PRESLAUGHTER TREATMENT AFFECTING INTRAMUSCULAR
AND PLASMA LIPIDS
I. E F F E C T O F A C T H I N R A B B I T S 1
John R. Romans, 2 Ivan S. Palmer, a Donald R. Wenger, 4
William J. Costello, s Harold J. Tuma 6 and
Richard C. Wahlstrom s ,7
South Dakota Agricultural Experiment Station, Brookings 5 7006
ment, resulting in a significant (P<.01) quadratic relationship with time of ACTH infusion.
All classes of rabbit intramuscular lipids increased slightly during the stress period. When
the six long-treatment rabbits (11 or 12 hr.)
were considered separately, only the PL mean
increase was significant (P<.05). Inclusion of
data from the three short-treatment rabbits (2
to 6 hr.) resulted in significant (P<.05) mean
increases in TG, C and TL.
Summary
The effect of ACTH-induced stress on
intramuscular and plasma lipids was studied in
rabbits. Nine mature rabbits were subjected to a
constant infusion of ACTH (1.5 mU/min./kg of
body weight). A significant relationship existed
between duration of ACTH infusion and the
concentration of all plasma lipid classes. After 1
hr. of ACTH infusion, plasma free fatty acid
(FFA) values were increased 11-fold over initial
values. Following the initial peak, a slight
decrease in FFA occurred during the ensuing
treatment period. A significant (P<.01) quadratic relationship existed between time of
ACTH infusion and plasma triglyceride (TG)
levels. TG levels generally showed a slight
decrease at 1 hr., and then gradually increased,
culminating in a fourfold increase after 11 or
12 hours. Cholesteryl esters (CI~) and cholesterol (C) responded significantly (P<.01) as
quadratic expressions of time of ACTH infusion, CE decreasing with time and C showing
a twofold increase from initial to final values.
Phospholipids (PL) decreased in the first half of
the treatment period but returned to initial
levels by termination, a significant (P<.01)
quadratic relationship. Total lipids (TL), the
sum of the five classes, increased during treat-
Introduction
Several studies with species other than meat
animals provide evidence that intramuscular
lipids supply the substrate for oxidative metabolism during fasting or exercise. Neptune et al.
(1960) found that phospholipids (PL) and
triglycerides (TG) decreased significantly in rat
diaphragm muscle during a 48-hr. fast. Havel,
Maimark and Borchgrevink (1963) measured
free fatty acid (FFA) turnover and oxidation
rates in six members of a wrestling team while
exercising on a treadmill, finding evidence that
intramuscular lipids furnish some fuel for contracting muscle in the postabsorptive state.
Similar results have been reported by Issekutz
et al. (1964) and Issekutz and Paul (1968) with
exercised dogs. Contrary to these findings,
Masoro, Rowell and McDonald (1966a), Masoro
et al. (1966b) and Masoro (1967) have provided
solid evidence that exercise in monkeys does
not cause a significant decrease in any component of the intramuscular lipid. These workers
also showed that fasting generally did not cause
depletion of rat skeletal muscle lipids. More
recently, Therriault et al. (1973) showed with
dogs that muscle TG levels increased, decreased
or remained unchanged, depending upon the
work load imposed by exercise.
1Published with the approval of the Director of the
South Dakota Agricultural Experiment Station as
Publication No. 1166a of the Journal Series.
2Present address: Department of Animal Science,
University of Illinois, Urbana/Champaign 61801.
3Department of Station Biochemistry.
4Present address: U.S. Department of Agriculture,
Animal Disease Laboratory, Ames, Iowa 50010.
s Department of Animal Science.
6Present address: Department of Animal Science,
Kansas State University, Manhattan 66502.
qThe authors express gratitude to W. L. Tucker
and H. W. Norton for aid in the statistical analysis.
32
J O U R N A L OF A N I M A L S C I E N C E , vol. 38 n o . 1, 1 9 7 4
INTRAMUSCULAR AND PLASMA LIPIDS
If intramuscular lipid (marbling) is broken
down in response to stress in meat animals,
lower marbling scores and thus lower quality
grades could result. In this study, adrenocorticotrophic hormone (ACTH), a known
adipokinetic agent, was administered to rabbits
to investigate the possible loss of marbling in a
laboratory animal physiologically similar to our
major meat producing species.
Materials and Methods
Rabbits were used in this investigation since
they are intermediate between the small laboratory animal (rat) and our meat producing
species. Furthermore, rabbits have a longissimus
muscle of sufficient size to permit accurate
sampling. In a preliminary feeding, 12 closely
related female New Zealand white rabbits were
fed a complete rabbit diets ad libitum from
weaning. Four rabbits were slaughtered 2, 10
and 18 weeks after weaning. Ether-extracts of
the longissimus muscle for the three periods
were 1.72, 2.35 and 3.13%, respectively, indicating that lipid was deposited in this muscle
and that rabbits would be suitable for the
proposed fat mobilization studies.
Nine mature male and female rabbits from
six litters of mixed breeds were given free
access to a complete rabbit diet 8 and water for
at least 6 months prior to experimentation.
Final weights ranged from 3.3 to 5.0 kilograms.
Stress was simulated by constantly infusing
ACTH, a proven mobilizer of FFA in rabbits
(Woods, Freeman and Kellner, 1962).
One hour before anesthesia each rabbit
received a subcutaneous injection (lOml/kg of
body weight) of physiological saline containing
5% dextrose to stabilize the FFA which otherwise might be mobilized under the stress of
anesthesia and operation (Hirsch et al., 1963).
It was anesthetized with sodium pentobarbital
(30 mg/kg of body weight), and a cannula was
inserted into the right external jugular vein for
withdrawing blood samples and infusing ACTH.
A cross-section containing approximately 5 g of
tissue was removed from the right longissimus
muscle just posterior to the 13th rib. The rabbit
was then placed in a restraining box and
A C T H 9 w a s infused into the jugular cannula at
a constant rate of 1.5 mU/min./kg of body
weight by means of a syringe pump 1~
33
Time of stress was calculated from the
beginning of ACTH infusion. Five-milliliter
blood samples were drawn at 0, 1, 3, 6, 9 and
12 hr., into heparinized syringes. The samples
were centrifuged for 15 min. at 800 x g and the
plasma was removed and stored at -20 C.
Immediately after the final blood sample was
taken, the rabbit was killed by injecting additional sodium pentobarbital into the jugular
vein. The final muscle sample was immediately
removed from the left longissimus muscle just
posterior to the 13th rib. The two muscle
samples thus came from the same longitudinal
location but from different sides of the animal.
Muscle samples were trimmed of all exterior fat
a n d fascia, sealed in airtight plastic bags and
stored at -20 C.
Data from those rabbits which did not
survive the full 12-hr. treatment are included,
"final" blood and muscle samples having been
taken immediately upon death.
Lipids were extracted from plasma and
muscle with a 2:1 chloroform-methanol (v]v)
solution as described by Folch, Lees and
Stanley, (1957). Total lipid (TL) thus extracted
was separated by thin-layer chromatography
into PL, cholesterol (C), FFA, TG and cholesteryl esters (CE). The C, FFA, TG and CE
f r a c t i o n s were measured by a photorefiectometric technique (Romans and Palmer,
1972). Micro determinations of phosphorus
were made (Bartlett, 1959) and the PL concentration computed assuming that PL are 4%
phosphorus.
The data were analyzed by the method of
least squares.
Results and Discussion
Plasma Lipids
Free Fatty Adds. It was essential in this
study that the stress agent have a definite FFA
mobilizing effect. It had been shown (Woods et
al., 1962; Hirsch et al., 1963) that ACTH had
such an effect in rabbits and this study supported those findings (figure 1). At the end of 1
hr. of ACTH infusion, FFA levels had increased
significantly (P<.01) six- to 18-fold over initial
values. Peak values were reached in 1 hr. and
were followed by a slow decline. However, in
the six rabbits which survived for 11 or 12 hr.,
the ratio of initial level of FFA to that at any
aRabbit Chow Checkers, Ralston Purina Company. time in the treatment period never fell below
1:6. The three animals which died after 2, 4 or
9Acthar, List 1025, Armour PharmaceuticalCo.
10Sage, Model 255-2 Variable Speed Syringe 6 hr. of the treatment all exhibited sharp
Pump.
decreases in their final samples. Neither sex nor
34
ROMANS ET AL.
80'
60'
40,
20
0
4
8
fo
f2
HOURS
Figure 1. Effect of continuous ACTH infusion (1.5
mU/min./kg) on plasma FFA in rabbits. Each symbol
represents 1 rabbit: o litter A littermates; [] litter B
littermates; ~ unrelated.
littermates were significant, but the indication
of littermates on the regression plots in figures
1 through 6 permits judgment of genetic
effects.
Triglycerides. Plasma TG changed significantly (P<.O1) with time (T) of continuous
ACTH infusion (figure 2). Generally a slight
drop in TG concentration of plasma occurred at
1 hour. In all surviving animals, plasma TG
began to increase after 3 hr. of infusion and
continued to termination. Increases in TG from
initial to ffmal samples varied from three- to
eightfold in the six rabbits which survived 11 or
12 hours. It has already been shown that FFA
were mobilized rapidly by ACTH infusion.
Furthermore, the plasma FFA levels remained
elevated throughout the experiment. Approximately 30% of such FFA may be taken up in
one passage through the liver (Steinberg, 1966).
Once in the liver the FFA can be oxidized for
energy or reesterified to TG. The newly synthesized TG will then be released into the plasma
in association with low density lipoproteins
thereby increasing plasma TG levels.
Cholesteryl Esters. There was a general
decrease (P<.01) in plasma CE level. A reason
for this decrease in the transport form of
cholesterol is not apparent, but perhaps there
was hydrolysis to free cholesterol.
Cholesterol. A gradual increase in plasma C
was noted, the change from initial to final
samples averaging 15 mg/lO0 ml of plasma,
approximately a twofold increase in the six
long-treatment rabbits. The significant (P<.0I)
quadratic effect of time of ACTH infusion on
plasma C level is shown in figure 4. The high
plasma FFA levels undoubtedly resulted in
increased FFA oxidation which in turn would
increase the concentration of acetyl coenzyme
A, a precursor of C. Maintaining high plasma
FFA levels could then result in the gradual
increase in the rate of C synthesis which was
observed. Similarity of littermates is apparent
in figure 4.
Phospholipids. A gradual decline in PL occurred up to approximately the sixth hour of
ACTH infusion, when an increase began, and at
12 hr. they were very near initial levels.
Statistical analysis found a significant (P<.01)
quadratic regression of PL on time (figure 5).
The PL response to ACTH infusion was somewhat similar to the TG response and might be
similarly explained. Perhaps ACTH initially
stimulated hydrolysis of PL thus causing a
disappearance of PL and an increase in FFA.
Eventually the resulting increased plasma FFA
might stimulate the liver to release more lipoproteins. Since such lipoproteins contain PL as
well as TG and C, a buildup of plasma PL
would result.
Total Lipids. Total lipid increases from
initial to final samples averaged twofold in the
six long-treatment rabbits, appearing (figure 6)
300
250
~ . 60.6
- 4.6T
* 1.3 T z
ZOO
~a
8
(.9
I00 (
8
1'o
HOURS
Figure 2. Effect (P<.01) of continuous ACTH
infusion (1.5 mU/min.lkg) on plasma TG in rabbits.
Each symbol represents 1 rabbit: o litter A littermates;
[] litter B littermates; ~ unrelated.
INTRAMUSCULAR AND PLASMA LIPIDS
35
160 '
140"
120
"~ " 4 4 . 3 - 4 . 7 1 T + 0 . 2 8 3 T
1401
2
~ . 100.6
- 9.3T
+ O. T 4 4 T
z
I
I00
--I
o
.rJ
0
so
o
o
"-,..9
120"
I001
(..9
60
80
409
6o !
o
fi
4
g
~3
i'o
,'z
H OURS
o
z
4
~
~
i'o
~
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Figure 3. Effect (P<.01) of continuous ACTH
infusion (1.5 mU/min./kg) on 'plasma CE in rabbits.
Each symbol represents 1 rabbit: o litter A littermates;
[] litter B littermates; * unrelated.
similar to the TG response. In all cases, finalsample plasma was lactescent.
Intramuscular Lipids
Intramuscular lipids were approximately
50% TG, 45% PL, 5% C and less than 0.5% FFA
by analysis. No CE were detectable. Because
FFA appeared in such minute amounts, they
were not included in the reported values of TL.
Therefore, as referred to in the discussion of
50"
= 20.4
-
1.29 T * 0.18t
T~
40-
o
o_
:
~-9 ZO~
IO~
0
~
~
8
ro
f2
HOURS
Figure 4. Effect (P<.01) of continuous ACTH
infusion (1.5 mU/min./kg) on plasma C in rabbits.
Each symbol represents 1 rabbit: o litter A littermates;
[] litter B littermates; ~ unrelated.
Figure 5. Effect (P<.01) of continuous ACTH
infusion (1.5 mU/min./kg) on plasma PL in rabbits.
Each symbol represents 1 rabbit: o litter A littermates;
[] litter B littermates; * unrelated.
intramuscular lipids, TL includes only TG, C
and PI.
Table 1 lists the changes in intramuscular
lipid which occurred in each rabbit during the
stress period. The same symbols are used to
identify rabbits in table 1 as were used in
figures illustrating plasma lipid response. Of the
six rabbits which survived 11 or 12 hr. of stress,
at least five showed increases (table 1) for each
lipid class the significance probabilities being
about .13, .058, .018 and .065 for TG, C, PL
and TL, respectively.
Including the short-treatment rabbits, the
increase in each lipid component was significant
(P<.02). That the level of ACTH-induced stress
in these rabbits reached a degree of severity
causing early death may be related to this
a u g m e n t e d intramuscular fat deposition.
Therriault (1973) has shown that muscle TG
levels change in relation to the work load
imposed in dogs.
A general increase in all intramuscular lipids
occurred in response to the administration of
ACTH, but in TL amounted to only about
one-tenth of a percent. Nevertheless, only five
of the 36 individual changes, for nine rabbits
and four lipid classes, were negative, a strong
indication that ACTH really caused positive
change.
Masoro et al. (1966a) showed that vigorous
muscle contraction, caused by electrical stimulation, for 5 hr. in monkeys did not significantly decrease the concentration of any intra-
36
ROMANS ET AL.
.
.
.
.
.
.
.
.
0
.
.
.d
o
o
(S'
o
~
d
~
"~
o
<
0
0
2
4
6
8
HOURS
I0
12
03
Figure 6. Effect (P<.01) of continuous ACTH
infusion (1.5 mU/min./kg) on plasma TL in rabbits.
Each symbol represents 1 rabbit: o litter A littermates;
[] litter B littermates; * unrelated.
~x
.<
muscular lipid ester class. Conversely, Issekutz
et al. (1964) and Issekutz and Paul (1968)
showed, by evaluating O~ uptake and CO~
output, that intramuscular lipids must serve as
an energy source for exercising dogs. They
suggested that each of the glyceride esters (TG,
PL) which store the muscle fatty acids and
provide energy are not present in large enough
amounts to show a significant change by
chemical analysis. This may be the case also for
these rabbit data.
The increase in all fractions of intramuscular
lipid caused by the continuous infusion of
ACTH was not expected, although the theoretical basis for such increases accompanying
elevated plasma FFA levels has been discussed
(Steinberg, 1966). Thus, subcutaneous or
visceral adipose tissue rather than intramuscular
lipids may have contributed to the increased
plasma FFA levels. The FFA available in plasma
in excess of immediate energy requirements
could be deposited in muscle as esters, resulting
in a net increase of TL in muscle.
It appears from these data that the high
plasma FFA levels were not produced by
mobilization of intramuscular lipids, though it
o.
o
z
o
o
[]
eq
r~
I
o.
~V
ut(-
~2
2<
.<
[-
#
I N T R A M U S C U L A R A N D P L A S M A LIPIDS
m a y be that m o b i l i z a t i o n occurred initially
f o l l o w e d b y rapid replacement. In any event,
there was very little net change o f muscle lipids
during the e x p e r i m e n t .
37
createctomized dogs. Amer. J. Physiol. 215:197.
Masoro, E. J., L. B. Rowell and R. M. McDonald.
1966a. Intracellular muscle lipids as energy sources
during muscular exercise and fasting. Fed. Proc.
25:1421.
Masoro, E. J., L. B. RoweU, R. M. McDonald and B.
Steiert. 1966b. Skeletal muscle lipids. II. Nonutilization of intracellular lipid esters as an energy
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