solution, 0.1% SDS, 1 mM EDTA, 50 mM

xiv

Figure 4.1.

Figure 8.1.

Figure 3.2.

sodium phosphate buffer (pH 6.5), and 0.5
mg/ml tRNA. RNA was hybridized with
beta-tubulin or beta-actin cDNA probes at
42 °C for 16 hours. Composition of the
hybridization solution was identical to
the prehybridization solution except that
the hybridization solution contained only
1X Denhardt’s and contained two million
cpm of probe/ml. Following
hybridization, blots were washed with
three cycles of 0. 2X SSC and 0.1% SDS at
55°C and then subjected to
autoradiography. Visualization of 18S and
288 RNA following methylene blue staining
of Northern blots is also shown. Each
lane represents one pig (n=6
pigs/treatment)........................ ...... 93

Diagram showing possible mechanisms by
which IGF-I may mediate growth hormone-
or ractopamine-induced skeletal muscle
hypertrophy in pigs. IGF-I may mediate
growth hormone or ractopamine actions by
1).acting in an endocrine manner, in
which case additional liver IGF-I is
transported to the skeletal muscle where
it elicits its effect on muscle protein
metabolism or 2) acting in a paracrine
and(or) autocrine manner, in which case
growth hormone or ractopamine acts
directly on the muscle resulting in local
production of IGF-I which effects protein
metabolism..................................101

Maps of the plasmid vector (top) and IGF-

I cDNA insert (bottom) that were used to
synthesize the IGF-I RNA probe. The cDNA

was subcloned into the EcoRI site of the
plasmid vector, pGEM-I (Promega, Madison,

WI), and generously provided by Dr. Frank
Simmen (Tavakkol et a1., 1988)..............114

Dose-response relationship between
quantity of total RNA used and
hybridization signal obtained in the
solution hybridization-nuclease
protection assay. A porcine IGF-I cDNA
was utilized 3? a template to synthesize
an antisense P-labeled RNA probe. The
probe was hybridized to 50,75, 100, and
150 ug of porcine liver RNA at 65°C for
14 hours. Following hybridization, 133
units of nuclease S1 were added and
samples incubated for an additional 1

hour at 37°C. Protected RNA fragments
were electrophoretically separated in 5%
acrylamide/8 M Urea gels and then
subjected to autoradiography. Transfer
RNA (tRNA) was included as a negative
control to account for nonspecific
background hybridization. The signals
obtained in the 580-base pair nuclease-
protected fragments were quantitated by
liquid scintillation analysis. Results
have been corrected for nonspecific
hybridization by subtracting the signal
obtained from hybridization to tRNA (y-
intercept=80; slope=7.7; r squared=.978)....116

LIST OF APPENDICES

Page
Appendix A. Acid guanidine phenol chloroform RNA -
extraction protocol....... ....... ...... ..... 103
Appendix B. IGF-I solution hybridization-nuclease
proteCtion assay.......OCOOOOOOOOOIOO ....... 107

xvii

LITERATURE REVIEW

Introduction

Development of strategies to improve the efficiency of
lean meat production from domestic livestock is a major
goal of animal scientists. Such strategies have included
dietary manipulation and, more recently, administration of
exogenous agents (e.g., growth hormone and beta adrenergic
agonists). Growth hormone and beta adrenergic agonists
increase muscle growth and decrease fat deposition in meat
animals. However, the extent to which these compounds
alter composition is dependent upon the nutritional status
of the animal. The above manipulations create useful
animal models in which the cellular and molecular
mechanisms involved in skeletal muscle growth can be
studied. Currently, mechanisms responsible for the
increased muscle growth resulting from implementation of
the above strategies are unclear.

Since insulin-like growth factor (IGF-I) has been
demonstrated to have positive effects on skeletal muscle
growth and protein accretion in cell cultures and in vivo,
this growth factor has been implicated in mediating the

actions of growth hormone and beta adrenergic agonists on

2
muscle growth. The following sections will describe the
biosynthesis of IGF-I and will review studies that have
been designed to examine the role of IGF-I in skeletal

muscle growth.

WM“ fLLLico- m 168 mwgd
11.132135123221419

Insulin-like growth factor-I (IGF-I; also referred to
as somatomedin-C) is a mitogenic single-chain polypeptide
structurally similar to insulin. The mature peptide
consists of 70 amino acids and has a molecular weight of
approximately 7500 daltons. IGF-I is synthesized as a
prepro-peptide and then processed to the mature peptide
similiar to insulin (Steiner, 1978; Steiner et a1., 1980:
Vassilopoulou-Sellin and Phillips, 1982; Rotwein et a1.,
1987). The peptide consists of 4 domains (B-C-A-D) flanked
by an amino-terminal peptide (signal peptide) and a
carboxyl-terminal extension (E domain) (Gammeltoft, 1989).
Hansen et al.(1983) and Rotwein (1986) have found from cDNA
cloning studies that two different IGF-I precursor peptides
exist. One contains 153 amino acids and the other contains
195 amino acids. The two mRNAs that encode these two
precursor peptides encode the mature peptide sequence (70
amino acids), amino-terminal peptide (48 amino acids), and
a carboxyl-terminal peptide of 19 or 61 amino acids. The
smaller precursor peptide (153 amino acids) is designated
IGF-IA, whereas the larger peptide (195 amino acids) is

designated IGF-IB. Studies using rat and mouse cDNAs have

3
also revealed IGF-IA and IGF-IB precursors (Bell et a1.,

1986; Casella et a1., 1987; Murphy et a1., 1987; Roberts et
al.,1987; Shimatsu and Rotwein, 1987). A cDNA encoding IGF-
IA has been isolated from porcine liver (Tavakkol et a1.,
1988 ) and chicken liver (Kajimoto and Rotwein, 1989).

The 70 amino acid sequence of the mature IGF-I peptide
is identical among the porcine, human, and bovine species
but differs from sheep, rat, mouse, and chicken by 1, 3, 4,
and 10 residues, respectively (Francis et a1., 1989;
Kajimoto and Rotwein, 1989). Amino acid sequences of the
IGF-IA carboxyl-terminal and amino-terminal peptides are
also highly conserved (Tavakkol et a1., 1988: Kajimoto and
Rotwein, 1989).

The two carboxyl-terminal peptide extensions of IGF-IA
and IGF-IB have no amino acid homology. The synthesis of
these two forms was believed to be the result of
alternative processing since only one IGF-I gene has been
identified in the human genome (Brissenden et a1., 1984:
Tricoli et a1., 1984). The human and rat genes that encode
IGF-I are 45 and 73 kb, respectively, and contain five
exons and four introns (Rotwein et a1., 1986; Shimatsu and
Rotwein, 1987). Subsequently, it was found that, in rats
and humans, exons 1 and 2 encode portions of the signal
peptide, exons 2 and 3 encode the mature IGF-I peptide,
exons 3,4, and 5 encode the carboxyl-terminal region and
3’- untranslated sequences (Shimatsu and Rotwein, 1987;

Rotwein et a1., 1986). IGF-IA (153 amino acids) is encoded

4
by exons 1,2,3, and 5, whereas IGF-IB (195 amino acids) is

encoded by exons 1,2,3, and 4 (Jansen et al., 1983;
Rotwein, 1986 and 1987). Tavakkol et al. (1988) have
compared human, rat, and pig exons 2,3, and 5 and found
that within these exons nucleotides corresponding to the
leader peptide (75 nt upstream from sequences encoding the
mature peptide), mature peptide, E peptide, and 3'-
untranslated region (first 85 nt) were 79, 86.2, 88, and
63.5% identical.

Processing of the primary IGF-I gene transcript has
been reviewed by Daughaday and Rotwein (1989). Not only
does alternative mRNA splicing occur at 5'- and 3'-ends of
the IGF-I gene, but there are several polyadenylation sites
at the 3’-end of exon 5. As a result, several IGF-I mRNA
species are generated ranging in size from 0.7 to 8.0 kb.

Traditionally it was thought that liver was the
primary site for the synthesis of IGF-I (Daughaday et al.,
1972), but many studies have provided evidence that IGF-I
is synthesized in multiple organs and tissues. The most
definitive studies first supporting this were those of Han
et al. (1987), Murphy et al. (1987a) and Han et
al.(1988),in which IGF-I mRNA was detected in various
organs and tissues. Prior to these studies, the
observation that tissue concentrations of IGF-I in several
tissues increased before an increase in serum IGF-I
following administration of growth hormone to rats
(D’Ercole et al., 1984) led to the proposal that IGF-I may

act on tissues in a paracrine or autocrine manner.

5
Orlowski and Chernausek (1988) found that administration of

rat growth hormone to hypophysectomized rats increased
liver and kidney IGF-I concentrations relative to
hypophysectomized controls, but had no effect on serum
concentrations. These observations suggested a discordance
of serum and tissue somatomedin levels and supported a
paracrine or autocrine action of IGF-I in rats administered
growth hormone. However, lack of a response in serum IGF-I
concentrations to growth hormone may be due in part to the
short half life of IGF-I in serum from hypophysectomized
rats (Zapf et al., 1986). In a recent review, Daughaday
and Rotwein (1989) discuss how IGF-binding proteins may be
involved in serum IGF-I stability. In hypophysectomized
rats, most serum IGF-I is associated with a 35 kd protein
which may potentiate IGF-I action, whereas in normal rats
most serum IGF-l is bound to a 150 kd complex which may
inhibit IGF-I action (Elgin et al., 1987). Hall et al.
(1988) have proposed that the smaller binding complex (also
referred to as insulin-like growth factor binding protein-I
or IBP-I; rev. by Holly and Wass, 1989) may serve to
transport IGF-I from the larger complex in the circulation
to the IGF-I target cells.

Blum et al. (1989) have concluded that the binding
proteins may function as a reservoir for the continuous
~release of low amounts of IGF-I. Slow release of IGF-I may
prevent down regulation of the IGF-I receptor and thus be a

more effective mitogenic stimulus than high concentrations

6
of IGF-I. Six IGF-I binding proteins have been found in

porcine serum and at least two forms have been found
secreted by muscle cell lines (McCusker et al., 1989a and
1989b). Physiological significances of IGF-1 binding
proteins are not clearly understood. Studies designed to
examine their role in IGF-l action are complicated since
these binding proteins are regulated hormonally,

nutritionally and developmentally (McCusker et al., 1989b).

nngnlnninn n; IGF-i gnne Expression

Several factors may regulate IGF-I gene expression in
tissues. These include tissue—specific, hormonal,
developmental, and nutritional factors (fasting, protein
and energy intake). Growth hormone increases liver IGF-I
transcription (Mathews et al., 1986) in growth hormone-
deficient mice and IGF-I mRNA abundance in many tissues
(rev. by Daughaday and Rotwein, 1989). Furthermore, the
multiple species of IGF-I mRNA in rat heart, kidney, lung,
and liver are decreased in hypophysectomized rats and
restored after growth hormone repletion (Roberts et al.,
1986 and 1987). Lowe et al.(1987) found that rat IGF-I
transcripts contained different alternative 5'-untranslated
regions which were expressed in a tissue-specific manner
and were differentially regulated by growth hormone.
Growth hormone also altered the size distribution of liver
IGF-I mRNA species in growth hormone-deficient rats.
(Matthews et al., 1986). Hepler et al. (1989) found that

when growth hormone was administered to hypophysectomized

7

rats, various IGF-I mRNA size classes (resulting from
differences in length of 3'-untranslated regions) were
differentially regulated. They suggested that the greater
stability and(or) more efficient utilization of
polyadenylation sites may account for more rapid rates of
growth hormone-stimulated induction.

Hormonal regulation of IGF-I gene expression is not
limited to growth hormone. Estrogens increased IGF-I mRNA
abundance in the uterus of pituitary-intact and
hypophysectomized ovarectomized rats, however, estrogens
had no effect on hepatic or renal IGF-l mRNA abundance
(Murphy et al., 1987b). Later it was found that estrogens
actually inhibited growth hormone-stimulated hepatic IGF-I
gene expression in ovariectomized, hypophysectomized rats
(Murphy and Friesen, 1988) suggesting that interaction of
hormones is important in regulation of IGF-I gene
expression. Interactions between thyroid hormones and
growth hormone-stimulated IGF-I synthesis and secretion
have been observed (Wolf et al., 1989) as well as
interactions between dexamethasone and growth hormone-
induced IGF-I mRNA (Luo and Murphy, 1989).

IGF-I gene expression is also regulated by plane of
nutrition. Fasting has been found to decrease serum IGF-I
concentrations and hepatic IGF-I mRNA abundance (Clemmons
et al., 1981; Maes et al., 1983; Elmer and Schalch, 1987).'
Protein or energy restriction produce similiar results

(Prewitt et al., 1982; Isley et al., 1983 and 1984; Maes et

al., 1984; Moats-Staats et al., 1989; VandeHaar et al.,
1989). The decreased IGF-I mRNA abundance associated with
fasting is due at least in part to decreased transcription
of the IGF-I gene (Straus and Takemoto, 1989). Reduction
in IGF-I mRNA abundance in response to altered nutritional
status may involve various mechanisms, such as regulation
of growth hormone receptor number and post-receptor events
(Maes et al., 1983; Bornfeldt et al., 1989; Maiter et a1,
1989; Thissen et al., 1989). Phillips (1986) has proposed
that, during restricted nutrition, the decreased growth
hormone-induced generation of somatomedins results in
decreased negative feedback of somatomedins on growth
hormone secretion. This results in a rise in growth
hormone secretion which in the presence of low
concentrations of somatomedins may allow metabolic fuels to
be diverted away from growth and toward vital organs
(brain, heart, lungs,etc.).

IGF-I gene expression is regulated developmentally and
appears to be regulated independently of growth hormone in
fetal stages of growth. For example, liver IGF-I mRNA in
rats increases 8.6-fold between days 11 and 13 of gestation
which is prior to the ontology of growth hormone synthesis
and secretion by the pituitary and before the appearance of
cell surface receptors for growth hormone (Rotwein et al.,
1987). Slabaugh et al. (1982), Flandez et al. (1986), Lund
et al. (1986), and Hynes et al. (1987) have all detected
IGF-I mRNA in fetal tissues. The developmental increase in

IGF-I mRNA also appears to parallel the developmental

9
increase in liver immunoassayable IGF-I during gestation.

However, since multiple tissues and organs synthesize IGF-
I, it is difficult to determine whether the tissue
concentration represents local synthesis or that from the
circulation.

IGF-I was first found to be synthesized by myoblasts
by Hill et a1. (1984). Subsequently, IGF-I gene expression
was also found to be regulated during myogenesis. IGF-I
mRNA levels are low in proliferating cultured myoblasts and
increase transiently by 6- to 10-fold within 48-72 hours
during myogenic differentiation (Tollefsen et al., 1989).
During muscle cell differentiation, there was a 2.5-fold
increase in IGF-I in the culture medium, a 2¥fold increase
in IGF-I receptor number, and greater than a 30-fold

increase in the secretion of an IGF binding protein.

EIIQQSS 2; 152:1 QR MESELQ SIERRA

Most of the actions of the IGF-I on muscle appear to
be primarily mediated by the IGF-I receptor (Ewton et al.,
1987; Kiess et al., 1987; Ballard et al., 1988). The IGF-I
receptor is a ligand-activated tyrosine-specific protein
kinase structurally related to the insulin receptor
(Rechler and Nissley, 1986: Ullrich et al., 1986). The
expression of IGF-I receptors in rat skeletal muscle is
greatest in fetal and early postnatal life and then begins
to gradually decline at 4 weeks of age to adult levels

which is 25% of fetal levels (Alexandrides et al., 1989).

10
Many studies have examined the effect of IGF-I on

cultured muscle cells. IGF-I has been demonstrated to
accelerate amino acid uptake in cultured human skeleton
myoblasts (Hill et al., 1986), L6 rat skeletal myoblasts
(Ewton et al., 1987), and myotubes derived from ovine
satellite cells (Roe et al., 1989). Glucose uptake was
stimulated by IGF-I in cultures of L6 myoblasts (Wang et
al., 1987), BC3H-1 myocytes (Farese et al., 1989) and
myocytes derived from ovine satellite cells (Roe et al.,
1989). Proliferation was enhanced by IGF—I in cultures of
human myoblasts (Hill et al, 1986), L6 myoblasts (Ewton et
al., 1987), and rat skeletal muscle satellite cells (Dodson
et al., 1985; Allen, 1986). Protein synthesis was
increased and degradation decreased by including IGF-I in
muscle cultures of L8 myotubes (Gulve and Dice, 1989), L6
myoblasts and myotubes (Beguinot et al., 1985), and
differentiated ovine satellite cells (Roe et al., 1989).
Differentiation, measured by increases in creatine kinase
activities, by fusion percentages, and by formation of
myosin heavy chain and myomesin, was increased by IGF-I in
cultures of L6 myoblasts (Ewton and Florini, 1980; Florin
and Ewton, 1986; Ewton et al., 1987). In rat skeletal
muscle satellite cells, IGF-I increased differentiation as
indicated by myotube nuclei density (Allen, 1986).
Although muscle cell fusion is cell density-dependent in
culture, IGF-I-induced differentiation is not a result of
greater cell density from the mitogenic effect of IGF-I.

Ewton and Florini (1981) demonstrated that IGF-I stimulated

11

differentiation over wide ranges of final cell densities.
IGF-I is unique in that, unlike most mitogens, it
stimulates both proliferation and differentiation at
physiological concentrations. However, Florini et al.
(1986) have demonstrated that at greater (nonphysiological)
concentrations, differentiation is inhibited. Mechanisms
by which IGF-I induces the above effects on cultured muscle
cells have been reviewed by Florini (1987) and Florini and
Magri (1989). Possible signal transducers include i
oncogenes (Ong et al., 1987; Schneider and Olson, 1988),
cytoskeletal polypeptides that are receptor tyrosine kinase
substrates (Beguinot et al., 1988), and polyamines (Ewton
et al., 1984; Multhauf and.Lough, 1986).

The effect of IGF-I on muscle growth was also examined
in isolated rat soleus muscles. IGF-I was found to enhance
RNA synthesis and the rate of polypeptide chain initiation
(Monier and LeMarchand-Brustel, 1984) and to increase
glucose and amino acid uptake (Poggi et al., 1979; Yu and
Czech, 1984).

Although IGF-I has been demonstrated to increase
weight gains in hypophysectomized rats (Shoelne et a1.,
1982), growth hormone-deficient dwarf rats (Skottner et
al., 1989), and normal rats (Hizuka et al., 1986), few
studies have been designed to examine the effects of IGF-I
on muscle growth in vivo. Jacob et al. (1989) were able to
reduce protein breakdown in fasted rats by administering

IGF-I. Pell and Bates (1989) administered IGF-I to dwarf

12
mice and observed increases in body weights and increases

in muscle protein synthesis and synthetic capacity
(measured as RNA/protein).

Inyolyement 91 L§§:; 1n ngdiating Growth Hormone-Induced
nnscln growth in Pig;

It has been known for some time that growth hormone
can alter carcass composition and improve feed efficiency
in pigs. Lee and Schaffer (1934) found that growth hormone
resulted in accretion of proportionately more muscle and
less fat tissue in pigs. Since then many laboratories have
examined the effect of growth hormone on animal
performance, carcass composition, and tissue growth.
Machlin (1972), Chung et al. (1985), Campbell et al.
(1989), and Smith et al. (1989) have all observed increases
in muscle growth in pigs in response to administration of
porcine somatotropin. The stimulatory effect of growth
hormone on growth is assumed to be mediated, at least in
part, by IGF-I.

Chung et al. (1985) administered 22 ug porcine growth
hormone/kg body weight/day i.m. for 30 days to barrows
initially weighing 32 kg. Barrows were fed a 16% crude
protein corn-soy diet. Growth rate was increased 10%, feed
efficiency was increased 4%, and muscle mass was increased
by 6% relative to controls. These changes were
accompanied by increases in serum IGF-I concentrations (55%
over control concentrations three hours after injection).

Etherton et al. (1987) conducted a dose-response

experiment with 0, 10, 30, 70 ug porcine growth hormone/kg

13

BW/day for 35 days i.m. in growing barrows and found a dose
response in carcass composition changes (increased muscle
and decreased fat with greater amounts of growth hormone)
and serum IGF-I (greater IGF-I concentrations with greater
amounts of growth hormone). Etherton et al. (1986),
Buonomo et al. (1987), and Walton and Etherton (1989) also
obServed increases in serum IGF-I concentration following
porcine growth hormone administration. Administration of
100 ug pituitary-derived porcine growth hormone/kg body
weight/day to intact males and females from 60 to 90 kg
body weight increased plasma IGF-I concentrations up to 2-
fold relative to controls (Owens et al., 1990).
Furthermore, IGF-I concentrations were less in females than-
males which may explain slower growth rates of female than
male pigs (Campbell and Taverner, 1988; Campbell et al.,
1989). However, Owens et al. (1990) did not find greater
plasma IGF-I concentrations in faster growing strains than
slower growing strains of male or female pigs. Such a
discrepancy may be due to other factors. Recently,
Mathison et al. (1989) have conducted IGF-I receptor
studies on satellite cell-derived myotube membranes from
two lines of rams selected for growth rate. They found
that membranes derived from rams selected for greater
growth rates had more IGF-I receptors than those from
slower growing rams. Owens et al. (1990) and Walton and
Etherton (1989) also observed greater concentrations of

IGF-I binding proteins in plasma of growth hormone-treated

14
pigs which could partially explain the greater

concentrations of plasma IGF-I since IGF-I bound to binding
proteins has a longer half life than free IGF-I in pigs,
sheep, and rats (Zapf et al.,1986; Francis et al., 1988:
Walton et al., 1989). The above results suggest that
mechanisms at the target tissue site may be more important
in modulating IGF-I action than plasma IGF-I concentrations
alone.

Evock et al. (1988) demonstrated with growing barrows
that 70 ug/kg BW/day of pituitary-derived porcine growth
hormone was equipotent to recombinant porcine growth
hormone in increaseing serum IGF-I concentrations and in
increasing skeletal muscle growth. Novakofski et al.
(1988) also found that recombinant and pituitary-derived
porcine somatotropin were equipotent in enhancing muscle
growth in pigs. A

Dietary protein concentration plays a major role in
the growth hormone-induced muscle growth in pigs. Smith et
al. (1989a) found that dietary crude protein concentration
had to be greater than 14% for recombinant porcine
somatotropin to increase muscle growth. Stoner et al.
(1989) also found that growth performance of porcine
somatotropin-treated pigs was dependent on the energy
density of the diet, but that lean tissue growth was not
significantly effected by energy density. Unfortunately,
no measurements of IGF-I were made in these studies.

There are no reports of measurements of tissue

concentrations of IGF-I (except serum IGF-I concentrations)

15 . .
or IGF-I mRNA in pigs administered growth hormone. Since

growth hormone receptors are present in skeletal muscle
(Satyanarayama et al., 1988: Zanelli et al., 1989) and
muscle cells (Adaofio and Kostyo, 1988; Lev and Holland,
1986), it seems plausible that growth hormone may act
directly on muscle to increase IGF-I synthesis. This
muscle IGF-I may mediate the growth hormone signal on
muscle growth. Although administration of growth hormone
has never been reported to decrease muscle protein
degradation in vivo in fed states, it has been reported to
increase protein synthesis in mice (Pell and Bates, 1989)
and steers (Eisemann, 1989). If IGF-I were mediating the
actions of growth hormone on muscle growth, then one would
expect muscle protein degradation to be decreased as well,
since IGF-I administration has been demonstrated to affect
both the synthesis and degradation of muscle protein in
vivo (as discussed earlier). Such discrepancy is difficult
to interpret, but may be due to other factors that are
induced by growth hormone which prevent degradation rates
from changing.

Measurements of skeletal muscle IGF-I mRNA levels
could provide clues to possible autocrine and(or) paracrine
roles of IGF-I in growth hormone-induced muscle growth.
Such studies have been conducted with rodents. Turner et
al. (1988) implanted adult female Wistar-Furth rats with
growth hormone-secreting GH3 cells and examined liver and

muscle growth and IGF-I mRNA abundance 80 days later.

Compared with control normallfihts, liver and gastrocnemius
muscle weights were increased 314% and 55%, respectively.
Respective changes in skeletal muscle and liver IGF-I mRNA
abundance were 2.4 and 8-fold greater in treated rats.
Increases in both liver and muscle IGF-I mRNA with
concommitant increases in muscle growth suggests that IGF-I
may play both endocrine and paracrine/autocrine roles in
skeletal muscle growth: however, differential roles of
liver IGF-I and muscle IGF-I were not distinguished in this
study. Matthews et al. (1988) studied the role of IGF-I in
mediating growth hormone-induced growth in transgenic mice
carrying GH fusion genes. Although circulating growth
hormone concentrations were greater in transgenic mice than
in normal mice at birth, hepatic IGF-I mRNA concentrations
and circulating IGF-I levels were not greater until 2 weeks
of age. Furthermore, accelerated growth was not observed
until three weeks of age, after the changes in IGF-I
status. Results of this study led Matthews and his
colleagues to conclude that IGF-I may be directly involved
in mediating the growth hormone effect on growth.
Unfortunately, skeletal muscle IGF-I mRNA abundance
was not quantitated in the above study, so it is unknown if
abundance of IGF-I mRNA was altered in response to
increased circulating levels of growth hormone. Isgaard et
al.(1989) have demonstrated that administration of growth
hormone to hypophysectomized male rats induces expression
of IGF-I mRNA in skeletal muscle, but effects of similiar

treatments on intact rats were not tested. Subsequently,

17
it was demonstrated (Isgaard et al., 1988) that single

injections i.v. every 8 hours or s.c. every 12 hours
(pulsatile treatment) were more effective in increasing
abundance of skeletal muscle IGF-I mRNA than continuous
infusions. Although growth hormone increased hepatic IGF-I
mRNA levels, pulsatile treatments were no more effective
than continuous infusions. However, body weight gain was
25% greater in rats subjected to the pulsatile injections
which implies that growth rate may correlate better with
abundance of IGF-I mRNA in muscle than in the liver.

Future studies of this type need to include measurements of
muscle growth so that the relationship between liver and
muscle IGF-I mRNA abundance and muscle growth can be

established.

Warmmmmwm
1292229 M29212 fizgzth

Beta adrenergic agonists increase skeletal muscle
growth and reduce fattening in many species (for reviews,
see Hanrahan, 1987; Williams, 1987, and Thorton and Tune,
1988). Yang and McElligott (1989) have discussed studies
designed to determine the mechanism of action of beta
adrenergic agonists; An indirect mechanism for the
anabolic action of beta adrenergic agonists has not been
identified. Many studies have demonstrated that various
circulating hormones are probably not involved in beta-

adrenergic agonist-induced muscle hypertrophy. For

18
example, although plasma growth hormone concentrations

increased 2- to 3-fold in sheep fed the beta adrenergic
agonist, cimaterol, for six weeks (Beermann et al., 1987),
cimaterol has been demonstrated to increase muscle growth
in beta agonist-fed hypophysectomized rats (Thiel et al.,
1987). Clenbuterol also stimulates growth in castrated and
adrenalectomized rats demonstrating that effects of
clenbuterol in male rats occur in the absence of a gonadal
or adrenal hormones (Rothwell and Stock, 1988).
Futhermore, serum from rats treated with clenbuterol and
serum from normal rats had similiar effects on protein
synthesis and degradation and proliferation in cultured L8
myoblasts (McElligott and Chaung, 1987). Beermann et al.
(1987) also found that plasma IGF-I concentrations were
depressed by 34% after administration of cimaterol.
However, the IGF-I concentrations decreased during the 6
hour sample collection period. This trend was probably due
to the withholding of feed during the collection period.
Fasting has been demonstrated to reduce plasma IGF-I
concentrations (Clemmons et al., 1981) and therefore makes
the above cimaterol-induced alterations in IGF-I
concentrations difficult to interpret. If one examines the
IGF-I measurements at the zero-hour time, IGF-I
concentration is greater in cimaterol-treated lambs than in
controls. There have been no other reports of plasma IGF-I
measurements in animals fed beta adrenergic agonists.
Results of studies designed to examine the effects of

beta adrenergic agonists on muscle protein turnover have

19

been inconsistent. This has been due to the use of
_different agonists, doses, and routes of administration in
different systems designed to quantitate changes in muscle
protein synthesis and degradation. Since skeletal muscle
contains betal and betaz adrenergic receptors (Apperly et
al. 1976; Stiles et al., 1984; Waldech et al., 1986;
Watson-Wright and Wilkinson, 1986; Rothwell et a1., 1987;
Liggett et al, 1988), it is possible that beta adrenergic
agonists act directly on muscle to exert their anabolic
actions. The increased fractional synthesis rate of
skeletal muscle protein in pigs fed ractopamine (Bergen et
al., 1989; Helferich et a1., 1990), in lambs fed
clenbuterol (Claeys et al., 1989), and in rats fed
clenbuterol (Maltin et al., 1987; MacLennan and Edwards,
1989) is consistent with reports of increased muscle
protein synthesis in cultures of muscle cells containing
ractopamine or clenbuterol (Anderson et al., 1990),
ractopamine (Adeola et al., 1989), and cimaterol (Young et
al., 1990). Furthermore, ractopamine increases the
relative abundance of skeletal muscle alpha actin mRNA in
pigs (Helferich et al., 1990) and myosin.light chain mRNA
in steers (Smith et al., 1989a) which indicates that
ractopamine enhances protein synthesis pretranslationally.
There have also been reports that beta adrenergic agonists
reduce muscle protein degradation in vivo and in cultures
of muscle cells (Forsberg and Merrill, 1986: Reeds et al.

1986; Bohorov et al., 1987; Morgan et a1., 1988: Young et

al., 1990). It is plausiblezghat IGF-I mediates the beta
adrenergic agonist-induced increase in skeletal muscle
protein synthesis and decreased protein degradation. IGF-I
has been demonstrated to increase muscle growth by reducing
protein degradation in rats (Jacob et al., 1989) and by
increasing protein synthesis in dwarf mice (Pell and Bates,
1989). IGF-I has also had similiar effects in muscle cell
cultures (Gulve and Dice, 1989; Beguinot et al., 1985; Roe
et al., 1989). If IGF-I does mediate beta adrenergic
agonist activity, then it most likely acts in a paracrine
or autocrine manner rather than in an endocrine manner. If
IGF-I acted in an endocrine manner in response to beta
adrenergic agonists, then circulating levels of IGF-I would
likely be increased. Increased serum IGF-I would result in
greater anabolic activity in cultures of L8 myoblasts
containing such serum. As stated earlier, McElligott and

Chaung (1987) detected no such difference.

mumm vew

The above review has described the expression of IGF-I
in mammalian tissues and has summarized some of the studies
that have been designed to determine the role of IGF-I in
growth and development of skeletal muscle. Many of these
studies have been conducted in hypophysectomized rats in
order that the interaction between growth hormone and IGF-I
expression can be characterized. Unfortunately, very few

studies in meat-producing animals (cattle, pigs, sheep, and

21

poultry) have included measurements of IGF-I tissue peptide
concentrations and mRNA abundance. Two animal models that
are useful for studying biological mechanisms involved in
growth of skeletal muscle can be created by 1)
administration of growth hormone to finishing swine and 2)~
oral administration of beta adrenergic agonists to pigs.
Both of these manipulations are associated with changes in
skeletal muscle mass which are due at least in part to
alterations in muscle protein synthesis or accretion
(Machlin, 1972; Chung et al., 1985; Maltin et al., 1987:
Evock et al., 1988; Novakofski et al., 1988; Bergen et al.,
1989; Campbell et al., 1989; Claeys et al., 1989; MacLennan
and Edwards, 1989; Smith et al., 1989b; Helferich et al.,
1990). Since IGF-I status has been demonstrated to be
altered by the above perturbations in various species, it
is possible that IGF-I mediates the changes in muscle
protein metabolism. IGF-I is synthesized by many tissues
and has been demonstrated to play endocrine and paracrine
and (or) autocrine roles in growth and development of
various tissues and organs, including skeletal muscle and
liver. Measurements of changes in IGF-I mRNA abundance in
skeletal muscle and liver in the animal models described
above could provide clues to the possible endocrine and
paracrine and (or) autocrine roles of IGF-I in muscle

hypertrophy in growing pigs.

CHAPTER 1
.DEVELOPMENT OF A METHOD TO DETERMINE THE
RELATIVE ABUNDANCE OF IGF-I mRNA IN LIVER AND

SKELETAL MUSCLE 0F PIGS
Abstract

To determine the relative abundance of IGF-I mRNA in
liver and skeletal muscle of market weight pigs, it was
necessary to develop a sensitive solution hybridization-
nuclease protection assay. Utilization of Northern
blotting techniques was sufficient to detect IGF-I mRNA in
uterine tissue of pregnant pigs (12 days of gestation), but
was not sufficient to detect differences in abundance of
IGF-I mRNA in liver and skeletal muscle of market weight
pigs. An antisense RNA probe was synthesized from a
porcine IGF-I cDNA and utilized in a solution hybridization
assay. It was necessary to purify the probe via agarose
gel electrophoresis and electroelution prior to
hybridization to liver and muscle RNA in order to remove
traces of the DNA template. Hybridization temperature at
65°C was found to reduce background relative to

hybridization at 42°C. Following hybridization, nuclease

22

31 was utilized to digest sifigle-stranded nonhybridized
RNA. Optimum digestion conditions were 133 U of nuclease
$1 per 100 ug RNA at 37°C for 1 hour. Protected fragments
were then electrophoresed in 8 M urea/5% acrylamide gels
under denaturing conditions. After gels were dried and
subjected to autoradiography, protected fragments were
excised and radioactivity was quantitated via liquid
scintillation analysis. Results were corrected for
nonspecific hybridization by subtracting the signal
obtained with tRNA. Intrassay and interassay coefficients
of variations were 2.8 and 2.7%, respectively. A linear
response was obtained using from 50 to 150 ug of liver RNA.
The sensitivity of this assay was sufficient to detect
increases in IGF-I mRNA abundance in liver and skeletal

muscle of market weight pigs treated with growth hormone.

Introduction

Since IGF-I status has been demonstrated to be altered
by administration of growth hormone or beta adrenergic
agonists in various species (Chung et al., 1985; Etherton
et al., 1986; Beermann et al., 1987; Buonomo et a1., 1987;
Etherton et al., 1987; Walton and Etherton, 1989; Owens et
al., 1990), it is possible that IGF-I mediates the changes
in muscle protein metabolism. IGF—I is synthesized by many
tissues and has been demonstrated to play endocrine and
paracrine and (or) autocrine roles in growth and
development of various tissues and organs, including
skeletal muscle and liver. Measurements of changes in IGF-
I mRNA levels in skeletal muscle and liver in the animal
models described above could provide clues to the possible
endocrine and paracrine and (or) autocrine roles of IGF-I
in muscle hypertrophy in growing pigs. The objective of
research presented in this chapter was to developan assay
that could detect any changes in abundance of IGF-I mRNA
following treatment with growth hormone or beta adrenergic

agonists.

24

Materials and Methods

Tissue gnnnln Collection nnn RNA Isnlation.

Following exsanguination during slaughter, samples of
liver and longissimus dorsi (LD) muscle were immediately
excised from newborn piglets and market weight barrows.
Samples were collected, cut into pieces approximately 8

3, frozen by submersion in liquid nitrogen and stored at

cm
-80°C. Samples of frozen (-80°C) uterine tissue were
obtained from Dr. Frank Simmen (University of Florida).
RNA was isolated from LD muscle and liver by an acid
guanidine thiocyanate phenol chloroform procedure
(Chomczynski and Sacchi, 1987; see Appendix A for details).
Under the conditions employed in the RNA isolation
procedure (extraction at pH 5 to 6), the final RNA
preparations do not contain DNA (Chomczynski and Sacchi,
1987; Wallace, 1987). Final RNA preparations were
resuspended in TE-8 [10 mM Tris (Boehringer Mannheim
Biochem., Indianapolis, IN), 1 mM EDTA (disodium EDTA,
Fisher Scientific, Fair Lawn, NJ; pH 8.0)] and stored at -
80°C. RNA solutions were scanned from 320 to 220 nm and

A260/A280 were determined to ensure that values greater

than 1.8 wére obtained. Concentrations of RNA in final

25

preparations were determinedzgrom A260 values. Poly(A)+
RNA was isolated from total RNA by batch absorption and
elution from oligo dT cellulose (Boehringer Mannheim
Biochem., Indianapolis, IN) as described by Maniatis et al.
(1982).

unnnnnnn 319; Analysis. Northern blot analysis was
conducted as described by Jump et al. (1984) with the
following modifications. Aliquots of total or Poly(A)+ RNA
were denatured and electrophoretically separated in 1.2%
agarose gels containing 2.2 M formaldehyde. RNA was
transferred to Zetabind membrane (Cuno, Inc., Meriden, CT)
with 50 mM sodium phosphate buffer, pH 6.5. Blots were
prehybridized in 50% formamide (Boehringer Mannheim
Biochem., Indianapolis, IN), 5X SSC (25X SSC is equivalent
to 3.75 M sodium chloride, 0.375 M sodium citrate), 5X
Denhardt’s solution [100x Denhardt’s solution is equivalent
to 2% bovine serum albumin (from fraction V, essentially
fatty acid-free, Sigma Chem. Co., St. Louis, MO), 2%
Polyvinylpyrrolidone—40 (Sigma Chem. Co., St. Louis, MO),
and 2% Ficoll-400 (Sigma Chem. Co., St. Louis, MO)], 50 mM
sodium phosphate buffer (pH 6.5), 1 mM EDTA (disodium EDTA,
Fisher Scientific Co., Fair Lawn, NJ), and 0.5 mg tRNA/ml
_(Boehringer Mannheim Biochem., Indianapolis, IN) for 2
hours at 42°C. The hybridization solution was identical to
.the prehybridization solution, except that the .
hybridization solution contained only 1X Denhardt’s
solution and contained two million cpm of probe/ml.

A 580-base pair porcine IGF-I cDNA subcloned into the

27
EcoRI site of the multiple cloning site of pGEM-l (Promega,

Madison, WI) was obtained from Dr. Frank Simmen (Figure
1.1; Tavvakol et al., 1988). The cDNA encodes a 25-amino
acid leader peptide, the mature (processed) 70-amino acid
porcine IGF-IA peptide, and a 35-amino acid carboxy-
terminal extension peptide. The cDNA insert was excised
with EcoRI (Boehringer Mannheim Biochem., Indianapolis, IN)
and purified by agarose gel electrophoresis and
electroelution as described by Maniatis et a1. (1982). The
purified cDNA was labeled with a Random Primed DNA Labeling
Kit (Boehringer Mannheim Biochem., Indianapolis, IN) using
[32PJdCTP (3000 Ci/mmol; Amersham) to a specific activity
of 4 X 108 cpm/ug DNA. After hybridization at 42°C for 16
hours, blots were washed with 2X SSC and 0.1% SDS
(Boehringer Mannheim Biochem., Indianapolis, IN) at room
temperature, then washed for three cycles in 0.1x SSC and
0.1% SDS at either 55° or 65°C. Dried blots were subjected
to autoradiography using XAR-S film (Eastman Kodak,
Rochester, NY) at -80°C with two intensifying screens
(DuPont, Wilmington, DE);

gynnhesis n; Antisense I§2;1 BEA gnobg. Orientation
of the cDNA insert in pGEM-l was determined by BamHI
restriction enzyme digestion. There is a BamHI site in the
multiple cloning sequence in pGEM-l and a BamHI site within
the cDNA insert 450 base pairs from the 5' end of the cDNA.
Following digestion, products were electrophoresed in

agarose gel, stained with ethidium bromide, and visualized

28 . 1

Figure 1.1. Maps of the plasmid vector (top) and IGF-I cDNA
insert (bottom) that were used for the synthesis of the
IGF-I cDNA and RNA probes. The cDNA was subcloned into the
EcoRI site of the plasmid vector, pGEM-I (Promega, Madison,
WI), and generously provided by Dr. Frank Simmen (Tavakkol
et al., 1988).

29

 

 

 

 

 

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mm 1 Gen!
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(2.9 Kb) ”m" a. 9'1.
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utilizing a uv light (302 an9 Orientation was determined
from the sizes of the BamHI fragments that were present
following digestion. It was determined that use of the SP6
promoter in pGEM-l would generate antisense transcripts
(discussed in Results and Discussion). The recombinant
plasmid containing the IGF-I cDNA was linearized with PvuII
and then utilized as a template in an SP6 Transcription Kit
(Boehringer Mannheim Biochem., Indianapolis, IN) with
[32PJUTP (400 Ci/mmol; Amersham Corp., Arlington Hts., IL)
for the synthesis of a [32PJUTP-labeled RNA probe (see
Appendix B for details). Due to the location of the PvuII
site within the plasmid, 98 base pairs of plasmid sequences
were also transcribed during the transcription reaction,
resulting in the synthesis of a 678-nucleotide probe. The
probe was purified by 1.2% agarose gel electrophoresis and
electroelution (Maniatis et al., 1982) and then used in a
solution hybridization assay as described by Krieg and
Melton (1987) with the following modifications. RNA
samples (100 ug) were annealed with two million cpm of
labeled probe for 14 hours at either 42° or 65°C in 80%
formamide, 0.4 M NaCl, 40 mM PIPES (pH 6.4; US Biochem.
Corp., Cleveland, OH), and 1 mM EDTA. Hybridizations were
conducted in 1.5-m1 microfuge tubes. For a negative
control, tRNA was included as a sample and subjected to the
same hybridization conditions as the tissue RNA samples.
Following hybridization, 13.3, 26.6, 133, or 266 units of
nuclease S1 (Boehringer Mannheim Biochem., Indianapolis,

IN) were added to each sample and the mixture was incubated

31

at 37°C for 1 hour in order to determine the optimum
concentration of nuclease $1 for digestion of single-
stranded nonhybridized labeled RNA. Samples were then
extracted with phenol-chloroform. The RNA was precipitated
from the aqueous phase with ethanol. RNA was
electrophoresed in 1.2% agarose as described for Northern
analysis above or resuspended in 90% formamide gel loading.
buffer, denatured at 90°C for 3 min and then loaded onto a
5% acrylamide/8M urea gel. Following electrophoresis,
agarose gels were subjected to autoradiography, whereas
acrylamide gels were dried and then exposed to film (XAR-S;
Eastman Kodak, Rochester, NY) at -80°C with intensifying
screens (DuPont, Wilmington, DE). The size of protected
RNA fragments was determined by including DNA molecular
weight markers in adjacent lanes. These marker lanes were
cut from the gels prior to drying, stained with ethidium
bromide, and visualized utilizing a uv light (302 nm) for
measurements of migration distances. Protected fragments
580 base pairs in length were excised from the sample lanes
of the gels and radioactivity quantitated by liquid
scintillation analysis. Results were corrected for
nonspecific hybridization by subtracting the values

obtained from tRNA negative controls.

Results and Discussion

Results of Northern blot analyses are presented in
Figure 1.2. Analysis of 40 ug of total RNA from uterine
tissue of pigs at 12 days of gestation resulted in
hybridization of the IGF-I cDNA probe to mRNA species of
1.2 and 8 kilobases. These results are consistent with
those of Tavakkol et al. (1988). Hybridization was
detectable with 23 ug of market weight pig liver and 38.3
ug of newborn pig liver, but the signal was very low
relative to the background. Hybridization to 1.2 and 8
kilobase species was evident with the newborn pig liver,
but not with the market weight pig liver. Due to the high
background on the autoradiogram, small changes in IGF-I
mRNA abundance would not be detectable with this method. A
disadvantage with using poly (A)+ RNA is that quantitation
is not accurate due to incomplete removal of poly (A)-
RNAduring the isolation procedure. Furthermore, large
quantities of RNA would be necessary in order to generate
sufficient quantities of Poly(A)+ RNA.

The solution hybridization-nuclease protection assay
descibed by Krieg and Melton (1987) provides a very

sensitive method to determine the relative abundance of

32

33

Figure 1.2. Autoradiograms from Northern blot analysis of
IGF-I mRNA in 40 ug of total RNA from uterine tissue of a
pregnant pig (12 days of gestation), 23 ug of Poly(A)+ RNA
from liver of a market weight pig, and 38. 3 ug of Poly(A)+
RNA from liver of a newborn pig. RNA was denatured and
electrophoretically separated in 1. 2% agarose gels
containing 2. 2 M formaldehyde and then transferred to
Zetabind (Cuno). Blots were prehybridized at 42 °C for 2
hours in 50% formamide, 5X SSC, 5X Denhardt's solution,
0.1% SDS, 1 mM EDTA, 50 mM sodium phosphate buffer (pH

6. 5), and 0. 5 mg/ml tRNA. RNA owas hybridized with a
'porcine IGF-I cDNA probe at 42 °C for 16 hours. Composition
of the hybridization solution was identical to the
prehybridization solution except that the hybridization
solution contained only 1X Denhardt’s and contained two
million cpm of probe/ml. Following hybridization, blots
were washed with three cycles of 0.1x SSC and 0.1% SDS at
65° C. Uterine and liver blots were exposed to film with two
intensifying screens at -80 C for 3 and 7 days,
respectively.

4
3

 

oawuoub . , 11’.

_
S
8
2

Figure 1.2

mRNAs in various tissues. ng sensitivity of this method
is greater than that of Northern blot or dot blot analyses
with cDNA probes because RNA-RNA hybrids are much more
stable than RNA-DNA hybrids (permitting hybridization under
conditions of greater stingency) and the RNA can be labeled
to higher specific activities (Krieg.and Melton, 1987).
IGF-I mRNA abundance has been determined in rat liver and
muscle with this technique in numerous studies, but there
are no reports in which this method has been used to
determine IGF-I mRNA abundance in liver or muscle of pigs.
Orientation of the cDNA in the pGEM-l plasmid vector
was determined by BamHI restriction enzyme digestion.
Digestion with BamHI resulted in a 3298 and a 147 base pair
fragment (Figure 1.3) indicating that synthesis of
transcripts with the SP6 promoter and SP6 polymerase would
generate antisense transcripts that would be complementary
to tissue IGF-I mRNA. Digestion of the recombinant plasmid
DNA with EcoRI, PvuII, and SacI each resulted in expected
fragment sizes. To minimize the amount of plasmid sequence
that was transcribed, the recombinant plasmid was
linearized with PvuII prior to transcription. The assay
represented in Figure 1.4 was conducted without purifying
the probe via agarose gel electrophoresis prior to
hybridization to tissue RNA. This assay was performed
using plasmid template DNA as a standard. Plasmid DNA
containing the IGF-I cDNA insert serves as a useful
standard since the antisense IGF-I RNA probe should

hybridize to it in a dose-dependent manner. Presence of

36

Figure 1.3. Agarose gel electrophoresis of restriction
enzyme digestion products. Following digestion of pGEM-
1/IGF-I plasmid DNA with restriction enzymes, products were
electrophoresed in 1% agarose, stained with ethidium
bromide, and visualized utilizing a uv light (302 nm).
Lambda and PhiX174 cut DNA were used as DNA size markers.
Marker fragment sizes are indicated in base pairs (bp). The
580-bp IGF-I cDNA insert was also electrophoresed.

37

UHOMCH <20“. H -mUH

 

Haem\H-eoH\H-zmee A
HH=>A\H-eeH\H-zmoe
Hmoom\H-eeH\H-=moe
H=Eee\H-eeH\H-zmee

HHHoe=\e~sz

Hmoem+HHHeeem\eenaeu

7
D. 2
..D 0

2

Figure 1.3

38

Figure 1.4. Autoradiogram from a solution hybidization
assay utilizing an IGF-I RNA probe contaminated with
endogenous template DNA. A porcine IGF-I ggNA was utilized
as a template to synthesize an antisense P-labeled RNA
probe. The probe was hybridized with 0 to 1000 pg of
exogenous plasmid template DNA at 42°C for 14 hours.
Following hybridization, 133 units of nuclease S1 were
added and samples incubated for an additional 1 hour at
37°C. Protected RNA fragments were electrophoretically
separated in 1.2% agarose gel containing 2.2 M formaldehyde
and then subjected to autoradiography. The probe was also
electrophoresed alone [Probe; 678 nucleotides (nt)in length]
nuclease $1 ($1).

39

onoum

Hm nlllr

mmo .Illfi

3.03 Am.

mecca A
$53 lfi
mecca IH
9.83 A

'- 678nt

Figure 1.4

40
endogenous template DNA in the probe solution led to

hybridization signals that appeared in reactions containing
0 ug of added DNA. The endogenous DNA rendered it
impossible to accurately quantitate abundance of tissue
IGFrI mRNA. Once steps were taken to remove the
contaminating DNA, expected results were obtained (Figure
1.5). The probe alone appeared as a 678-nucleotide
fragment and the RNA-RNA hybrids appeared as 580-base pair
fragments. IGF-I mRNA was detected in porcine liver and LD
muscle. The assay represented in Figure 1.5 includes
electrophoresis in 8 M urea/5% acrylamide. Acrylamide was
used rather than agarose in order to improve the resolution
of the RNA fragments. RNA fragments less than 700 base
pairs can be resolved more effectively in acrylamide than
in agarose. Once the resolution was improved,
hybridization to many smaller RNA species other than the
580-nucleotide mRNA was observed, indicating the need to
determine optimum hybridization and nuclease digestion
conditions.

Various amounts of nuclease 81 were tested to
determine the amount necessary for digesting single-
stranded nonhybridized labeled RNA, but an amount that
would minimize degradation of RNAéRNA hybrids (Figure 1.6).
When 81 was included in the assay at 266 units, the RNA
hybids appeared to be partially degraded, whereas amounts
less than 133 U resulted in incomplete digestion of RNA.

'Future assays were conducted using 133 U of nuclease $1 per

41

Figure 1.5. Autoradiogram from a solution hybridization
assay utilizing an IGF-I RNA probe purified by gel
electrophoresis. Fifty and 100 ug of RNA from liver (A)
and LD muscle (C) of a fed finishing pig and from liver of
a 48 hour fasted pig (B) was included in the assay. A
porcine IGF-I gDNA was utilized as a template to synthesize
an antisense 3 P-labeled RNA probe. The probe was
hybridized to RNA at 42°C for 14 hours. Following
hybridization, 133 units of nuclease 81 were added and
samples incubated for an additional 1 hour at 37°C.
Protected RNA fragments were electrophoretically separated
in 5% acrylamide/8 M Urea gels and then subjected to
autoradiography. Transfer RNA (tRNA) was included as a
negative control to account for nonspecific background
hybridization. The $1 nuclease—protected RNA fragments are
580 bases in length. The IGF-I probe was also included
alone and is 678 bases long.

42

-'580

bases

 

_
8%
W s
a
b

 

Figure 1.5

43

Figure 1.6. Autoradiogram from an IGF-I solution
hybridization assay in which four amounts of nuclease 81
were used following hybridization. Each hybridization
reaction contained 100 ug of finishing pig liver RNA and 2
million cpm of IGF-I RNA probe. A porcine IGF-I cDNA was
utilized as a template to synthesize an antisense P-
labeled RNA probe. The probe was hybridized to RNA at 42°C
for 14 hours. Following hybridization, 266, 133, 26.6 or
13.3 units of nuclease 81 were added and samples incubated
for an additional 1 hour at 37°C. Protected RNA fragments
were electrophoretically separated in 5% acrylamide/8 M
Urea gels and then subjected to autoradiography. The 580-
base pair (bp) protected RNA fragment is shown.

Figure 1.7. Autoradiogram from an IGF-I solution
hybridization assay in which hybridization was conducted
with 100 ug of pig liver RNA at either 42 or 65 C. A
porcine IGF-I EDNA was utilized as a template to synthesize
an antisense 3 P-labeled RNA probe. The probe was
hybridized to RNA at 42° or 65°C for 14 hours. Following
hybridization, 133 units of nuclease 81 were added and
samples incubated for an additional 1 hour at 37°C.
Protected RNA fragments were electrophoretically separated
in 5% acrylamide/8 M Urea gels and then subjected to
autoradiography. Transfer RNA (tRNA) was included as a
negative control to account for nonspecific background
hybridization. The 81 nuclease-protected RNA fragments are
580 bases in length. The IGF-I probe was also included
alone and is 678 bases long.

44

3 5? o
a a I ' .D d. U 0
‘°"" 3 S 8 E N m
fi H m u e' w
s -’n 2!
h ,‘ 580
580- '2 ibases
'I
bases I ‘.

 

   

Figure 1.6 Figure 1.7

 

 

100 ug reaction. When 42° agé 65°C were compared for
hybridization temperatures, 65°C resulted in much less
hybridization to smaller RNA fragments and greater
hybridization to the 580-nucleotide species (Figure 1.7).
All future assays were conducted at 65°C.

A dose-response study was conducted using 50, 75, 100,
and 150 ug of liver RNA (Figure 1.8). The assay was linear
within this range (Figure 1.9); therefore, future assays
were conducted with 100 ug of total RNA under the optimal
conditions described above. The intra- and inter—assay
coefficients of variation were 2.8 and 2.7%, respectively.
A detailed protocol for the assay is included in Appendix
B. .

The assay represented in Figure 1.8 also included RNA
from liver and LD muscle of growth hormone-treated pigs
(150 ug growth hormone/kg body weight daily; tissue was
obtained from Dr. D. Beermann at Cornell University).
After correction for nonspecific hybridization by
subtraction of the signal obtained with tRNA, it was
determined that growth hormone increased the abundance of
IGF-I mRNA in liver and skeletal muscle by 1.4 and 1.2
times relative to controls. These results indicate that
the sensitivity of the solution hybridization assay
descibed above is sufficient to detect changes in IGF-I
mRNA abundance in liver and skeletal muscle of finishing

pigs treated with anabolic agents.

46

Figure 1.8. Autoradiogram from an IGF-I solution
hybridization assay in which RNA from liver (LIV) and LD
muscle (LD) of a control pig (C) and a growth hormone-
treated pig (GH) was utilized. Fifty, 75, 100, and 150 ug
of C-LIV RNA was included in the assay for examination of a
dose-response relationship. A porcine IGF-I cDNA was
utilized as a template to synthesize an antisense P-
labeled RNA probe. The probe was hybridized to RNA at 65°C
for 14 hours. Following hybridization, 133 units of
nuclease 31 were added and samples incubated for an
additional 1 hour at 37°C. Protected RNA fragments were
electrophoretically separated in 5% acrylamide/8 M Urea
gels and then subjected to autoradiography. Transfer RNA
(tRNA) was included as a negative control to account for
nonspecific background hybridization. The 81 nuclease-
protected RNA fragments are 580 bases in length. The IGF-I
probe was also included alone and is 678 bases
long.Transfer RNA (tRNA) was included as a negative control
to account for nonspecific hybridization. The IGF-I RNA
probe was included alone in the gel and is 678 nucleotides
(nt) long. Protected fragments are 580 base pairs (bp) in
length.

47

 

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48

Figure 1.9. Dose-response relationship between quantity of
total RNA used and hybridization signal obtained in the
solution hybridization-nuclease protection assay. A porcine
IGF-I cDNA 3gas utilized as a template to synthesize an
antisense3 P-labeled RNA probe. The probe was hybridized
to 50, 75, 100, and 150 ug of porcine liver RNA at 65 °C for
14 hours. Following hybridization, 133 units of nuclease
81 were added and samples incubated for an additional 1
hour at 37° C. Protected RNA fragments were
electrophoretically separated in 5% acrylamide/8 M Urea
gels and then subjected to autoradiography. Transfer RNA
(tRNA) was included as a negative control to account for
nonspecific background hybridization. The signals obtained
in the 580-base pair nuclease-protected fragments were
quantitated by liquid scintillation analysis. Results have
been corrected for nonspecific hybridization by subtracting
the signal obtained from hybridization to tRNA (y-
intercept=80; slope=7.7; r squared=.978).

49

1400 Corrected cpm 1n protected fragment

, 1200 e '
1000 -
eoo -
500 -

400 *-

 

 

200 1 1 1 1 J J
0 25 50 75 100 125 150

Quantity of RNA in assay, ug

Figure 1 . 9

Summary

Sensitivity offered by Northern blot analyses is not
sufficient to detect changes in IGF-I mRNA abundance in
market weight pig liver and skeletal muscle. A sensitive
solution hybridization-nuclease protection assay allowed
the detection of changes in IGF-I mRNA levels in liver of
market weight pigs administered growth hormone. This assay
will be useful to monitor changes in abundance of IGF-I
mRNA in liver and skeletal muscle of pigs administered
growth hormone and beta adrenergic agonists during the

finishing phase of growth.

50

CHAPTER 2
'ADMINISTRATION OF GROWTH HORMONE TO PIGS ALTERS
THE RELATIVE ABUNDANCE OF IGF-I mRNA

IN LIVER AND SKELETAL MUSCLE
Abstract

Abundance of IGF-I mRNA was determined in liver and
skeletal muscle of market weight crossbred barrows using a
solution hybridization-nuclease protection assay. Pigs
were intramuscularly administered either 50 ug recombinant
porcine growth hormone (GH) per kg body weight or vehicle
daily for 24 days. Pigs were fed either 14% or 20% crude
protein corn-soybean meal diets. Percent muscling of I
carcasses from pigs administered GH was greater (P < 0.01)
than controls. RNA was isolated from liver and longissimus
dorsi muscle that was obtained during slaughter. Relative
to controls, GH increased (P < 0.05) the abundance of liver
IGF-I mRNA 2.7-fold in pigs fed the 14% protein diet and
3.0-fold in pigs fed the 20% protein diet. Muscle IGF-I
mRNA abundance in GH-treated pigs was only 77% and 84% of
abundance in control pigs fed the 14% and 20% protein

diets, respectively (P < 0.08). GH increased (P < 0.001)

51

serum IGF-I concentration 1.33fold in pigs fed the 14%
protein diet and 2.0-fold in those fed the 20% protein
diet. These results indicate that administration of GH to
pigs influences the relative abundance of liver and
skeletal muscle IGF-I mRNA and that the these alterations
are dependent upon dietary protein concentration.

Increased abundance of liver IGF-I mRNA and increased serum
IGF-I concentrations suggests that IGF-I plays an endocrine

role in mediating GH-induced muscle hypertrophy in pigs.

Introduction

Administration of porcine growth hormone to pigs
increases skeletal muscle growth (Machlin, 1972; Chung et
al., 1975; Etherton et al., 1987; Evock et al., 1988;
Campbell et al., 1989; Smith et al., 1989a). Growth
hormone-induced hypertrophy of skeletal muscle has been
accompanied by increased serum or plasma insulin-like
growth factor-I (IGF-I) concentrations in pigs (Chung et
al., 1985; Etherton et al., 1987) and increased abundance
of liver and skeletal muscle IGF-I mRNA in rats (Turner et
al.,1988). Since IGF-I also increases protein synthesis .
when included with cultured skeletal muscle cells (Ewton
and Florini, 1980; Gulve and Dice, 1989; Roe et al., 1989)
and increases skeletal muscle protein synthesis when
administered to dwarf mice (Pell and Bates, 1989), it is
possible that IGF-I mediates the action of growth hormone
on skeletal muscle growth.

Recently, Smith et al. (1989a) found that the dietary
crude protein concentration had to be at least 14% in order
for recombinant porcine growth hormone to increase muscle
growth in finishing pigs. Dietary protein restriction

(i.e., 5% crude protein in rat diets) has been demonstrated

53

to reduce serum or plasma IGFSI concentrations and liver
IGF-I mRNA abundance in humans and rats (Prewitt et al.,
1982; Isley et al., 1984; Maes et al., 1984b; Moats-Staats
et al., 1989; VandeHaar et a1, 1989) which suggests that
dietary protein concentration may modulate the anabolic
actions of growth hormone on porcine muscle growth via
IGF-I.

The objectives of this study were to 1) determine
whether administration of porcine growth hormone to
finishing pigs increases the abundance of IGF-I mRNA in
liver and skeletal muscle and 2) examine the effects of

dietary protein concentrations on abundance of IGF-I mRNA.

Materials and Methods

nninnlnL gnnn nnn Treatments. Thirty-two crossbred
barrows were divided into two blocks based upon initial
body weight. Average initial body weight of pigs in the
heavy and light blocks were 81.7 and 66.8 kg, respectively.
Within each block, pigs were assigned to four pens so that
minimal differences in body weight existed among pens. All
pigs were fed a 14% crude protein corn-soybean meal diet
for 11 days to allow the pigs to become acclimated to the
facilities and to ensure that pigs were in good health.
Temperature of the swine facility was maintained between
12° and 18°C throughout the entire experimental period.
Following the preliminary feeding period, four treatments
were randomly assigned to the four pens of each block so
that there was a total of two pens (8 pigs) per treatment.
The four treatments were: 1) somatotropin plus a 14% crude
protein corn-soybean meal diet (GH14), 2) injection vehicle
plus a 14% crude protein corn-soybean meal diet (C014), 3)
somatotropin plus a 20% crude protein corn-soybean meal
diet (GH20), and 4) injection vehicle plus a 20% crude
protein corn-soybean meal diet (C020). Diet compositions

are shown in Table 2.1. Somatotropin was a recombinant

55

56

Table 2.1. Composition and calculated analyses
of diets fed to crossbred barrows

 

Ingredients 14% CP 20% CP

 

---% of dry matter--

Corn 80.75 63.75
Soybean meal (44% CP) 16.00 33.00
Mono-di-calcium phos. 1.00 1.00
Calcium carbonate 1.00 1.00
Vitamin-mineral premixa 1.00 1.00

 

Calculated analysis

 

Crude protein 14 20
L-Lysine 0.65 1.10

- ------- kcal/kg ------
ME 3175 3157

 

aPremix provided per kg of diet: Vitamin A,
3,300 10; Vitamin D3, 600 IU; riboflavin, 3.3
mg; nicotinic acid, 17.6 mg; d-pantothenic
acid, 13.2 mg; choline, 110 mg; Vitamin 812,
19.8 ug; an, 74.8 mg; Fe, 9.4 mg; Mn, 37.4 mg;
Cu, 9.9 mg; I, 0.5 mg; Se, 0.1 mg.

porcine growth hormone synthggized and generously supplied
by the Upjohn Co. (Kalamazoo, MI). Somatotropin was mixed
with sterile vehicle (2.5 mg/ml 0.03 M NaHCOB, 0.15 M NaCl,
pH 9.5) daily and administered intramuscularly in the right
side of the neck once daily (0900) at an amount equal to 50
ug/kg body weight/day. Pigs were weighed weekly to adjust
somatotropin dosages and were administered the final
injection on day 24 of the treatment period. Pigs were fed
ad libitum and had access to water via automatic water
dispensers. On day 25 of the treatment period, 24 hours
after the last injection, pigs were weighed, transported to
the Michigan State University Meat Laboratory, and
slaughtered (under USDA Meat Inspection supervision) for
collection of tissue samples and measurement of carcass
variables. Muscle samples were collected from the left
side of the carcass as described below. Percent carcass
muscle was estimated using the right side of the carcass
according to the NPPC (1983) equation. Longissimus dorsi
(LD) muscle area was determined with a grid at the tenth
rib on the right side of the carcass.

tissue sample collection, BEA Lsolation, and Analysis.

Following exsaguination, samples of LD muscle from the
left side of the carcass and samples of liver were
immediately excised and weighed. Samples were collected,

cut into pieces approximately 8 cm3

, frozen by submersion
in liquid nitrogen and stored at -80°C. The remaining LD
muscle on the left side of the carcass and the remaining

liver was weighed in order that total left LD muscle and

58

liver weights could be calculated.

RNA was isolated from LD muscle and liver of four pigs
per treatment (selected at random; 2 pigs/pen) as described
in Chapter 1 and Appendix A.

Relative abundance of IGF-I mRNA in the liver and
skeletal muscle RNA was quantitated using a sensitive
solution hybridization-nuclease protection assay as
described in Chapter 1 and Appendix B. An internal control
hybridization standard (RNA isolated from liver of a
somatotropin-treated pig) was used in all hybridization
assays to aid in the normalization of hybridization data
among assays. Data presented are normalized treatment
means.

Abundance of beta-tubulin mRNA was also determined on
the liver and skeletal muscle RNA samples by Northern blot
analysis. Northern blot analysis was conducted as
described by Jump et al. (1984) with the following
modifications. Twelve ug aliquots of LD muscle RNA and 20
ug aliquots of liver RNA were denatured and
electrophoretically separated in 1.2% agarose gels
containing 2.2 M formaldehyde. RNA was transferred to
nitrocellulose paper (Schleicher and Schuell, Keene, NH)
with 10X SSC. Blots were prehybridized in 50% formamide,
5X SSC, 5X Denhardt’s solution, 0.1% SDS, 1 mM EDTA, 50 mM
sodium phosphate buffer (pH 6.5), and 0.5 mg/ml tRNA for 2
hours at 42°C. The hybridization solution was identical to

the prehybridization solution, except that the

hybridization solution contaiged only 1X Denhardt’s
solution and contained two million cpm of probe/ml. A
mouse beta-tubulin cDNA (m-beta-5 tubulin) subcloned into
the EcoRI site of pUC9 was obtained from Dr. Donald
Cleveland at John Hopkins University (Sullivan and
Cleveland, 1986). The cDNA was random primed (Boehringer
Mannheim Biochemicals) using [32P1dCTP (3000 Ci/mmol:
Amersham Corp., Arlington Hts., IL) to a specific activity
of 4 x 108 cpm/ug DNA. After hybridization at 42°C for 16
hours, blots were washed with 2X SSC and 0.1% SDS at room
temperature, then washed for three cycles in 0.2x SSC and
0.1% SDS at 55°C. Under these hybridization conditions,
the beta-tubulin cDNA hybridizes to mRNA species of 1800
and 2800 nucleotides, sizes consistent with beta-tubulin
mRNA from other species (Lewis et al., 1985). Following
autoradiography, hybridization of RNA to beta-tubulin cDNA
was quantified by densitometry. Blots that contained liver
RNA were stripped with 0.01X SSC and 0.5% SDS for 2 hours
at 70°C and then re-hybridized with a rat beta-actin cDNA
probe under the same conditions as above, except that the
blots were washed with 0.1x SSC and 0.1% SDS following
hybridization. The beta-actin cDNA was obtained from Dr.
Larry Kedes laboratory (University of Southern California
School of Medicine) and has been characterized by P.
Gunning (unpublished). Under the hybridization conditions
employed above, the beta actin probe hybridizes to a 2100-
nucleotide mRNA species. Later, blots were stained with

methylene blue as described by Maniatis et al. (1982) to

60

visualize the 188 and 28S ribosomal bands in order to
confirm that equivalent amounts of RNA were loaded onto
gels and that the transfer efficiency was similar among
lanes.

Blood was collected from two pigs of each treatment
two hours after the final injections on day 24 of the
treatment period. Serum was harvested, stored at -20°C,
and analyzed for growth hormone concentration as described
by Smith and Kasson (1990) and for IGF-I concentration as
described by Dahl et al. (1990).

Statistical Asalysis. All data were statistically
analyzed using the General Linear Models Procedure of SAS
(1987). A randomized complete block design was utilized.
Effects of block, dietary crude protein concentration,
growth hormone, and the interactions were tested using
variation among pens (i.e., block X protein X growth
hormone) as the error term. Due to the low number of pens
per treatment and degrees of freedom for the error term,
variation among pens was pooled with variation among pigs
and used as the error term in cases in which the F
statistic for variation for pens was less than 2Fo.50 as
outlined by Gill (1989); however, there is potential for
bias from pooling the sum of squares (and corresponding
degrees of freedoms) for pens and animals. Contrasts
between means were analyzed by Bonferonni t-tests as

outlined by Gill (1978).

Results and Discussion

In order to ensure that the barrows selected for the
present study were healthy, all pigs were fed a
conventional 14% crude protein finishing diet for 11 days
prior to the treatment period. Due to the large range in
initial body weights among the pigs, two blocks were
formed, a heavy block which consisted of pigs with an
average weight of 81.7 kg, and a light block which
consisted of pigs with an average body weight of 66.8 kg.
Following the 11-day preliminary feeding period, all pigs
were observed to be in excellent health. Average body
weights on day 1 of the treatment period for pigs selected
for RNA analyses were 90.6 and 76.7 kg for the heavy and
light blocks, respectively. Respective slaughter weights
on day 24 of the treatment period were 109.3 and 98.7 kg.
Weight gains during the treatment period, liver weights at
slaughter, and various carcass measurements are presented
in Table 2.2. Initial body weight (block factor in the
statistical model) had no statistically significant effect
on any variable, except for LD muscle weight. The mean
weight of LD muscle from pigs of the heavy block was 11.4%

greater (P < 0.01) than LD muscle from pigs of the light

61

62

Table 2.2. Effects of growth hormone for 24 days on average

daily gains, liver weights, and carcass measurements of

pigs fed 14% and 20% crude protein diets

 

 

 

Treatmenta
Variable c014 6814 0020 0320 SBM
Avg. daily gain g/d 885 819 814 914 101
Liver weight, g5 1573 1875 1726 2082 127
Right LD area, cm2 28.8 27.9 27.4 31.7 1.9
Left LD weight., g 2240 2056 2162 2355 121
Carcass muscle, % 54.4 55.7 52.7 56.7 0.9

 

‘Co=control; Gn=growth hormone; 14=14% CP; 20=20% CP.

ben effect (P < 0.025).
°Gn effect (9 < 0.01).

block. Estimated percent caggass muscle was 5% greater (P '
< 0.01) in pigs administered growth hormone than in control
pigs. Greater percent carcass muscle in treated pigs is
due to both an increase in muscle mass and a decrease in
backfat deposition. Stimulation of muscle growth may have
been greater if a larger dose of growth hormone had been
administered. Several workers (reviewed by Steele et al.,
1989) have conducted dose-response experiments in growing
pigs and have found optimum dosages on most carcass
variables from 70 to 120 ug/kg body weight/day. The
improvement in percent carcass muscle, LD muscle area, and
LD muscle weight between growth hormone-treated pigs and
control pigs tended to be greater in the group of pigs fed
the 20% protein diet (Table 2.2). This finding was not
surprising since Smith et al. (1989a) reported that the
dietary crude protein concentration had to be greater than
14% in order for growth hormone to improve growth and feed
efficiency. Smith (1989a) observed reductions in average
daily gain and increased feed to gain ratios in growth
hormone-treated pigs fed diets containing only 14% crude
protein. Similar trends were observed in the present study
for average daily gain, LD muscle area, and LD muscle
weight; however, these changes were not statistically
significant in the present experiment.‘ Newcombe et al.
(1988) reported that in market weight pigs administered 3
mg of recombinant porcine growth hormone per day the LD
muscle area increased linearly when dietary crude protein

concentration was increased from 14% to 26%. Furthermore,

64
Steele et al. (1989) found that the improvement in daily

gain, feed:gain and LD muscle area resulting from the
administration of growth hormone to pigs fed an 11% crude
protein diet was only 50% of the improvement observed in
treated pigs fed diets containing at least 15% protein.

Growth hormone also increased (P < 0.025) mean liver
weight by 20% relative to controls. Administration of
growth hormone has been demonstrated to increase liver
weight in pigs (Etherton et al., 1987), steers (Eisemann et
al., 1989) and dwarf mice (Pell and Bates, 1989).
Hypersomatotropism has been shown to increase the weights
of several tissues and organs in rats (Turner et al.,
1986).

Autoradiograms from the IGF-I solution hybridization
assays are shown in Figure 2.1. Hybridization of the 678-
nucleotide [32P]-labeled IGF-I probe to tRNA was
negligible. Hybridization to liver and muscle RNA resulted
in 580-base pair protected fragments. Negligible
hybridization to other mRNA species occurred under the
conditions of the assay. To quantitate relative adundance
of IGF-I mRNA, the 580-base pair fragments were excised
from the gels and subjected to liquid scintillation
analysis. Relative to controls, growth hormone increased
(P < 0.05) the abundance of liver IGF-I mRNA 2.7-fold in
pigs fed the 14% protein diet and 3.0-fold in pigs fed the
20% protein diet (Table 2.3). Although the induction of

IGF-I mRNA tended to be greater in pigs fed the high

65

Figure 2.1. Autoradiograms of IGF-I mRNA abundance in liver
and LD muscle from growth hormone-treated (GH) and control
(C0) pigs fed 14% crude protein (14) or 20% crude protein
(20) diets. Liver and muscle RNA was isolated and analyzed
via a solution hybridization-nuclease protection assay. A
porcine IGF-I EDNA was utilized as a template to synthesize
an antisense 3 P-labeled RNA probe. The probe was
hybridized to 100 ug of RNA at 65°C for 14 hours.
Following hybridization, 133 units of nuclease 81 were
added and samples incubated for an additional 1 hour at
37°C. Protected RNA fragments were electrophoretically
' separated in 5% acrylamide/8 M Urea gels and then subjected
to autoradiography. Transfer RNA (tRNA) was included as a
negative control to account for nonspecific background
hybridization. The 81 nuclease-protected RNA fragments are
580 base pairs (bp) in length. RNA from liver of a pig from
GH20 was included in all hybridization assays as an
internal standard (Std.) to aid in the normalization of
data among assays. Each lane represents one pig (n=4
pigs/treatment).

66

0mm!

3

 

VNX3

 

 

H.N cuswum

 

ONIU

MAUmDZ GA

 

MM>HA

 

omoo

67

Table 2.3. Relative abundance of IGF-I mRNA in liver and LD
muscle from growth hormone-treated (GH) and contrgl (CO)
pigs fed 14% (14) or 20% (20) crude protein diets

 

 

 

Treatment
Variable C014 0814 C020 0820 SEM
Liver IGF-I mRNAa 0.215 0.582 0.241 0.721 0.122
LD IGF-I mRNAb 0.304 0.234 0.276 0.230 0.029

 

‘Abundance is expressed relative to an internal control
hybridization standard (RNA from liver of a growth hormone-
treated pig).

“an effect (9 < 0.05).

ban effect (9 < 0.08).

protein diet, statistical siggificance could not be
detected. Protein deficient diets (5% crude protein) have
been demonstrated to reduce liver IGF-I mRNA abundance in
rats (Moats-Staats et al., 1989; VandeHaar et al., 1989);
however, the 14% crude protein diet used in this experiment
is not considered to be a protein-deficient diet for market
weight pigs. A diet with less than 14% protein (e.g., 5%
crude protein) presumably would have been associated with
lower abundance of liver IGF-I mRNA and may have prevented
the growth hormone-induced increase in IGF-I mRNA.

Muscle IGF-I mRNA abundance in growth hormone-treated
pigs was only 77% and 84% of the abundance in control pigs
fed the 14% and 20% protein diets, respectively (Table
2.3). Since abundance data are reported relative to total
RNA, then an increase in ribosomal RNA relative to mRNA
could contribute to the decrease in abundance of IGF-I
mRNA. Muscle RNA content has not been quantitated in this
study, but growth hormone has been demonstrated to increase
muscle RNA content in steers (Eisemann et al., 1989).
Despite this possibility, decreased (P < 0.08) abundance of
IGF-I mRNA in muscle was not expected since Turner et al.
(1988) observed increases in IGF-I mRNA abundance in both
“liver and skeletal muscle of rats implanted with growth
hormone-secreting GH3 cells. This difference could be a
result of greater concentrations of serum growth hormone in
rats implanted with GH3 cells. Serum concentrations of
growth hormone in these treated rats are greater than 2000

ng/ml compared to normal concentrations of 10 to 100 ng/ml

69 .
(Turner et al, 1986). Serum growth hormone was quantitated

in 8 pigs (2 pigs/treatment) of the present experiment 2
hours after injection and was found to be 69.9 ng/ml in
growth hormone-treated pigs relative to 4.1 ng/ml in
controls (Figure 2.2). To briefly address the question of
whether greater amounts of growth hormone may have
increased muscle IGF-I mRNA abundance, RNA was isolated
from liver and LD muscle of a market weight pig
administered 150 ug growth hormone/kg body weight daily
(three times more than that administered to the pigs of the
present study) and a control pig from a study conducted at
Cornell University (tissue was obtained from Dr. D.
Beermann at Cornell University). Relative to the control,
abundance of IGF-I mRNA was 1.4 times greater in liver and
1.2 times greater in LD muscle of the treated pig.
Although these results suggest that larger doses of growth
hormone may increase muscle IGF-I mRNA abundance, such
interpretation is highly speculative due to the limited
amount of data.

Turner et al. (1988) did not examine muscle IGF-I mRNA
abundance until 40, 60, and 80 days following implantation
of the GH3 cells. In the present study, porcine IGF-I mRNA
abundance was examined after a 24-day treatment period in V
tissues that were collected 24 hours after the final
injection of growth hormone. Isgaard et al. (1989)
conducted a time-course study in which IGF-I mRNA abundance

was examined in gastrocnemius muscle of hypophysectomized

70

Figure 2.2. Concentrations of growth hormone (GH; open
bars) and IGF-I (hatched bars) in serum from growth
hormone-treated (GH) and control (C) pigs fed 14% CP (14)
or 20% CP (20) diets. Each bar represents the mean of two
pigs. Standard errors of individual treatment means are
indicated by the error bars. Pooled standard errors of the
means are 2.73 and 30.6 ng/ml for GH and IGF-I,
respectively. GH effect for both serum GH and serum IGF-I
(P < 0.01).

 

 

 

 

 

 

 

N .\t

s 8 \. a
m r

 

 

 

 

§

 

80 89mm GH. ng/ml [:1

+1

 

 

50-
40--

L
0 0.
2

GH14 C020 GH20

C014

Treatment

72
rats at 0, 1, 3, 6, l2, and 24 hours after a single

injection of growth hormone. Maximum increases in IGF-I
mRNA abundance were noted between 6 and 12 hours after
injection. Abundance at 24 hours was approximately 50% of
the maximum abundance. The rats of Isgaard et al. (1989)
were hypophysectomized, whereas intact barrows were used in
the present study. The results of these studies emphasize
the need to include measurements of IGF-I mRNA abundance at
several timepoints in growth hormone studies. .

Growth hormone increased (P < 0.001) serum IGF-I
concentration 1.6-fold in pigs fed the 14% protein diet and
2.0-fold in pigs fed the 20% protein diet (Figure 2.2).
These increases are consistent with those observed by
Etherton et al. (1987) and Owens et al. (1990) when growing
pigs were administered growth hormone. This increased
serum IGF-I concentration in growth hormone-treated pigs is
most likely due to the increased synthesis of liver IGF-I.
Although the increased abundance of liver IGF-I mRNA cannot
necessarily be interpreted as an increase in IGF-I
synthesis, it would seem reasonable to suggest that this
increased message resulted in the increased serum IGF-I
concentration. This increased serum IGF-I may act in an
endocrine manner in which it is transported to the skeletal
muscle where it acts to increase protein synthesis.
Furthermore, the increased serum IGF-I may have been
responsible for the decreased abundance of IGF-I mRNA in
the LD muscle via a feedback inhibition mechanism.

Increased serum IGF-I concentrations may reduce the

73
synthesis of muscle IGF-I by decreasing muscle IGF-I mRNA

abundance. Whether these changes in IGF-I mRNA abundance
are due to changes in transcription rates, processing of
mRNA, or mRNA stability is unknown. Mathews et al. (1986)
have reported that administration of growth hormone to
growth hormone-deficient mice increases liver IGF-I
transcription, but whether this occurs in
nonhypophysectomized barrows is unknown. It is also
unknown whether administering growth hormone to pigs alters
the size distribution of liver IGF-I mRNA species as has
been reported in mice (Mathews et al., 1986) and rats
(Hepler et al., 1989). The solution hybridization assay
utilized in this study does not permit quantitation of the
various IGF-I mRNA species.

The role of IGF-binding proteins in regulating IGF-I
action is unclear. Six IGF-binding proteins have been
identified in porcine serum and at least two forms have
been found secreted by muscle cell lines (McCusker et al.,
1989a and 1989b). Since these binding proteins are
regulated hormonally, nutritionally, and developmentally
(McCusker et al., 1989b), it is possible that they play
major roles in mediating the actions of IGF-I in growth
hormone-induced muscle hypertrophy. Furthermore, IGF-
binding proteins may serve as a mechanism by which dietary
protein concentrations modulate growth hormone actions on
skeletal muscle.

To determine whether the changes in IGF-I mRNA

abundance in the liver and Lquuscle were a result of a
specific effect of growth hormone on IGF-I mRNA or a
generalized effect of growth hormone on tissue hypertrophy
and increases many mRNAs, liver and LD muscle RNA were
subjected to Northern blot analyses for determination of
beta-tubulin and beta-actin mRNA abundance (Figure 2.3).
Beta-tubulin and beta-actin are constitutive proteins that
act in concert and contribute to the cytoskeletal structure
of eukaryotic cells (Sullivan and Cleveland). Abundance of
beta-actin mRNA was not determined in the skeletal muscle
RNA due to the low abundance of beta-actin mRNA and cross-
hybridization of the beta-actin cDNA probe to alpha-actin
mRNA, an extremely abundant message in porcine skeletal
muscle. Abundance of liver beta-tubulin and beta-actin
mRNA and LD beta-tubulin mRNA was not altered by growth
hormone in pigs fed the 20% protein diet; however, within
the 14% protein diet, abundance of liver beta-tubulin and
beta-actin mRNA was 1.9- and 1.8-fold greater (P < 0.05),
respectively, in pigs administered growth hormone than in
controls. Abundance of LD beta-actin mRNA was 1.5-fold
greater (P < 0.05) in pigs administered growth hormone than
in controls (Table 2.4). Examination of Northern blots .
following methylene blue staining indicated that transfer
of RNA among lanes was similar (Figure 2.4). These results
indicate that growth hormone may alter the abundance of
several mRNAs and that this effect is dependent upon
dietary protein concentration. The effect of growth

hormone on muscle growth appeared to be different in pigs

75

Figure 2.3. Autoradiograms from Northern blot analyses of
beta-tubulin and beta-actin mRNA abundance in liver and LD
muscle from growth hormone-treated (GH) and control (CO)
pigs fed 14% crude protein (14) or 20% crude protein (20)
diets. Twelve ug of muscle RNA and 20 ug of liver RNA were
denatured and electrophoretically separated in 1. 2% agarose
gels containing 2. 2 M formaldehyde and then transferred to
nitrocellulose. Blots were prehybridized at 42 0C for 2
hours in 50% formamide, 5X SSC, 5X Denhardt's solution,
0.1% SDS, 1 mM EDTA, 50 mM sodium phosphate buffer (pH 6. 5),
and 0. 5 mg/ml tRNA. RNA was hybridized with beta-tubulin
or beta-actin cDNA probes at 42 °C for 16 hours.

Composition of the hybridization solution was identical to
the prehybridization solution except that the hybridization
solution contained only 1X Denhardt’s and contained two
million cpm of probe/ml. Following hybridization, blots
were washed with three cycles of 0. 2X SSC and 0.1% SDS at
55°C and then subjected to autoradiography. Visualization
of 188 and 288 RNA following methylene blue staining of the
Northern blots is also shown. Each lane represents one pig
(n=4 pigs/treatment).

76

m.w curmfim

 

oumo

88 3:8

mqombx GA

 

 

 

omzo

owou

mm>HA

¢H=o

GHNIVLS VNH

V138

NILOV

NI1flflfll-V138

77

Table 2.4. Relative abundance of liver beta-tubulin and
beta-actin mRNA and LD muscle beta-tubulin mRNA in growth
hormone-treated (GH) and control (CO) pigs fed 14% (14) or

20% (20) crude protein diets

 

 

 

Treatment
Variable c014 6814 0020 GH20 83M
Liver tubulin mRNA“ 0.548 1.065 0.558 0.607 0.147
Liver actin nnna‘ 1.280 2.282 1.076 1.120 0.174
LD tubulin mama“ 1.079 1.610 1.373 1.192 0.135

 

1Abundance is expressed in densitometer units.
aWithin the 14% protein diet, GB effect (P < 0.05).

78
fed the 14% protein diet than in pigs fed the 20% protein

diet (Table 2.2) in that growth hormone tended to improve
muscle growth in pigs fed the 20% protein diet, but was
detrimental to muscle growth in pigs fed the 14% protein
diet. Since expression of beta-tubulin is developmentally
regulated in a variety of tissues, including liver and
skeletal muscle (Lewis et al., 1985; Wang et al., 1986),
‘these differential changes in muscle growth may explain the
difference in beta-tubulin and beta-actin expression among
the two groups of pigs. In a study by Murphy et al.
(1987b) in which effects of estrogen on IGF-I expression
was studied in rats, beta-actin mRNA abundance was used as
a constitutive marker. Estrogen was found to increase the
relative abundance of beta—actin mRNA. These studies
demonstrate that perturbations in hormonal status to alter
tissue growth results in changes in abundance of several

mRNA species.

Summary

Administration of 50 ug recombinant porcine
somatotropin/kg body weight/day to market weight pigs for
24 days increased abundance of liver IGF-I mRNA and
decreased abundance of skeletal muscle IGF-I mRNA. These
changes appeared to be influenced to some degree by dietary
protein concentration. Greater abundance of liver IGF-I
mRNA and serum IGF-I concentrations in growth hormone-
treated pigs in this study would support an endocrine role
for IGF-I in growth hormone-induced muscle growth. Since
IGF-I has been demonstrated to increase muscle protein
synthesis when administered to mice (Pell and Bates, 1989),
it is possible that the increased circulating levels of
IGF-I may act on the skeletal muscle to increase protein
synthesis. Expression of IGF-I needs to be examined in
muscle and liver at various times following administration
of different doses of growth hormone so that the role of
IGF-I in growth hormone-induced muscle growth can be more

completely characterized.

79

CHAPTER 3
EFFECTS OF THE PHENETHANOLAMINE, RACTOPAMINE, ON
THE RELATIVE ABUNDANCE OF IGF-I mRNA IN LIVER

AND SKELETAL MUSCLE 0F PIGS
Abstract

Relative abundance of liver and skeletal muscle IGF-I
mRNA was determined in crossbred barrows (average body
weight 93.6 kg) that were fed a 16% crude protein corn-
soybean meal diet supplemented with 0 or 20 ppm of
ractopamine for 28 days. Carcasses from pigs fed
ractopamine had a 26% greater (P < 0.05) mean longissimus
.dorsi muscle (LD) area and a 19% greater (P < 0.001) mean
LD muscle weight than controls. RNA was isolated from
liver and LD muscle that was obtained during slaughter and
used in a solution hybridizationénuclease protection assay
to determine the relative adundance of IGF-I mRNA.
Ractopamine had no apparent effect (P > 0.20) on liver or
muscle IGF-I mRNA abundance. 'Although liver and skeletal
muscle both synthesize IGF-I, ractopamine appears to have
little effect on IGF-I synthesis at the mRNA level after a

28-day feeding period in market weight pigs.

80

Introduction

The phenethanolamine, ractopamine, increases muscle
growth in market weight pigs. Bergen et a1. (1989) have
previously demonstrated that ractopamine increases the
fractional rate of skeletal muscle protein synthesis.
Increases in fractional synthesis rates of alpha-actin
synthesis were accompanied by increases in the abundance of
alpha-actin mRNA, indicating that ractopamine increases
actin synthesis pretranslationally (Helferich et al.,
1990). Insulin-like growth factor-I (IGF-I) may mediate
these actions of ractopamine, since IGF-I increases protein
synthesis and decreases protein degradation in cultured
muscle cells (Ewton and Florini, 1980; Gulve and Dice,
1989; Roe et al., 1989), and it increases skeletal muscle
protein synthesis when administered to dwarf mice (Pell and
Bates, 1989). Furthermore, growth hormone-induced .
hypertrophy of skeletal muscle is accompanied by increases
in liver and muscle IGF-I mRNA abundance in rats (Turner et
al., 1988). Ractopamine may act on the liver to increase
the synthesis of IGF-I. Liver IGF-I may then act On
skeletal muscle in an endocrine manner to increase protein

synthesis. Alternatively, ractopamine may increase

81

82
synthesis of skeletal.musc1e IGF-I which may act in a
paracrine or autocrine manner to increase skeletal muscle
growth. The objective of this study was to determine

whether ractopamine increases the abundance of IGF—I mRNA

in liver and skeletal muscle of pigs.

Materials and Methods

Asisslss Case asd Trestmests. Twelve crossbred pigs

with an average initial body weight of 72 kg were randomly
assigned to four pens. Two dietary treatments were
randomly assigned to the four pens so that there were two
pens per treatment. The treatments were 1)l6% crude
protein corn-soy diet (Table 3.1) and 2)16% crude protein
corn-soy diet supplemented with 20 ppm of the
phenethanolamine, ractopamine (1-[4-hydroxypheny11-2-[1
methyl-3 (4-hydroxyphenyl) propylamino] ethanol; generously
provided by Eli Lilly and Co., Indianapolis, IN). Pigs
were fed ad libitum and had access to water via automatic
water dispensers. On day 28 of the feeding period, pigs
were slaughtered at the Michigan State University Meat
Laboratory (under USDA Meat Inspection supervision) for
collection of tissues and measurements of carcass
variables. Muscle samples were collected from the left
side of the carcass as described below. Longissimus dorsi
(LD) muscle area was determined with a grid at the tenth

rib on the right side of the carcass.

83

84

Table 3.1. Composition and calculated analyses
of diet fed to barrows

 

Ingredients % of dry matter

 

Corn

Soybean meal (48% CP)
Calcium phoshate (dibasic)
Calcium carbonate

NaCl

Vitamin-mineral premixa

ps1
OOOHQO
e e e e e e

““000”

 

Calculated analysis

 

Crude protein 16
L-Lysine 0.70

hssllfig
ME 3185

 

aPremix provided per kg of diet: Vitamin A,
3,300 10; Vitamin D3, 600 IU; riboflavin, 3.3
mg; nicotinic acid, 17.6 mg; d-pantothenic
acid, 13.2 mg; choline, 110 mg; Vitamin 812,
19.8 ug; Zn, 74.8 mg; Fe, 9.4 mg; Mn, 37.4 mg;
Cu, 9.9 mg; I, 0.5 mg; Se, 0.1 mg.

85

Tissue sam 18 collection, RNA IsslsgissL ssg
883118.12-

Following exsanguination, samples of LD muscle from
the left side of the carcass and samples of liver were
immediately excised and weighed. Samples of each were
rapidly cut into pieces approximately 8 cm3, frozen by
submersion in liquid nitrogen and stored at -80°C. The
remaining LD muscle on the left side of the carcass and the
remaining liver was weighed in order that total left LD
muscle and liver weights could be calculated.

RNA was isolated from L0 muscle and liver of all pigs
as described in Chapter 1 and Appendix A.

Relative abundance of IGF-I mRNA in liver and skeletal
muscle RNA was quantitated using a sensitive solution
hybridization-nuclease protection assay as described in
Chapter 1 and Appendix B. An internal control
hybridization standard (RNA isolated from liver of a
somatotropin-treated pig) was used in all hybridization
assays to aid in the normalization of hybridization data
among assays. Data presented are normalized treatment
means.

Abundance of beta-tubulin mRNA was also determined in
the liver and skeletal muscle RNA samples by Northern blot
analysis as described in Chapter 2. Following Northern
analysis, blots were stained with methylene blue as

described by Maniatis et al. (1982) to visualize the 188

and 28S ribosomal bands in order to confirm that equivalent

86
amounts of RNA were loaded onto gels and that the transfer

efficiency was similar among lanes.

fitstistical Asalysis. All data were statistically
analyzed using the General Linear Models Procedure of SAS
(1987). Effects of ractopamine were tested using variation
among pens (i.e., pens within treatment) as the error term.
Due to the low number of pens per treatment and degrees of
freedom for the error term, variation among pens was pooled
with variation among pigs and used as the error term in
cases in which the F statistic for variation for pens was
less than 2F0.50 as outlined by Gill (1989); however, there
is potential for bias from pooling the sum of squares (and

corresponding degrees of freedoms) for pens and animals.»

Results and Discussion

Administration of ractopamine for 28 days increased
muscle growth as assessed by a 26% greater (P < 0.05) mean
LD muscle area and a 19% greater (P < 0.001) mean LD muscle
weight in barrows fed 20 ppm ractopamine than in those fed
the basal control diet (Table 3.2). Accompanying the
increased muscle mass in treated pigs was a 41% increase (P
< 0.01) in average daily gain. Slaughter weights were 97.6.
and 89.5 kg for the ractopamine-fed and control pigs,
respectively. These changes are consistent with those
observed by Bergen et al. (1989) in which market weight
pigs were fed ractopamine for up to 42 days. Bergen et al.
(1989) demonstrated via [14C] tyrosine continuous infusions
that the increased muscle mass in pigs fed ratopamine was
due at least in part to an increased fractional rate of
skeletal muscle protein synthesis. Furthermore, Helferich
et al. (1990) has recently reported an increased fractional
synthesis rate of skeletal muscle alpha actin in pigs fed
ractopamine. The increase in actin synthesis was
accompanied by increased abundance of skeletal muscle alpha
actin mRNA indicating that ractopamine induces actin

synthesis pretranslationally. Utilizing in vitro

87

88

Table 3.2. Effects of ractopamine for 28 days on skeletal
muscle and body weight gains of crossbred pigs

 

 

 

Treatment
Variable Control Ractopamine SEM
Right Ln area, c 2‘ 29.5 37.2 1.3
Left LD weight, :b 1842 2192 43.8
Average daily gain, g/dc 636 895 129

 

:Treatment effect (P < 0.05).
Treatment effect (P < 0.001).
cTreatment effect (P < 0.01).

translation assays, Helfericgget al. (1990) have also
demonstrated that ractopamine induces the synthesis of
other myofibrillar proteins at the pretranslational level
of regulation. Skjaerlund et al. (1989) have examined
abundance of skeletal muscle alpha actin mRNA in LD muscle
of pigs after a 14-, 28-, and 42-day feeding period.
Maximum increases in the relative abundance of alpha-actin
mRNA occurred at the 28-day time point, which corresponded
to the time of the maximum increase in muscle mass.
Autoradiograms from the IGF-I solution hybridization
assays are shown in Figure 3.1. Hybridization of the 678—
nucleotide [32P1-labeled IGF-I probe to tRNA was
negligible. Hybridization to liver and muscle RNA resulted
in 580-base pair protected fragments. Negligible
hybridization to other mRNA species occurred under the
conditions of the assay. To quantitate relative adundance
of IGF-I mRNA, the 580-base pair fragments were excised
from the gels and subjected to liquid scintillation
analysis. Ractopamine had no effect (P > 0.20) on IGF-I
mRNA abundance in either liver or LD muscle (Table 3.3).’
Abundance of beta-tubulin mRNA was determined in liver and
LD muscle RNA via Northern analyses and was not altered by
administration of ractopamine (Figure 3.2 and Table 3.3)
indicating that lack of an effect of ractopamine on message
abundance was not limited to IGF-I mRNA. Staining of
Northern blots with methylene blue confirmed that the
transfer of RNA among all lanes was similar (Figure 3.2).

Lack of an effect of ractopamine on liver IGF-I mRNA

90

Figure 3.1. Autoradiograms of IGF-I mRNA abundance in liver
and LD muscle from ractopamine-treated (R) and control (C)
pigs. Liver and muscle RNA was isolated and analyzed via a
solution hybridization-nuclease protection assay. A porcine
IGF-I cDNA gas utilized as a template to synthesize an
antisense 3 P-labeled RNA probe. The probe was hybridized
to 100 ug of RNA at 65°C for 14 hours. Following
hybridization, 133 units of nuclease 81 were added and
samples incubated for an additional 1 hour at 37°C.
Protected RNA fragments were electrophoretically separated
in 5% acrylamide/8 M Urea gels and then subjected to
autoradiography. Transfer RNA (tRNA) was included as a
negative control to account for nonspecific background
hybridization. The $1 nuclease-protected RNA fragments are
580 bases in length. The IGF-I probe was also included
alone and is 678 bases long. Each lane represents one pig
(n=6 pigs/treatment).

LIVER

91

LD MUSCLE

 

:31: Probe

 

1-
.5

I?

" "" ...)... Probe

E'

tRNWL

0
.0
O
H
9.4

Figure 3.1

 

92

Figure 3.2. Autoradiograms from Northern blot analyses of
beta-tubulin mRNA abundance in liver and LD muscle from
ractopamine (R) and control (C) pigs. Twelve ug of muscle
RNA and 20 ug of liver RNA were denatured and
electrophoretically separated in l. 2% agarose gels
containing 2. 2 M formaldehyde and then transferred to
nitrocellulose. Blots were prehybridized at 42 °C for 2
hours in 50% formamide, 5X SSC, 5X Denhardt's solution,
0.1% SDS, 1 mM EDTA, 50 mM sodium phosphate buffer (pH

6. 5), and 0. 5 mg/ml tRNA. RNA was hybridized with beta-
tubulin or beta-actin cDNA probes at 42° C for 16 hours.
Composition of the hybridization solution was identical to
the prehybridization solution except that the hybridization
solution contained only 1X Denhardt’s and contained two
million cpm of probe/ml. Following hybridization, blots
were washed with three cycles of 0. 2x SSC and 0.1% SDS at
55°C and then subjected to autoradiography. Visualization
of 18S and 288 RNA following methylene blue staining of
Northern blots is also shown. Each lane represents one pig
(n=6 pigs/treatment).

93

LD MUSCLE

LIVER

 

 

zHADmDH

 

-<Hmm

2HHO<-<Hmm

 

 

nmzH<Hm <zm

Figure 3.2

94

Table 3.3. Relative abundance of IGF-I mRNA and beta-
tubulin mRNA in liver and LD muscle of ractopamine-treated
(R) and control (C) pigs

 

 

 

Treatment
Variable Control Ractopamine SEM .
Liver IGF-I mans 0.181 0.163 0.029
Liver beta-tubulin mRNA 0.547 0.553 0.087
Liver beta-actin mRNA 1.219 1.108 0.258
LD IGF-I mRNA 0.238 0.248 0.019
LD beta-tubulin mRNA .1.500 , 1.560 0.169

 

‘Abundance of IGF-I mRNA is expressed relative to an
internal control hybridization standard (RNA from liver of
a growth hormone-treated pig); abundance of beta-actin and
beta-tubulin mRNA is expressed in densitometer units.

95
abundance is consistent with the following observations.

First, an increased abundance of liver IGF-I mRNA would
likely result in greater liver IGF-I synthesis, and
subsequently greater circulating concentrations of IGF-I;
Beermann et al. (1987) were unable to detect any increase
in plasma IGF—I concentrations in sheep fed the beta
adrenergic agonist, cimaterol. Second, if circulating
levels of IGF-I were elevated, then serum from treated
animals would result in greater anabolic activity when
included in muscle cell cultures because IGF-I increases
proliferation and protein synthesis in cultures of muscle
cells (Ewton and Florini, 1980; Gulve and Dice, 1989; Roe
et al., 1989). McElligott and Chaung (1987) demonstrated
that serum from rats treated with clenbuterol and serum
from normal rats had similar effects on protein synthesis
and degradation and cell proliferation in cultures of L8
myoblasts. Given these data, hepatic or circulating IGF-I
does not appear to play major roles in mediating
ractopamine-induced muscle hypertrophy.

Since ractopamine has been demonstrated to directly
increase protein synthesis in cultures of L6 myotubes
(Adeola et al., 1989; Anderson et al., 1990), beta
adrenergic agonists may act directly upon the skeletal
muscle in Vivo to increase muscle growth. A direct action
of ractopamine on muscle protein metabolism may be mediated
by local synthesis of IGF-I. This locally synthesized IGF-

I could then act upon the muscle to increase protein

synthesis. Administration of96IGF-I to dwarf mice has been
demonstrated to increase skeletal muscle protein synthesis
and mhscle growth (Pell and Bates, 1989). It is possible
that changes in IGF-I mRNA abundance may change prior to
the 28-day time point that was selected in the present
study. Although abundance of IGF-I mRNA was not altered,
ractopamine may alter the synthesis of IGF-I at the
translational or post-translational levels. It is unknown
whether ractopamine results in more translationally active
IGF-I mRNA. Quantitation of tissue and serum IGF-I peptide
concentrations would provide additional information;
however, such measurements do not distinguish between
endocrine and paracrine and(or) autocrine roles of IGF-I
since the presenCe of muscle IGF-I peptide may be the
result of local synthesis or contribution from other
tissues. Changes in IGF-I receptor number and affinity in
skeletal muscle and alterations in the distribution of IGF-
binding proteins among tissues of pigs fed ractopamine have
not been examined and may also represent mechanisms of
growth regulation. These changes could occur without
changes in IGF-I mRNA of peptide concentrations.
Alternatively, it is possible that IGF-I does not play a
role in mediating ractopamine-induced muscle hypertrophy in
pigs. The actions of ractopamine may be mediated by other
growth factors or involve a number of different signal

transduction mechanisms.

Summary

Administration of ractopamine for 28 days to market
weight pigs does not appear to have a significant effect on
liver or skeletal muscle IGF-I mRNA abundance. Results of
the present study do not support the hypothesis that IGF-I
mediates the actions of ractopamine on skeletal muscle. If
ractopamine increases skeletal muscle growth in pigs by
changing liver or muscle IGF-I status after 28 days, then
it must occur by mechanisms other than changes in abundance
of IGF-I mRNA. Other mechanisms may include regulation at
the tanslational, post-translational, receptor and post-
receptor levels, or may involve changes in tissue

distributions of IGF-binding proteins.

97

CONCLUSIONS AND RECOMMENDATIONS

Each of the two experiments that were conducted has
provided us with a different model in which various
biological mechanisms involved in skeletal muscle growth
could be investigated. The objective of this dissertation
was to determine if liver and skeletal muscle IGF-I mRNA
was altered when market weight pigs were subjected to these
perturbations. To accomplish this objective, it was first
necessary to develop an assay with sufficient sensitivity
to detect changes in abundance of market weight pig tissues
in which IGF-I mRNA is in extremely low abundance, compared .
with tissues of younger pigs and those of rats. This is
the first report in which the relative abundance of IGF-I
mRNA has been quantitated in liver and skeletal muscle of
market weight pigs using a solution hybridization-nuclease
protection assay. Results from the experiments indicate
that regulation of relative abundance of IGF-I mRNA is
dependent upon dietary protein and that it is altered by
administration of growth hormone to pigs. Furthermore,
ractopamine-induced muscle hypertrophy was not associated
with changes in abundance of IGF-I mRNA.

Mechanisms by which growth hormone or ractopamine may

98

induce skeletal muscle hyperggophy are shown in Figure 4.1.
IGF-I may mediate the actions of growth hormone or
ractopamine in an endocrine manner or in a
paracrine/autocrine manner. For example, growth hormone or
ractopamine may act at the liver to increase IGF-I
synthesis. Increased liver IGF-I may be accomplished by
increased IGF-I mRNA and subsequent IGF-I synthesis. This
additional liver IGF-I may then be secreted by the liver
and transported to the skeletal muscle where it may
increase protein synthesis. Alternatively, growth hormone
or ractopamine may act directly on the skeletal muscle
(myofibers or nonmuscle cells) resulting in increased local
production of IGF-I synthesis. The additional IGF-I may
then act on myofibers in a paracrine or autocrine manner to
increase protein synthesis.

Results of the present experiments would support an
endocrine role of IGF-I in growth hormone-induced muscle
growth in pigs. In the growth hormone-treated pigs, the
increased abundance of liver IGF-I mRNA presumably
contributed to the increased circulating concentrations of
IGF-I. It is tempting to speculate that the greater
circulating concentrations of IGF-I contributed to the
muscle hypertrophy that was observed in the treated pigs.
Lack of an increase in muscle IGF-I abundance in growth
hormone-treated pigs suggests that IGF-I does not mediate
growth hormone action in a paracrine and(or) autocrine
manner. Furthermore, lack of responses in liver and muscle

IGF-I abundance in pigs treated with ractopamine suggests

100

Figure 4.1. Diagram showing possible mechanisms by which
IGF-I may mediate growth hormone- or ractopamine-induced
skeletal muscle hypertrophy in pigs. IGF-I may mediate
growth hormone or ractopamine actions by 1) acting in an
endocrine manner, in which case additional liver IGF-I is
transported to the skeletal muscle where it elicits its
effect on muscle protein metabolism or 2) acting in a
paracrine and(or) autocrine manner, in which case growth
hormone or ractopamine acts directly on the muscle
resulting in local production of IGF-I which effects
protein metabolism.

101

ENDOCRINE EABAQBINEZAUTOCRINE

Growth hormone

u/<:::;.Ractopamine~\;::\\\a

 

 

 

1169-1 mRNA. Tier-I mRNA

Tier-I Tcirc. Tier-I

II‘II“-9

IGF-I TProtein Synthesis

 

 

 

 

 

LIVER ' SKELETAL MUSCLE

Figure 4.1

102
that IGF-I may not mediate ractopamine-induced muscle

growth.

Several factors must be considered during
interpretation of these results. For example, it is
critical that in future studies tissues be collected at
various times following the initiation of the various
treatments. Selecting a single timepoint to quantitate
variables does not provide a complete picture of the
changes and adaptations that may occur during such
treatments. Doses of pharmacological agents, composition
of diets, sex, and breed all need to be considered when
designing future studies. The possible interaction of
circulating levels of IGF-I on tissue IGF-I synthesis
suggests that several variables need to be examined,
including tissue IGF-I peptide levels and circulating and
tissue concentrations of IGF-I binding proteins. Future
studies should be designed to investigate changes in IGF-I
receptor number and affinity and alterations in post-
receptor mechanisms that may be influenced by growth
hormone or ractopamine. Such measurements are critical in
determining the endocrine and paracrine/autocrine roles of

IGF-I in muscle growth.

APPEN DI CES

103

APPENDIX A

ACID GUANIDINE PHENOL CHLOROFORM RNA EXTRACTION PROTOCOL
(modification of Chomczynski and Sacchi, 1987)

REAGENIS;

4 M Guanidine thiocyanate:

7

2

3

guanidine thiocyanate (Kodak) 94.60 g.
sodium citrate 0.74 g.
B-mercaptoethanol (14 M) 1.42 ml
10% Na sarcosyl 10.0 ml

Total volume of 200 ml: use sterile GD-HOH.
Adjust pH to 7.0 with 1 M HCl or 1 N NaOH.

Filter through 0.45 um millipore.

Store in sterile brown bottle at room temperature.

Guanidine Hydrochloride

guanidine-HCl (Sigma, practical grade) 668.6 g.
'Na-acetate (trihydrate) 2.7 g.
dithiothreitol 150.0 mg.
iodoacetamide 1.84 mg.
0.2 M EDTA (pH 8.0) 5.0 ml.

Add GD-HOH to 900 ml.

Dissolve all reagents by heating in a 50°C water bath.
When all reagents are dissolved, adjust to pH 7.0 with
glacial acetic acid or 5 N NaOH.

Adjust final volume to 1 1.

Filter through 0.45 um millipore.

Store in sterile bottle in the dark at room
temperature.

Na-acetate, pH 5.0

Dissolve 272 g. Na-acetate (trihydrate) in 600 ml
sterile GD-HOH.

Adjust to pH 5.0 with glacial acetic acid.

Adjust final volume to 1.0 1.

Filter through 0. 45 um millipore.

Store at 4 0C. in sterile bottle.

Na-acetate, pH 5.0

Na-acetate (trihydrate) 409.8 g.
iodoacetamide 1.84 g.

Dissolve reagents in 300 ml glacial acetic acid and
300 ml sterile GD-HOH. Adjust to pH 5.0 with glacial

104
.acetic acid. Adjust final volume to 1. 0 1. Filter
through 0. 45 um millipore filter and store in sterile
bottle at 4 °C.

ETOH-Na-acetate, pH 5.0.

Absolute Ethanol 660 ml.
3 M Na-acetate, 10 nM iodoacetamide, (pH 5. 0) 11 ml.
Filtration of this solution is not necessary, store in
sterile bottle at —20 oC.

TE-8.0:
1.0 M Tris-H01, pH 8.0 (20°C) 10.0 ml.
0.2 M EDTA, pH 8.0 5.0 m1.

Adjust final volume to 1.0 l. with sterile GD-HOH.
Store at room temperature. .

10% Na-sarcosyl:

Dissolve 10 g. Na-sarcosyl (ICN) in 100 ml HOH, filter
through 0.45 millipore and store at room temperature.
Note: sarcosyl is a carcinogen, so take the
appropriate precautions.

EZIBAQIIQEL

Use sterile glassware and plasticware throughout the
procedure.

Add 1 9 frozen pig LD muscle or liver to a sterile 30 ml.
Corex tube containing 10 ml 4 M guanidine thiocyanate
homogenization buffer. Homogenize using a Polytron at a
setting of 6 for 20 seconds or until the tissue is
completely homogenized.

Add 1 ml 2 M Na acetate, pH 5, stopper, and vortex.
Add 10 ml HOH-saturated phenol, vortex.

Add 2 ml chloroform:isoamyl (24:1).

Mix vigorously, put on ice for 15 minutes.

Centrifuge at 10,000 x g for 20 minutes at 4°C.

Remove the aqueous layer with a sterile Pasteur pipet and
put it into a sterile 30 ml Corex tube.

To the organic phase, add 2 ml 4 M guanidine
thiocyanate and 0.2 ml 2 M Na acetate and vortex.

Add 2 ml HOE-saturated phenol and 0.4 ml
chloroform:isoamyl.

105
Vortex and put on ice for 15 minutes.

Centrifuge at 10,000 x g for 20 minutes at 4°C.

Add this aqueous layer to the previously collected aqueous
layer.

Extract the aqueous composite with 5 m1 HOH-saturated
phenol and 5 ml chloroform: isoamyl.

Centrifuge at 10,000 x g for 20 minutes at 4°C.

Remove the aqueous layer and put it into a sterile 30 ml
Corex tube.

Add 1 volume of isopropanol to the aqueous layer, cover
the tube with Parafilm and mix.

Store at -20°C for at least 2 hours to allow precipitation
of RNA.

Centrifuge at 10,000 x g for 20 minutes at 4°C.

Transfer pellet to a 1.5 ml microfuge tube with 750 ul

7 M guanidine HCl. Add 75 ul 2 M Na acetate, pH 5 and 450
ul absolute.ethanol and allow RNA to precipitate at -20°C
for 1 hour.

Microfuge at 12,000 x g for 5 minutes. Decant and
drain.

Resuspend the pellet in 300 ul of 3 M Na acetate, pH 5.
Sediment and drain as described above.

Resuspend the RNA pellet in 300 ul of EtOH-Na acetate, pH 5
and sediment and drain as described above.

Resuspend the RNA pellet in 300 ul of absolute ethanol
and sediment and drain as described above.

Resuspend the RNA pellet in of TE-8.0 (100 ul if LD
muscle RNA or 400 ul if liver RNA).

Dilute 5 ul of RNA solution to 1 ml with TE-8 and scan in a
spectrophotometer from 320 to 220 nm. Record the optical
density at 260 and 280 nm. The ratio of 260/280 should be
greater than 1.8.

106

RNA concentration of solution is equal to the optical
density at 260 nm multiplied by 200 (dilution factor)

and divided by 25 (RNA extinction coefficient). DNA
contamination is not a problem with this extraction
procedure because extractions are carried out at pH 5 to 6
(Chomczynski and Sacchi, 1987; Wallace, 1987).

RNA samples should be labeled and stored at -80°C.

107

APPENDIX B

Ififizl EQLEIIQN EXBBLDIZAIIQN:NEQLEA§§ EBQIEQILQH A§§AI
BEAQEEEfi AER fiQLQIIQH.=

Pvu II, 24 U/ul (BMB) with buffer M. Store at -2o°c.
Chloroform:Isoamyl alcohol, 24:1. Store at RT.
Phenol:Chloroform:Isoamyl, 1:1. Store at 4°C.
Ethanol, absolute. Store at -20°C.
Transcription Kit (BMB). Store at -20°C.
TE-8, filter and autoclave. Store at -20°C.
tRNA,lO ug/ul. Store at -2o°c.
5 M NH4Ac, filter and autoclave. Store at RT.
4 M NaCl, filter and autoclave. Store at RT.
RNA denaturing solution (for Northern gel):
20 ul 10X MAE, pH 7
70 ul deionized formaldehyde
200 ul deionized formamide
Make up fresh.
4X RNA loading buffer: 50% glycerol
0.4% bromophenol blue
0.4% xylene cyanol

1 mM EDTA
Store at RT.

108
Agarose/formaldehyde mini gel (Northern):

10 ml 10X MAE, pH 7
1.2 g agarose (low EEO)

72.4 ml HOH
Melt agarose in HOH and MAE in microwave
on high setting for 2.5—3 min. Cool to
approx 60°C, add 17.6 ml deionized
formaldehyde and pour gel.

Mini gel electrode buffer:

25 ml 10x MAE, pH 7
180 ml HOH
45 ml deionized formaldehyde

Hybridization solution:

100 ul 10 X hybridization buffer
100 ul HOH

800 ul formamide

Make up fresh.

10X hybridization buffer:
400 mM PIPES, pH 6.4
4 N NaCl
10 mM EDTA
12.09 g PIPES in 50 ml HOH, adjust pH to
6.4 with 5 N NaOH. Add 23.38 g NaCl and
5 ml 0.2 M EDTA. Bring up to 100 ml with
HOH. Filter, autoclave, store at RT.

SI solution:

367.5 ul 4 N NaCl (0.28 M)
87.5 ul 3 M NaAc, pH 4.5 (50 mM)
236.3 ul 100 mM ZnSO4 (4.5 mM)
5.0 ul Nuclease SI (400 U/ul from BMB;
final conc. is 381 U/ml or
.133.36 U/.35ml)
5.§§§ mlHOH
5.25 ml total volume

Proteinase K, 25 mg/ml HOH. Store at -20°C.
10% SDS. Store at RT.
Denaturing loading buffer:
I 2 mg bromophenol blue (0.02%)
2 mg xylene cyanol (0.02%)
9 ml formamide (90%)

1 ml 10X TBE (1X)
Store at RT.

109
5% Polyacrylamide/8 M urea gel:

48 g urea

4.75 g acrylamide

0.25 9 bis
40 ml HOH
10 m1 10X TBE
Heat on stir plate gently (#2) until
urea is in solution (about 30 min.).
Filter through 0.45 um filter unit,
bring up to 100 ml vol. with HOH.
Degas for 20-30 min. Add 0.5 ml of 10%
APS and 50 ul TEMED and pour gel
immediately with 10 ml pipet and pipet-
aid. Allow gel to polymerize for 1.5 h.
Flush wells with 1X TBE and pre-run for
1.5-2 h. at 380 V (constant voltage).
Use 1X TBE as electrode buffer.

CO °
Linearizatign sf 298821119521 plasmid DNA:
Maps of the plasmid vector and cDNA insert that are

utilized in the following assay are shown in Figure 8.1.

The following is combined in each of 2 microfuge tubes;
24.5 ul HOH
3.0 ul buffer M
0.5 01 DNA (2.75 ug)
2.0 ul Pvu II (24 U)

30.0 ul total vol.
Incubate at 37°C for 2.5-3 h (vortex after 1 h).
Bring volume up to 100 ul with TE-8 in each tube.
Extract with phenol/chloroform/isoamyl alcohol and then
with chloroform/isoamyl alcohol (referred to as PIC/IC
extract) and ppt. DNA from aqueous phase with 5 ul 4 M NaCl
and 200 ul EtOH.
Allow DNA to ppt. for 30 min at -20°C.

Centrifuge for 8 min and dry pellets in Speed-Vac.

110
§¥n£h§§i§ sf riboprobe (using BMB §P6117 Issnscsiption

8111:

To each tube of dried linearized DNA add:

12 ul 32P-UTP (10 uCi/ul; 400 Ci/mmol; final conc. of
UTP=15 uM)
3 ul ATP, CTP, GTP mixture (1:1:1, i.e. 1 ul of each nt,
10 mmol/l; final conc. of each=500 uM)
Resuspend pellet, then add:
2 ul 10X buffer
3 ul RNase inhibitor (55 U/ul; not kit's, purchased
1 ul SP6 Polymerase (10 U/ul) . separately)

Vortex well and incubate at 37°C for 1 h (vortex after 30
min).

After 1 h, add 2 ul DNase I (20- 30 U) and 1 ul RNase
inhibitor (55 U) to each tube, vortex, and incubate an
additional 15 min at 37° C.

Add 76 ul TE-8 to bring volume of each tube to 100 ul and
then PIC/IC extract.

Then add: 10 ul tRNA (10 ug/ul)

27 ul 5 M NH4Ac (1/4 vol.)
330 ul EtOH (3 vol.)

Allow RNA to ppt. for 30 min at -20°C (during ppt'n, pour
mini 1.2% agarose-formaldehyde gel).

Centrifuge 8 min, resuspend pellets in 100 ul TE-8, and re-
ppt. with 25 ul 5 M NH4Ac and 300 ul EtOH at -200 C for 30
min.

After centrifugation for 8 min, resuspend each pellet in 11
ul TE-S.

Add 29 ul RNA denaturing solution and heat to 65°C for 10
min.

Add 10 ul of 4X RNA loading buffer and load wide well of
mini 1.2% agarose-formaldehyde gel (Northern).

Electrophorese for 1. 5 h at 65 V.

Briefly blot gel dry, wrap in saran wrap and autoradiograph
for 10 min.

Riboprobe band should appear midway between the tracking
dyes.

111

Cut out riboprobe band from the gel and electroelute RNA
using 1 ml TE- -8 for 1.5 h at 80 V in fresh electrode
buffer.

After squeezing the gel out of the dialysis tubing, remove
TE-S and place into a microfuge tube.

Centrifuge for 1 min to pellet any residual agarose.

Remove TE-8 and divide among 2 or 3 tubes and then ppt. the
RNA; e. 9., if 265 ul/tube, then add 5 ul tRNA, 68 ul 5 M
NH4Ac (1/4 vol), and 810 ul EtOH (3 vol) and allow RNA to
ppt. for 30 min at -20° C.

Centrifuge 8 min.

Resuspend pellets in a total of 200 ul of TE- 8 (combined)
and re-ppt. with 50 ul 5 M NH4Ac and 600 ul EtOH for 30 min
at -20 oC.

Centrifuge 8 min and resuspend the pellet in 200 ul of
hybridization solution.

Quantitate 0.5 ul by liquid scintillation by ppt'n. of 0.5
ul with 1 ml of 10% TCA/lo mM Na pyrophosphate containing
25 ul tRNA and filtratiOn through a glass fiber filter.

Solutisn hybsidisstion:

To 100 ug of total RNA dried in microfuge tubes, add 2
million cpm of probe and hybridization solution for a total
volume of 30 ul. Also include a tube containing 10 ug (1
ul) of tRNA plus probe and hybridization solution to serve
as a negative control.

Heat each tube to 90°C in a heating block for 10 min.

Place each tube directly into a 65°C shaking water bath for
14-16 hours.

Nusleass digessiog and slectsophoresis:

Centrifuge all tubes briefly to spin down any condensation.

Add 350 oul 81 solution to each tube, vortex, and incubate 1
h at 37° C (during incubation, prepare denaturing
acrylamide/urea gel sol’n, filter, and degas).

Centrifuge briefly and add 3 ul Proteinase K (25 mg/ml) and
20 U1 of 10% SDS.

Vortex and incubate an additional 15 min at 37°C (during
incubation pour acrylamide/urea gel).

112

PIC/IC extract and then ppt. RNA with 5 ul tRNAb 105 ul 5 M
NH4Ac, and at least 1 ml EtOH for 30 min at -20 C

Centrifuge 8 min.

Resuspend pellets in 100 ul TE- 8 and re-ppt. RNA with 25 ul
5 M NH4Ac and 300 ul EtOH for 30 min at -20° C (start pre-
running gel at this time).

Centrifuge 8 min and then dry pellets in Speed-Vac.

Resuspend pellets in 20 ul denaturing loading buffer and
heat to 90 C for 2-3 min.

Two ug (8 ul) of DNA mw marker III (BMB) should be dried
down, resuspended in loading buffer and heated for 2-3 min,
too. .

3900 cpm of probe alone should also be resuspended in 20 ul
loading buffer and heated.

Cool all samples on ice and then load 5% Polyacrylamide/S M
Urea gel.

Gel should be pre-run for 1-1.5 h prior to loading.

After loading gel, electrophorese for 2.5 h at 380 V
(constant voltage; current will be approx. 30-40 mA). The
first tracking dye will run off the gel; the second
tracking dye will end up at the bottom of the gel at
completion.

Cut off marker lane, stain with EtBr for 2 h, and then
photograph.

Dry remaining gel in gel dryer for 1.5 h at 60°C on PAGE
cycle.

Wrap gel in saran wrap and autoradiograph.

A dose-response curve is shown in Figure B.2. The.solution
hybridization-nuclease protection assay was conducted using
50, 75, 100, and 150 ug of liver RNA. Values were
corrected for nonspecific hybridization by subtracting the
hybridization signal obtained with tRNA (y-intercept=80;
slope=7.7; r square=.978). Intra- and inter-assay
coefficients of variation using 100 ug of total RNA are 2.8
and 2.7%, respectively, in this assay.

113

Figure B.1. Maps of the plasmid vector (top) and IGF-I cDNA
insert (bottom) that were used to synthesize the IGF-I RNA
probe. The cDNA was subcloned into the EcoRI site of the
plasmid vector, pGEM—I (Promega, Madison, WI), and
generously provided by Dr. Frank Simmen (Tavakkol et al.,

1988).

114

   

  
       
 
    

pGEM"-1

Riboprobe Gemini
Transcription Plasmid n

(2.9 Kb) ...... I

  

MI

%

Ive-.3; at 740:0"
I

 

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Site

‘1

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£35?

 

. g
‘ L——-—.—.-_.j—9'9cq03

F—.---

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. 9:-
..“.‘. —
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tn In vei in en an "e w an on an Me See no
$9 746 m 01:: 616 70: 1'76 CR 76: fit 4:: 806 7:7 8“ 46 ca GO
in in ate in an low in an no w an m eie tr en ”.1
90 CO .646 If: at 161 me 067 686 C16 :16 64: 0:: CT! 06 11! 676
nmmmmmmmmammmmam
mmmmaeocmutmucmmxautuunr
mmmumamaumammmmz
M 40: 467 a: 406 00: en 06 as a: IR 076 647 m 13 78 m
azammamzmmmammme
uaaamutmammwentumeucumm
an 5.: m 2! 1!: in 9:8 is: 61! a; in m 1!: 22 1:! in
790 cc; 0:8 06 73 0:6 (:86 it: fit on 8:: as at a: 48 686 876
a: m 11.! a; 9.2 an sec vei er. ele ale er; Ms "I see at
lflmuemucmcuflecn mucunmmuzm
we In ele 810 in I" tel 849 I00 in as an en en 6” ear
I‘ 764 064 u: u: u: 746 see 416 :mewmammsw
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“I mmaxmmmmxmfluxsawmucmuu7mufl
m xmmamccmufiw7uecnct44146471066244.4335 940

Figure 8.1.

847

 

115

Figure B.2. Dose-response relationship between quantity of
total RNA used and hybridization signal obtained in the
solution hybridization-nuclease protection assay. A porcine
IGF-I cDNA gas utilized as a template to synthesize an
antisense 3 P-labeled RNA probe. The probe was hybridized
to 50, 75, 100, and 150 ug of porcine liver RNA at 65°C for
14 hours. Following hybridization, 133 units of nuclease
S1 were added and samples incubated for an additional 1
hour at 37°C. Protected RNA fragments were
electrophoretically separated in 5% acrylamide/8 M Urea
gels and then subjected to autoradiography. Transfer RNA
(tRNA) was included as a negative control to account for
nonspecific background hybridization. The signals obtained
in the SSC-base pair nuclease-protected fragments were
quantitated by liquid scintillation analysis. Results have
been corrected for nonspecific hybridization by subtracting
the signal obtained from hybridization to tRNA (y-
intercept=80; slopes7.7; r squared=.978).

116

1400 Corrected cpm in protected fragment

1200 ..
1000 r
800 r-
600 _-

400 *-

 

200 l 1 1 l l 1
0 25 50 75 100 125 150

Quantity of RNA in assay, ug

Figure B.2. ‘

LITERATURE CITED

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11
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