Protein kinetics in callipyge lambs.

Abstract

The objectives for this experiment were to determine the effect of the callipyge phenotype on protein kinetics. We studied callipyge and normal lambs (n = 37) at 5, 8, and 11 wk of age (n = 4 to 7/ group) to determine how protein kinetics are altered by this trait. Total protein, DNA, and RNA and calpastatin activity were measured in five skeletal muscles and in the heart, kidneys, and liver, and protein accretion rates were calculated. At 8 wk, the fractional synthesis rates of proteins in these tissues were measured in vivo using a primed, continuous 8-h infusion of [2H5]phenylalanine. Fractional rates of protein degradation were estimated by differences. At 5 wk of age, muscle weights, protein mass, protein:DNA, RNA:DNA, and calpastatin activity were higher (P < .05) for callipyge, and protein mass differences continued to increase through 11 wk. At 8 wk, fractional rates of protein synthesis and degradation were lower (P < .05) in callipyge than in normal lambs. The organs of callipyge lambs exhibited reduced growth at 11 wk. Thus, enhanced muscle growth seems to be maintained in callipyge lambs by reduced protein degradation rather than increased protein synthesis. However, we cannot exclude the possibility that the initial onset of the callipyge condition may be caused by an increase in the fractional rate of protein synthesis.

Full text

Page 1

J. W. Savell and M. L. Fiorotto
C. L. Lorenzen, M. Koohmaraie, S. D. Shackelford, F. Jahoor, H. C. Freetly, T. L. Wheeler,
Protein kinetics in callipyge lambs
2000, 78:78-87.
J ANIM SCI
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Protein kinetics in callipyge lambs1,2
C. L. Lorenzen*, M. Koohmaraie†,3, S. D. Shackelford†, F. Jahoor‡, H. C. Freetly†,
T. L. Wheeler†, J. W. Savell*, and M. L. Fiorotto‡
*Department of Animal Science, Texas A & M University, College Station 77843; †Roman L. Hruska U.S.
Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933-0166; and ‡Children’s Nutrition Research
Center, ARS, USDA, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030
ABSTRACT:
to determine the effect of the callipyge phenotype on
protein kinetics. We studied callipyge and normal
lambs (n = 37) at 5, 8, and 11 wk of age (n = 4 to 7/
group) to determine how protein kinetics are altered by
thistrait.Totalprotein,DNA,andRNAandcalpastatin
activity were measured in five skeletal muscles and in
the heart, kidneys, and liver, and protein accretion
rates were calculated. At 8 wk, the fractional synthesis
rates of proteins in these tissues were measured in
vivo using a primed, continuous 8-h infusion of [2H5]-
phenylalanine. Fractional rates of protein degradation
were estimated by differences. At 5 wk of age, muscle
The objectives for this experiment were
Key Words: Calpastatin, Fiber, Sheep, Protein Synthesis, Protein Degradation
2000 American Society of Animal Science. All rights reserved.
J. Anim. Sci. 2000. 78:78–87
Introduction
A gene mutation, callipyge, that results in enhanced
skeletal muscle growth has been identified in sheep
(Koohmaraie et al., 1995; Carpenter et al., 1996; Jack-
son et al., 1997a). Lambs that eventually express this
1Mention of a trade name, proprietary product or specific equip-
ment is necessary to report factually on the available data; however,
the USDA neither guarantees nor warrants the standard of the prod-
uct, and the use of the name by the USDA implies no approval of
the product to the exclusion of others that may also be suitable.
2The authors acknowledge the technical assistance of P. Beska,
M.DelRosario,M.Doumit,P.Ekeren,C.Ernst,L.Frazer,B.Freking,
C.Haussler,andS.Hauver.Wealsogratefullyacknowlegethehelpful
discussions withHarry J.Mersmann andPeter J.Reeds in theprepa-
ration of this manuscript, and the editorial assistance of Leslie Lod-
deke. This project has been funded in part by the U.S. Department
of Agriculture, Agricultural Research Service, under Cooperative
Agreement number 58-6250-6001. The contents of this publication
do not necessarily reflect the views or policies of the U.S. Department
ofAgriculture,nordoesmentionoftradenames,commercialproducts
or organization imply endorsement by the U.S. Government.
3To whom correspondence should be addressed. Phone: 402/762-
4221; fax: 402/762-4149; E-mail: koohmaraie@email.marc.usda.gov.
Received February 26, 1999.
Accepted May 26, 1999.
78
weights, protein mass, protein:DNA, RNA:DNA, and
calpastatin activity were higher (P < .05) for callipyge,
and protein mass differences continued to increase
through 11 wk. At 8 wk, fractional rates of protein
synthesis and degradation were lower (P < .05) in calli-
pyge than in normal lambs. The organs of callipyge
lambs exhibited reduced growth at 11 wk. Thus, en-
hanced muscle growth seems to be maintained in calli-
pyge lambs by reduced protein degradation rather than
increased protein synthesis. However, we cannot ex-
clude the possibility that the initial onset of the calli-
pyge condition may be caused by an increase in the
fractional rate of protein synthesis.
phenotype are born with apparently normal muscle de-
velopment, and the callipyge-associated muscle hyper-
trophybecomesnoticeableat4to6wkofage(Koohmar-
aie et al., 1995; Jackson et al., 1997a). The increase in
muscle mass is not common to all muscles, and growth
of some muscles has been shown not to differ at all
(Koohmaraie et al., 1995; Jackson et al., 1997b).
The callipyge phenotype increases muscle growth via
hypertrophy and muscles from market-age lambs have
higher DNA content suggestive of increased protein
synthetic capacity and higher calpastatin activity sug-
gestive of reduced protein degradation (Koohmaraie et
al., 1995). However, the effect of the callipyge pheno-
type on protein turnover has not yet been studied.
Therefore, the objective of the present study was to
determine the relative roles of muscle protein synthesis
and protein degradation in callipyge-induced muscle
growth. To control for potential differences in general
systemic factors between phenotypes, comparisons
were made both for muscles whose weight is increased
bythecallipygephenotype(longissimusandbicepsfem-
oris) and for muscles whose weight is not increased by
the callipyge phenotype (infraspinatus and supra-
spinatus).
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Protein turnover in callipyge lambs
79
Table 1. Pelleted dieta
Ingredientg/kg diet (as fed)
17% Dehydrated alfalfa
Casein
Ground corn
Soybean meal (solvent extruded)
Methionine
Beet molasses
Soybean oil
Sulfur
Sodium chloride
Vitamin mixb
Dry matter
Crude protein
Energy, kcal/kg
250
180
412
80
4
20
50
2
1
1
905
286
3,090
aBeginning at 3 wk, lambs were allowed ad libitum access to the
pellet diet.
bContained: retinal acetate, 8.8 × 106IU/kg mix; cholecalciferol,
8.8 × 105IU/kg mix; α-tocopherol acetate, 8.8 × 102IU/kg mix.
Materials and Methods
Animals and Experimental Design
The protocol was approved by the Roman L. Hruska
U.S. Meat Animal Research Center Animal Care and
Use Committee. A breeding strategy that takes into
account the paternal polar overdominance inheritance
pattern of the callipyge gene was designed to yield 50%
normal and 50% callipyge lambs (Cockett et al., 1996;
Freking et al., 1998). All lambs were sired by Dorset
rams heterozygous for the callipyge gene and express-
ingthemusclehypertrophyphenotypeandfromnoncal-
lipyge Romanov ewes. Lambs were allowed to suckle
for the first day of life and then were transferred to a
nursery. The nursery consisted of pens with raised
floors,eachequippedwithanautomaticmilkdispenser.
The lambs were provided with supplemental heat for
the first few weeks and then were maintained at 20°C.
Lambs were allowed ad libitum access to a commercial
lamb milk replacer (Ultrafresh Lamb Milk Replacer,
LandO’Lakes,FortDodge,IA),whichwasdiscontinued
after approximately 4 wk of age. Beginning at 3 wk of
age, the lambs also were given ad libitum access to
a high protein/high energy pelleted diet designed to
support maximal rates of growth (Table 1). At 4 wk of
age,lambswereevaluatedformuscleexpressionindica-
tive of the callipyge phenotype. Body weights, without
feed deprivation, were determined weekly beginning at
4 wk. The lambs (n = 37) were assigned to one of three
groups to be studied at 5 (callipyge n = 4; normal n =
7), 8 (callipyge n = 7; normal n = 7), and 11 (callipyge
n = 7; normal n = 5) wk of age. Final verification of
each lamb’s phenotype was determined by experienced
evaluators on the basis of muscle definition of the
skinned carcass.
The lambs were killed at 5 and 11 wk by captive bolt
gun followed by exsanguination and at 8 wk with an
i.v.overdose ofsodiumpentobarbitalfollowed byexsan-
guination. Skeletal muscles and organ samples were
rapidly removed, frozen in liquid nitrogen, and stored
at−70°Cuntilanalysis.Samplesforcalpastatinactivity
were processed immediately without freezing. After all
the samples for analysis had been removed, remaining
muscles or organs were quantitatively dissected, and
the total weight was determined. The muscles and or-
gans that were collected included longissimus (LD), bi-
ceps femoris (BF), infraspinatus (IS), supraspinatus
(SS), diaphragm (DIA), heart (HT), liver (LIV), and kid-
ney (KID). The tissues and organs were analyzed for
total protein, DNA, and RNA concentrations and cal-
pastatin activity.
In the 8-wk-old group, the rate of protein synthesis
in these same muscles and organs was determined in
vivo. Two indwelling jugular catheters were placed per-
cutaneously, using aseptic procedures, 48 h before the
lambs were to be infused. The infusion catheter was
advanced through the left jugular by a distance that
would place the catheter tip in the superior vena cava;
the sampling catheter was inserted a short distance
into the right jugular so that it was above the infusion
catheter. The catheters were flushed with heparinized
saline, capped, and then secured to the skin with a
suture. Prior to the start of the infusion, each lamb
was transferred to an individual metabolic crate. To
minimize separation anxiety, two to three crates of
lambs were placed next to each other during an infu-
sion; these were housed within the nursery in close
proximity to the nursery pens. The lambs were allowed
to eat throughout the period of infusion. A blood sample
was collected, and then a primed, continuous infusion
of [2H5]phenylalanine was administered for 8 h. The
lambs then were killed, and tissue samples were taken
as described above. The amount of [2H5]phenylalanine
incorporated into total tissue proteins and in the cellu-
lar and plasma free amino acid pools was determined
by mass spectrometry. On the basis of these measure-
ments, total body phenylalanine flux and in vivo rates
of protein synthesis, accretion, and degradation were
estimated.
Protein, DNA, and RNA
Tissue samples were homogenized in chilled, deion-
izedwater. Aliquotsofthehomogenate weresolubilized
in .1 M NaOH at 37°C for 1 h and then analyzed for
protein concentration (Lowry et al., 1951) using BSA
for a standard. The DNA concentration was measured
using the combinedprocedures of Cesarone etal. (1979)
andLabarcaandPaigen(1980);calfthymusDNA(Frac-
tion V) was used as a standard. The remainder of the
homogenate was acidified to .2 M perchloric acid to
precipitate total RNA and protein. Total RNA was de-
termined as described by Munro and Fleck (1969). The
protein pellet and acid supernatant were used for the
estimation of [2H5]phenylalanine enrichment as de-
scribed below.
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Lorenzen et al.
80
Calpastatin Activity
The procedure was carried out as previously de-
scribed (Koohmaraie et al., 1995). Briefly, a fresh 5-g
sample of tissue was homogenized in five volumes of
homogenizationbuffer(50mMTris?HCl,10mMEDTA,
.1 g/L ovomucoid, 6 mg/L leupeptin, pH 8.3) and centri-
fuged, and the supernatant was dialyzed. The dialysate
was heated to inactivate calpain activity and centri-
fuged, and the calpastatin in the supernatant was puri-
fied using a DEAE-Sephacel column. Calpastatin activ-
ity was determined from the inhibition of purified m-
calpain activity by the calpastatin extracted from the
tissues. Data are reported as units of calpastatin activ-
ity per gram of tissue protein; a unit is defined as the
amount of calpastatin needed to inhibit one unit of m-
calpain activity.
Fiber Typing
Samples of the LD were frozen in liquid-nitrogen-
cooled isopentane; 10 ?m-thick transverse sections
were cut with a cryostat, air-dried, and then stained
formyofibrillaradenosinetriphosphatasewithacid(pH
4.35) and NADH-TR activities, a simultaneous combi-
nationstainingprocedure,asdescribedbySolomonand
Dunn (1988). A minimum of 200 fibers per lamb were
classifiedasslow-oxidative(β-red),fast-oxidative/glyco-
lytic (α-red), or fast-glycolytic (α-white). The areas of
the same fibers were measured with Micromp PM
(Southern Micro Instruments, Atlanta, GA) interactive
image analysis of planar morphometry.
Protein Synthesis In Vivo
A sterile solution of [2H5]phenylalanine (98% en-
riched, Cambridge Isotope Laboratories, Woburn, MA)
was prepared in 9 g/L NaCl. Lambs received an initial
priming bolus dose equivalent to a 1-h infusion, fol-
lowed by an 8-h constant infusion at the rate of 40
?mol?kg body weight−1?h−1. In a pilot study conducted
beforehand, we had determined that the priming dose
and infusion rate yielded a steady-state plasma
tracer:tracee ratio within 40 to 60 min of the bolus
injection, and this was maintained throughout the 8-h
infusion. Heparinized blood samples were drawn at 0,
4, 5, 6, 7, and 8 h after the start of the infusion; they
were centrifuged, and the plasma samples were snap-
frozen. At the end of the 8-h infusion, lambs were killed
and tissues were obtained as described above. Due to
equipment failure during the infusion, protein synthe-
sis measurements could not be obtained for one calli-
pyge lamb.
Phenylalanine enrichment in the plasma, the tissue-
free pool, and the protein-bound fraction were isolated
and prepared for gas chromatography-mass spectrome-
try (GCMS) as follows. Plasma was acidified with four
volumes of 1 M acetic acid. The protein-free superna-
tant from each acidified tissue homogenate described
above was neutralized with 4 M KOH and used for
the determination of the tissue-free amino acid pool.
Protein pellets were hydrolyzed in 4 M HCl (Ultrex
grade)at 110°Cfor 24h.After dryingand severalwash-
ings with water, the amino acids were resuspended in
1 M acetic acid. All amino acid samples were purified
using cation exchange chromatography (Dowex AG50,
8% crosslinked, H+form) before derivatization. The
amino acids derived from the plasma, tissue free pool,
and the protein hydrolysates were converted to the n-
propyl ester, heptafluorobutyramide derivative (Ha-
chey et al., 1991). Isotopic enrichment was measured
by negative chemical ionization GCMS by monitoring
the ions at a mass-to-charge ratio of 383 [m+0] to 388
[m+5].
Calculations
The total protein, DNA, and RNA masses of each
muscle and organ were calculated from their respective
concentrations inthe individual tissues andtotal tissue
weight. The ratios, RNA:DNA, protein:DNA, and
RNA:protein(indicativeofthetissue’sproteinsynthetic
capacity) also were calculated.
The fractional synthesis rate (FSR) of the constitu-
tive proteins was calculated from the tracer:tracee ra-
tios of the samples at the end of the 8-h infusion ac-
cording to the equation
FSR (%/d) = (Ebound× 24 × 100)/(Efree× t)
where Ebound is the enrichment of protein-bound
[2H5]phenylalanine at the end of the infusion, Efreeis
theintracellularfree[2H5]phenylalanineforeachtissue
at the end of the infusion, and t is the total time of the
infusion in hours. The tracer:tracee ratio of the tissue-
free phenylalanine at the start of the infusion was as-
sumed to be the same as for plasma phenylalanine,
whichwasverylow.Theabsoluterateofproteinsynthe-
sis was calculated as the product of fractional protein
synthesisrateandthetotalproteinmassofeachmuscle
or organ at 8 wk of age. The translational efficiency
(KRNA, mg protein synthesized/g RNA) of the cellular
ribosomes (which constitute 85 to 90% of total RNA),
was calculated from the quotient of fractional protein
synthesis rate and the RNA:protein ratio.
The total protein contents of each muscle and organ
were highly correlated to the live weight of the lamb
(r2> .96 for all muscles; r2> .92 for all organs) with no
separate,identifiablecontributionofagetothevariance
in total tissue protein content. Thus, prediction equa-
tionsweregeneratedforeachgenotypetoestimateindi-
vidualmuscleororganproteinmassesfromliveweight.
For those lambs studied at 8 wk, these prediction equa-
tions together with the individual lamb’s body weight
from 5 wk of age were used to derive the protein con-
tentsofmusclesororgansatearlierages.Proteinaccre-
tion rates for each muscle and organ were then derived
from the slope of the regression of protein mass vs age
for each individual lamb. Fractional accretion rates
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Protein turnover in callipyge lambs
81
(FAR, %/d) were determined by dividing the absolute
protein accretion rates by the protein mass of the tissue
at 8 wk.
Fractional protein degradation rates were estimated
from the difference between fractional protein synthe-
sis rate and fractional protein accretion rate. Absolute
protein synthesis and degradation rates (g/d) were cal-
culated by multiplying the fractional rates by the total
protein content of individual tissues.
Total body phenylalanine flux was calculated using
the following equation:
Flux (?mol?kg body weight−1?h−1) = ([Ei/Ep] − 1) × i
where Eiis the tracer:tracee ratio of the infusate; Epis
the value for the steady-state enrichment of the plasma
calculatedastheaverageofthe4,5,6,7,and8hvalues;
and i is the rate of infusion of phenylalanine in ?mol?kg
body weight−1?h−1.
Statistical Analysis
Preliminary analyses indicated that, for both normal
andcallipygelambs,dissectedtissueweightsweremore
stronglycorrelatedwithliveweightoftheanimalbefore
slaughterthan slaughterage.Therefore,all masstraits
(protein mass, DNA mass, and RNA mass) were ad-
justed to a common live weight within age group to
eliminate the error-inflating effects of variation in
growth rate among animals. Within each phenotype,
adjustment factors were derived by regressing each
mass trait against live weight across all age groups.
Because most experiments (Shackelford et al., 1998)
have indicated similar growth rates for normal and
callipyge,thenormalandcallipygelambswereadjusted
to a common live weight within each age group.
Protein turnover data were analyzed with one-way
ANOVA.Allothertraitswereanalyzedas2(phenotype)
× 3 (age group) factorially arranged, completely ran-
domized design. When the interaction was not signifi-
cant (P > .05), it was pooled into the error term. The
significance of differences between callipyge and nor-
mal lambs in measures of protein turnover conducted
only at 8 wk was tested with Student’s t-test (two-way).
Differences in response between muscles were deter-
mined with ANOVA using muscle and phenotype as
the main effects. Differences with P-values less than
.05 were considered significantly different. Values were
expressed as means either with the pooled standard
deviation used in the ANOVA when testing for age and
phenotype effects, or ± SEM for the protein turnover
data when only the phenotype effects are evaluated.
Results
Growth
Body weights of callipyge and normal lambs were not
significantly different at any age (3.14 ± .61 kg, 8.53 ±
.68, 15.12 ± 1.27, and 22.51 ± 2.80 kg at birth, 5, 8, and
11 wk of age, respectively) and increased linearly from
5 to 11 wk of age (2.20 ± 1.11 kg/wk). Similarly, all
muscle weights increased with age,as did their protein,
RNA, and DNA masses (Table 2). The callipyge pheno-
type was associated with increased (P < .001) protein
mass for longissimus and biceps femoris but not for
infraspinatus and supraspinatus. These results are
consistent with previous observations of the effect of
the callipyge phenotype on weights of those muscles in
market-weightlambs(Koohmaraieetal.,1995;Jackson
et al., 1997c). The increase in protein mass in callipyge
longissimus and biceps femoris was predominantly due
toanabsoluteincreaseintissueweight(17.1%increase,
P < .001 for LD; 21.4% increase, P < .001 for BF). To a
lesser extent, the increase in protein mass in callipyge
longissimus and biceps femoris was caused by an in-
crease in protein concentration (4.5% increase, P < .01
for LD; 3.4% increase, P < .03 for BF). There was a
phenotype × age interaction on longissimus and biceps
femoris protein mass such that the magnitude of the
difference in protein mass between normal and calli-
pyge increased with age. The interaction was particu-
larlypronouncedforbicepsfemoris,forwhichthediffer-
ence in protein mass between phenotypes increased
from a 2.0-g effect (17.9% change) at 5 wk to a 8.7-g
effect (28.5% change) at 11 wk. In market-age (169 d)
lambs, Koohmaraie et al. (1995) observed a 42% in-
crease in biceps femoris weight associated with the cal-
lipyge phenotype.
Muscle DNA (Table 2) content increased with age (P
< .001) for all muscles. Muscle DNA content was not
affected (P > .05) by phenotype. The protein:DNA ratio
(Table 3) increased with age for all muscles. In both
longissimus and biceps femoris, the protein:DNA ratio
was greater for callipyge relative to normal lambs.
Total RNA mass (Table 2) increased with age in all
muscles. For both longissimus and biceps femoris, RNA
content was greater (P < .001) in callipyge than normal
lambs. The increase in total RNA content associated
with the callipyge phenotype was proportionally
greaterforbicepsfemoristhanforlongissimus(P<.05).
For both longissimus and biceps femoris, RNA:DNA
ratio was higher for callipyge (Table 3). The callipyge
phenotype did not affect RNA:protein ratio in any
tissue.
Proteinmassofliverandkidneyswerelowerforcalli-
pyge lambs (Table 4); however, phenotype did not affect
heart protein mass. These results are consistent with
previous observations (Koohmaraie et al., 1995) of the
effect of the callipyge phenotype on weights of those
organs in market-weight lambs. For each organ, the
slope of the regression line between organ protein mass
and body weight was significantly lower for callipyge
lambs. The changes in organ protein masses tended to
be accompanied by parallel changes in DNA and total
RNA masses. Thus, values for protein:DNA and
RNA:proteinweresimilaramongphenotypes(Table5).
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Lorenzen et al.
82
Table 2. Total protein, DNA, and RNA contents of longissimus (LD), biceps femoris (BF), infraspinatus (IS),
supraspinatus (SS), and diaphragm (DIA) in normal (N) and callipyge (C) lambs at 5, 8, and 11 wk of age
Age
5 wkx
8 wkx
11 wkx
Pooled
SD
(df = 34)
Phenotype
×
Age
NCNCNC
Musclen = 7n = 4n = 7n = 4n = 7n = 4Phenotype Age
Total protein, g
34.6d
19.9d
9.8c
6.8b
6.8b
Total DNA, g
306c
178b
104b
62b
67b
Total RNA, g
244c
128c
58c
47c
54b
LD
BF
IS
SS
DIA
20.5e
11.2e
5.7d
4.4c
3.8c
24.4e
13.2e
5.5d
3.8d
3.8c
42.8c
24.6c
9.2c
6.6b
6.6b
55.4b
30.5b
16.3a
10.5a
9.7a
68.6a
39.2a
14.9b
10.1a
10.1a
3.5
2.0
1.1
.5
.6
***
***
†
*
NS
***
***
***
***
***
*
***
NS
NS
NS
LD
BF
IS
SS
DIA
178e
119c
52d
44c
38c
205e
120c
55d
39c
40c
255d
190b
80c
61b
63b
424b
303a
129a
87a
81a
469a
321a
125a
79a
80a
33
32
13
7
6
NS
NS
†
†
NS
***
***
***
***
***
**
NS
*
NS
NS
LD
BF
IS
SS
DIA
120e
63d
32d
32d
28d
150d
79d
34d
28d
29d
257c
141c
54c
41c
46c
305b
163b
88a
72a
68a
356a
211a
79b
65b
71a
25
15
5
6
7
***
***
*
**
NS
***
***
***
***
***
NS
*
*
NS
†
a,b,c,d,eWithin a row, means not bearing a common superscript letter differ (P < .05).
xAdjusted to a common live weight of 9.3, 15.3, and 22.9 kg at 5, 8, and 11 wk, respectively.
†P < .10.
*P < .05.
**P < .01.
***P < .001.
Table 3. Protein:DNA, RNA:DNA, and RNA:protein ratios of longissimus (LD), biceps
femoris (BF), infraspinatus (IS), supraspinatus (SS), and diaphragm (DIA) in normal
(N) and callipyge (C) lambs at 5, 8, and 11 wk of age
Age
5 wk8 wk 11 wk
NCNCNCpSD*
(34*)Musclen = 7n = 4n = 7n = 7n = 5n = 7Phenotype†
Age
Protein/DNA, g/mg
139
113
120
122
121
RNA/DNA, g/g
1.06
.75
.62
.71
.73
RNA/protein, mg/g
6.225.22
5.83 5.36
5.775.42
6.406.81
6.887.07
LD
BF
IS
SS
DIA
113
100
106
95
97
116
110
101
98
99
124
113
95
107
99
171
129
107
111
106
149
122
116
127
124
17
10
9
9
8
<.001
<.020
NS
NS
NS
<.001
<.001
<.001
<.001
<.001
LD
BF
IS
SS
DIA
.68
.58
.64
.69
.71
.74
.68
.62
.72
.74
.87
.72
.58
.74
.81
.72
.60
.65
.83
.86
.78
.66
.62
.81
.90
.16
.09
.05
.08
.07
<.05
<.05
NS
NS
NS
NS
NS
NS
<.001
<.001
LD
BF
IS
SS
DIA
6.05
5.83
6.08
7.31
7.37
6.15
6.19
6.17
7.11
7.45
7.12
6.36
6.09
6.93
8.12
5.20
5.41
5.39
6.44
7.26
.74
.45
.42
.80
.76
NS
NS
NS
NS
NS
<.001
<.001
<.001
NS
NS
†Phenotype × age interaction was not significant (P > .05).
*Pooled standard deviation with error degrees of freedom.
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Protein turnover in callipyge lambs
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Table 4. Total protein, DNA, and RNA contents of heart (HT), kidney (KID), and liver (LIV)
in normal (N) and callipyge (C) lambs at 5, 8, and 11 wk of age
Age
5 wkx
8 wkx
11 wkx
Pooled
SD
(df = 34)
Phenotype
×
Age
NCNCNC
Organn = 7n = 4n = 7n = 4n = 7n = 4Phenotype Age
Total protein, g
11.0b
10.3c
84.0c
Total DNA, g
135a
330c
1,309c
Total RNA, g
109b
272c
1,864c
HT
KID
LV
7.2c
6.8d
38.5e
8.1c
5.8d
32.3e
10.0b
9.6c
66.2d
14.1a
15.6a
118.2a
13.4a
13.5b
102.7b
1.1
1.0
6.4
NS
***
***
***
***
***
†
NS
†
HT
KID
LV
70c
285cd
1,040d
83bc
265d
1,047d
139a
307cd
1,268c
90b
527a
2,382a
85bc
455b
2,141b
16
43
163
NS
*
†
***
***
***
NS
NS
NS
HT
KID
LV
108b
221de
1,087e
137a
198e
984e
103b
244cd
1,641d
161a
438a
2,790a
147a
379b
2,514b
19
33
176
NS
**
**
***
***
***
*
NS
NS
a,b,c,d,eWithin a row, means not bearing a common superscript letter differ (P < .05).
xAdjusted to a common live weight of 9.3, 15.3, and 22.9 kg at 5, 8, and 11 wk, respectively.
†P < .10.
*P < .05.
**P < .01.
***P < .001.
Muscle Histomorphometry
The cross-sectional area of all fibers increased with
age and, for the fast-oxidative/glycolytic and fast-glyco-
lytic fibers, the areas were significantly greater in calli-
pyge lambs (Table 6). There was no age × phenotype
interaction.Therewasnoeffectofcallipygeontheslow-
oxidative fibers. There was a shift in the fiber-type dis-
tribution in callipyge lambs from fast-oxidative/glyco-
lytic to fast-glycolytic. This shift also was present by 5
Table 5. Protein:DNA, RNA:DNA, and RNA:protein ratios of heart (HT), kidney (KID),
and liver (LIV) in normal (N) and callipyge (C) lambs at 5, 8, and 11 wk of age
Age
5 wk 8 wk11 wk
NCNCNCpSD*
(37*) Organn = 7n = 4n = 7n = 7 n = 5n = 7Phenotype† Age†
Protein/DNA, g/mg
164
30
50
RNA/DNA, g/g
1.80
.83
1.17
RNA/protein, mg/g
11.0
28.0
23.5
HT
KID
LIV
105
23
35
104
22
34
91
32
66
84
32
57
154
30
48
12
2
5
NS
NS
NS
<.001
<.001
<.005
HT
KID
LIV
1.70
.77
1.02
1.70
.77
.99
.92
.85
1.47
.85
.83
1.34
1.67
.84
1.19
.22
.06
.11
NS
NS
NS
NS
<.05
<.05
HT
KID
LIV
16.2
33.2
29.5
16.3
34.8
29.3
10.1
26.6
22.4
10.1
25.5
23.5
10.8
28.0
24.9
1.3
2.2
1.7
NS
NS
NS
<.001
<.001
<.001
†Phenotype × age interaction was not significant (P > .05).
*Pooled standard deviation with error degrees of freedom.
wk of age. The effects of the callipyge phenotype ob-
served herein for 5-, 8-, and 11-wk-old lambs are consis-
tent with previous observations made in market-age
lambs(Koohmaraieetal.,1995;Carpenteretal.,1996).
Protein Turnover at 8 Weeks of Age
For lambs studied at 8 wk, absolute daily rates of
protein accretion (Table 7) were greater for longissi-
mus and biceps femoris of callipyge lambs. The differ-
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Lorenzen et al.
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Table 6. Muscle fiber areas and fiber-type distribution in longissimus of normal (N)
and callipyge (C) lambs at 5, 8, and 11 wk of age
Age
5 wkx
8 wkx
11 wkx
NCNCNCpSD*
(34*)Fiber typen = 7n = 4n = 7n = 7n = 5n = 7Phenotype†Age†
Cross-sectional area, ?m2
548785
1,126842
1,443 1,375
1,250 1,040
Fiber-type distribution, %
711
37 49
56 40
SO
FOG
FG
Average
510
573
977
755
545
888
672
771
755152
226
313
242
NS
<.001
<.005
<.001
<.005
<.005
<.005
<.005
1,399
1,868
1,598
1,275
1,075
1,132
940
SO
FOG
FG
12
40
48
10
36
55
883
7
8
NS
<.02
<.005
<.05
NS
<.05
41
51
39
53
xAdjusted to a common live weight of 9.3, 15.3, and 22.9 kg at 5, 8, and 11 wk, respectively.
*Pooled standard deviation with error degrees of freedom.
†Phenotype × age interaction was not significant (P > .05).
ence between callipyge and normal lambs was
greater, proportionally, for the biceps femoris mus-
cles. The difference in absolute rates of protein accre-
tion between phenotypes was approximately propor-
tional to the difference in protein mass between phe-
notypes; consequently, fractional protein accretion
rates were not different between phenotypes (Figure
1). In longissimus and biceps femoris, both the frac-
tional protein synthesis rate and the fractional pro-
tein degradation rate were reduced (P < .05) for calli-
Table 7. Absolute rates of protein accretion, synthesis, and degradation and
translational efficiency (KRNA) of longissimus (LD), biceps femoris (BF), infraspinatus
(IS), supraspinatus (SS), diaphragm (DIA), heart (HT), kidneys (KID), and liver
(LIV) in normal (N; n = 7) and callipyge (C; n = 6) lambs at 8 wk of age
Accretion,
g protein/d
Synthesis,
g protein/d
Degradation,
g protein/d
KRNA,
g/g
TissueNCNCNCNC
LD .69
(.09)a
.41
(.03)
.21
(.02)
.12
(.01)
.12
(.01)
.17
(.01)
.20
(.02)
2.12
(.16)
.93*
(.05)
.63*
(.06)
.21
(.01)
.13
(.01)
.13
(.01)
.14
(.02)
.16
(.02)
1.56*
(.17)
2.44
(.18)
1.24
(.05)
.55
(.04)
.39
(.02)
.40
(.02)
1.42
(.07)
5.16
(.31)
34.00
(1.01)
2.34
(.27)
1.40
(.06)
.51
(.04)
.48
(.04)
.39
(.01)
1.19
(.13)
4.68
(.25)
38.60*
(1.82)
1.76
(.15)
.82
(.06)
.34
(.04)
.27
(.02)
.28
(.01)
1.25
(.06)
4.96
(.30)
30.88
(1.14)
1.41
(.27)
.77
(.09)
.30
(.04)
.35
(.04)
.25
(.01)
1.04
(.11)
4.53
(.25)
37.04*
(1.84)
10.40
(.50)
10.27
(.49)
9.75
(.51)
8.74
(.32)
7.59
(.18)
13.12
(.25)
19.72
(.43)
18.96
(1.38)
8.41
(.90)
9.07
(.90)
8.93
(.65)
1.54
(.64)
8.09
(.13)
10.80*
(.96)
17.98*
(.62)
21.99
(.92)
BF
IS
SS
DIA
HT
KID
LIV
aParenthetical values are the SEM.
*Phenotypes differ (P < .05). Phenotypes tend to differ (P < .06).
pyge lambs. Thus, in longissimus and biceps femoris,
protein accretion seems to be much more efficient for
callipyge lambs.
In longissimus, the lower fractional protein synthe-
sis rate was associated with a decrease in KRNA(Table
7). For those muscles that did not exhibit a callipyge-
associated increase in protein mass (infraspinatus,
supraspinatus, and diaphragm), the fractional pro-
tein accretion, synthesis, and degradation rates were
similar among callipyge and normal lambs.
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Protein turnover in callipyge lambs
85
Figure 1. The fractional rates of protein synthesis, accretion, and degradation in (a) the longissimus (LD), biceps
femoris (BF), infraspinatus (IS), supraspinatus (SS), and diaphragm (DIA); (b) the heart (HT), kidneys (KID), and liver
(LIV), of normal (N, n = 6) and callipyge (C, n = 7) lambs at 8 wk of age. The error bars represent ± 1 SEM; *, P < .05
for normal vs callipyge.
The most marked change in protein turnover was in
the liver. In callipyge lambs, there was a decrease in
both the fractional and absolute rates of protein accre-
tion. Both the absolute and fractional rates of protein
synthesisandproteindegradation werehigherforcalli-
pyge liver. However, the increase in synthesis rate was
smaller than the increase in degradation rate, leading
to the net reduction in accretion.
Asmall,albeitsignificant,reductioninfractionalpro-
tein synthesis, degradation, and accretion was evident
for the heart. There was a tendency for protein accre-
tion, synthesis, and degradation to be lower in the kid-
ney of callipyge lambs. For both heart and kidney, KRNA
was lower in callipyge lambs.
Estimates of whole-body phenylalanine flux were not
different between normal and callipyge lambs (211 ± 7
and 185 ± 16 ?mol?kg−1?h−1, respectively; P > .1).
Calpastatin Activity
In longissimus and biceps femoris, calpastatin activ-
ity was greaterfor callipyge lambs (Table 8).For longis-
simus,therewasamagnitudinalphenotype ×ageinter-
action on calpastatin activity in which the effect of phe-
notype on calpastatin activity was particularly large at
5 wk of age. Calpastatin activity decreased with age in
all the organs, but there was no effect of callipyge.
Discussion
Results reported herein provide insight into the ef-
fects of the callipyge phenotype on protein metabolism
in a select group of skeletal muscles and organs. By
andlarge,inthosemuscleswhosemasswasnotaffected
by the callipyge phenotype, protein metabolism was
similar among phenotypes. However, for those muscles
whose mass was increased by the callipyge phenotype,
the fractional rates of protein synthesis and degrada-
tion were reduced. This finding suggests that the mus-
cling advantage of callipyge is maintained through a
reduction in the rate ofprotein degradation rather than
an increase in the rate of protein synthesis.
Because the present experiment was conducted at a
time point at which the protein masses of normal and
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Lorenzen et al.
86
Table 8. Calpastatin activity of longissimus (LD), biceps femoris (BF), infraspinatus (IS),
supraspinatus (SS), diaphragm (DIA), heart (HT), kidneys (KID), and liver (LIV) of
normal (N) and callipyge (C) lambs at 5, 8, and 11 wk of age
Age
5 wk8 wk11 wk
NCNCNC pSD*
(31*)Tissuen = 7n = 4n = 7n = 7n = 5n = 7 Phenotype†Age†
Calpastatin activity, U m-calpain/g protein
32b
15c
24 12
27 19
26 21
40 31
194142
36 35
108 57
LD
BF
IS
SS
DIA
HT
KID
LIV
17c
21
28
29
33
298
45
228
45a
26
29
33
49
321
47
177
21c
18
33
26
42
189
35
85
26b
20
21
32
38
154
33
71
9
8
<.001
<.03
NS
NS
NS
NS
NS
NS
<.02
<.02
NS
NS
NS
<.001
<.05
<.001
10
10
14
80
12
54
a,b,cWithin LD values that do not share a common superscript letter differ (P < .05).
†Phenotype × age interaction was significant for LD (P < 0.03); age effect present only in callipyge lambs.
*Pooled standard deviation with error degrees of freedom.
callipyge lambs had already begun to diverge, we do
not yet know whether the mechanism responsible for
theinitialdifferenceinproteinmassamongphenotypes
is the same as the mechanism responsible for main-
taining that difference. Although we did not detect a
difference among phenotypes in the fractional protein
accretion rate of longissimus or biceps femoris, there
must have been a difference among phenotypes at some
earlier age for the protein mass of those muscles to be
greater in callipyge at 8 wk. We cannot exclude the
possibility that earlier differences in the fractional rate
of protein accretion may have been caused by an in-
crease in the fractional rate of protein synthesis. It
would be of merit to investigate the effect of the calli-
pyge phenotype on protein metabolism in the perinatal
period. However, in preliminary attempts to study pro-
teinmetabolisminnewbornlambs,weobservednumer-
ous technical difficulties inherent to newborn lambs
that precluded us from studying newborn lambs in
this experiment.
Koohmaraie et al. (1995) showed that differences
among muscles in the level of callipyge-induced hyper-
trophy were strongly correlated with the level of calli-
pyge-induced increase in calpastatin activity. In the
present experiment, differences among muscles in the
level of callipyge-induced increase in muscle protein
mass was correlated with calpastatin activity (r = .96),
fractional degradation rate (r = −.75), and fractional
synthesis rate (r = −.79). The negative relationship be-
tweentheeffect ofthecallipygephenotype onfractional
synthesis rate and muscle hypertrophy supports the
hypothesis that the muscling advantage of callipyge is
maintained through a reduction in the rate of protein
degradation rather than an increase in the rate of pro-
tein synthesis.
The mechanism underlying the enhanced protein de-
position in the longissimus and biceps femoris of calli-
pyge lambs can only be inferred on the basis of the 8-
wk proteinturnover data, togetherwith thegrowth and
compositional measurements at 5 and 11 wk of age.
By 8 wk of age, the longissimus and biceps femoris in
callipyge lambs seemed to have attained a new steady
state in which higher muscle masses were sustained
by lower fractional protein degradation rate. This re-
sponse is similar to that observed in the skeletal mus-
cles of rats after chronicadministration of the β-agonist
clenbuterol (Reeds et al., 1986; Maltin et al., 1989;
Hesketh et al., 1992); after a transient period of en-
hanced muscle growth, a larger muscle protein mass
was associated with a proportionally higher RNA con-
tent, but no change in RNA:protein ratio, whereas frac-
tional protein synthesis rateand fractional protein deg-
radation rate were significantly lower than in controls.
In the β-agonist model, the decrease in fractional pro-
tein degradation rate seemed to be more protracted
than the increase in fractional protein synthesis rate
and contributed to the more chronic anabolic response.
Studies of β-agonist treatment in lambs have yielded
similar findings (Nash et al., 1994; Mersmann, 1998).
Thereductionsinproteindegradationhavebeenassoci-
atedwithreductionsinproteaseactivitiesandaneleva-
tion in calpastatin activity (Mersmann, 1998). Thus,
thereductionsinthefractionalratesofproteinturnover
in callipyge longissimus and biceps femoris at 8 wk
might represent an eventual adaptation to the hyper-
trophied condition, rather than the cause of the hyper-
trophy. The greater proportion of fast-twitch fiber mass
in the callipyge lambs may also have contributed to
the reduction in fractional protein synthesis rate and
fractional protein degradation rate because these fibers
have lower turnover rates than slow-twitch fibers (Gar-
lick et al., 1989).
Implications
The muscling advantage of callipyge seems to be
maintained through a reduction in the rate of protein
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Protein turnover in callipyge lambs
87
degradation rather than an increase in the rate of pro-
tein synthesis. This provides a basis for the develop-
ment of strategies to select for or to alter body composi-
tion in meat animals. Although decreasing the rate of
muscle protein turnover results in favorable effects on
the rate and efficiency of muscle growth, this may not
be the most desirable method to alter muscle growth
in meat animals given the associated negative effects
on meat tenderness.
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