International Journal of Obesity (2000) 24, 1040±1050
ß 2000 Macmillan Publishers Ltd All rights reserved 0307±0565/00 $15.00
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Functional and histological characteristics of
skeletal muscle and the effects of leptin in the
genetically obese (ob=ob) mouse
SA Warmington1*, R Tolan1 and S McBennett1
1
Department of Physiology, University of Dublin, Trinity College, Dublin 2, Ireland
BACKGROUND: Skeletal muscle mass in genetically obese (ob=ob) mice displays a reduced mass compared with their
normal lean counterpart mice. However, the functional capacity of the available skeletal muscle mass in these animals
has not yet been determined.
OBJECTIVE: To investigate the properties of skeletal muscle in ob=ob mice and determine the effects of leptin
administration on skeletal muscle in these mice.
METHODS: Following 4 weeks of i.p. leptin administration (or control treatment) anaesthetized ob=ob and lean mice
had their extensor digitorum longus and soleus muscles removed, and standard measures of isometric contractile
properties and fatigability were performed. Histochemistry was used to determine ®bre type proportions and
individual ®bre areas of all muscles.
RESULTS: Leptin had no effect on the morphology or function of ob=ob skeletal muscle despite reducing body mass in
ob=ob mice. Force production was unaltered in obese mice. However, a signi®cant prolongation of contraction and
relaxation times were evident. Obese skeletal muscle was also more fatigue resistant. Fibre proportions displayed a
more slow type pro®le in ob=ob skeletal muscle, and in conjunction with previous work a reduced ability to
hypertrophy.
CONCLUSION: Skeletal muscle from obese mice is morphologically and functionally different from lean mouse
skeletal muscle. Obese muscle is very similar to skeletal muscle from aged mice, and the speci®c contractile
properties examined appear to be determined by the ®bre make-up of these muscles.
International Journal of Obesity (2000) 24, 1040±1050
Keywords: leptin; skeletal muscle; isometric contractile parameters; ®bre type
Introduction
Obesity in the genetically obese mouse (ob=ob) is
caused by a mutation in the gene encoding leptin.1
Lack of leptin production by white adipocytes leads to
obesity in these mice. They also exhibit a number of
pathophysiological characteristics including over 50%
body fat, hyperinsulinaemia, hyperphagia, hyperglycaemia when fasting, and reductions in energy expenditure, O2 consumption, and thermogenic activity.2 In
contrast, human obesity is not characterized by a lack
of circulating leptin and is thus more likely to result
following some disruption in the messaging pathway
downstream from leptin binding to its receptor.3 Thus,
leptin administration in humans appears to be ineffective, while in the ob=ob mouse, leptin treatment
corrects the present obesity.
It has also been shown in genetically obese mice
(ob=ob) that there is a signi®cantly reduced mass of a
number of limb skeletal muscles.4 ± 9 This reduction
*Correspondence: SA Warmington, Department of Physiology,
University of Dublin, Trinity College, Dublin 2, Ireland.
E-mail: [email protected]
Received 10 September 1999; revised 1 March 2000; accepted
7 April 2000
may be caused by several factors including the elevated circulating levels of corticosteroid hormones
observed in these mice. This would promote protein
degradation while simultaneously limiting protein
synthesis.3,9 There is also a reduced long bone
length in ob=ob mice which will limit the growth
and development of skeletal muscle,8,11 and the reduction in DNA available for transcription in these
animals will also limit the synthesis of protein and
subsequently muscle mass accretion.8 However, to the
best knowledge of the authors, it is unknown whether
this reduction in skeletal muscle mass occurs with
human obesity.
In general, treatment of obesity, despite being
initially successful in reducing body mass, often
fails to result in a sustained reduction from the
obese state.12 ± 14
Treatment of obesity is often associated with various drug administrations13 and, more traditionally,
combinations of dietary restriction combined with
increases in energy expenditure through regular prescribed exercise programs. Thus, an alteration in
skeletal muscle mass may further limit the success
of these exercise treatment programs and the maintenance of weight loss through an increased energy
expenditure.
Skeletal muscle characteristics in ob/ob mice
SA Warmington et al
It is unknown whether, in these obese mice, the
available skeletal muscle has different functional
characteristics to skeletal muscle from non-obese
populations. There is evidence for leptin receptors in
skeletal muscle; however, the effects of leptin on
skeletal muscle have not yet been described. Therefore, in this study we evaluated the contractile and
histochemical properties of the limb skeletal muscles
extensor digitorum longus (EDL) and soleus from
ob=ob and lean mice. We also aimed to determine
the effects of a one-month intraperitoneal administration of leptin on these functional measures of skeletal
muscle in both lean and ob=ob mice.
Methods
All experiments performed in this study were conducted in accordance with the European Community
Directive 86=609=EC, and the Republic of Ireland,
Ministry of Health licensing requirements and guidelines of the Cruelty to Animals Act, 1876.
Animals
Thirteen genetically obese (Ob=Ob) male mice (5 1
month) of the inbred Aston strain, and 12 genetically
normal=lean control (CON) littermates were obtained
from an established colony in the Bio Resources Unit,
Trinity College, Dublin. Animals from each strain
were randomly divided into a leptin treated or phosphate buffered saline (PBS) treated group to provide
four experimental populations. CON-PBS (n ˆ 6),
CON-leptin (n ˆ 6), Ob=Ob-PBS (n ˆ 6), and
Ob=Ob-leptin (n ˆ 7). Animals were housed in pairs
within the animal holding facility of the Department
of Physiology, Trinity College, Dublin, with a CON
and Ob=Ob mouse sharing individual cages. This area
was maintained at 20 1 C and operated on a 12:12 h
normal-phase light=dark cycle. All animals were
allowed free access to food (standard laboratory
chow, Red Mills Ltd) and water for the duration of
the experimental trials.
Drug treatment
Animals assigned to receive leptin treatment (both the
CON-leptin and Ob=Ob-leptin groups) were injected
intraperitoneally with 0.1 ml of 25 mg=ml recombinant
murine leptin (Pepro Tech EC Ltd, London, UK) in
10 mM phosphate buffered saline twice a day (09:00
and 17:00 h), 6 days per week (excluding Sunday) for
4 weeks. During the same treatment period control
animals (both the CON-PBS and Ob=Ob-PBS groups)
received intraperitoneal injections of 0.1 ml of 2.5 mM
PBS at the same times each day.
Morphology
To ensure leptin was exerting an effect during the
treatment period, all animals were observed for any
changes in physical activity and, during the ®rst week
of treatment, body mass was recorded daily. For the
remainder of the study body mass was measured once
per week. Following treatment, a ®nal measure of
body mass was made and, following dissection, heart
mass and the mass of the individual hindlimb skeletal
muscles, soleus (slow-twitch) and extensor digitorum
longus (EDL, fast-twitch) were recorded.
1041
Muscle dissection
Following the last day of treatment CON and Ob=Ob
animals were anaesthetized with 100 mg=kg of
sodium pentobarbitone (RMB Animal Health Ltd,
Dagenham, UK) via an intraperitoneal injection. The
EDL and soleus muscles were then carefully dissected
free and intact from their insertion to origin and
placed in Krebs ± Henseleit solution (consisting of,
in mM; NaCl 118; KCl 4.75; MgSO4 7H2O 1.18;
NaHCO3 24.8 with CaCl2 2.5 and D-glucose 11, added
on the day of experimentation) bubbled with carbogen
(5% CO2 and 95% O2, BOC Gases, Ireland). While
continually bathed with Krebs solution, muscles were
then cleaned of any residual tissue (generally adipose
and connective tissue) with ®ne scissors. The tendons
from each muscle were then tied at each end using 5=0
braided, non-absorbable, surgical silk (Braun Surgical
GmbH, Melsungen, Germany), leaving small loops
for attachment to a force-recording apparatus. The
muscles were then stored in Krebs solution (< 30 min)
until used for the measurement of isometric contractile properties and histochemical analysis. Following
the muscle dissection the animals were killed by
cervical dislocation and removal of the heart.
Isometric contractile measurements
Isometric contractile properties were measured using
standard laboratory techniques to investigate muscle
function.15 The loop of suture from one end of the
muscle was hooked to a calibrated washington type D
force transducer (Bioscience, Kent, UK), while the
other end was connected to an immovable pin held
stable by a micromanipulator. The muscle was subsequently suspended in a bath containing a carbogen
bubbled Krebs solution. Flanking each side of the
muscle, and aligned in a parallel direction to the
muscles longitudinal axis, were a pair of platinum
stimulating electrodes. Isometric contractions were
evoked via supramaximal square wave pulses generated from a stimulator with isolation unit (S44 Model,
Grass Medical Instruments, Quincy, MA, USA). The
tension produced by the muscle was recorded via the
force transducer connected to a twin-channel
MacLab1 and stored on a Macintosh Power PC
running MacLab Chart software version 5.8. Single
(1 Hz) twitch stimulations, with 1 min rest periods
were used to determine the optimum length (L0) of
each muscle. This is the length at which maximum
twitch tension (Pt) is produced. The length of the
International Journal of Obesity
Skeletal muscle characteristics in ob/ob mice
SA Warmington et al
1042
muscle was adjusted via the micromanipulator and
this length was maintained for all subsequent stimulations. The following parameters were measured from
a twitch response: the maximum isometric twitch
tension (Pt); the time-to-peak twitch tension (TTP);
and the time taken to relax from maximum twitch
tension to 50% Pt (12RT).
A force ± frequency relationship was determined for
each muscle by recording the tension produced following stimulation at frequencies of 1, 5, 10, 30, 50,
70, 90 and 110 Hz for 1 s with a 3 min rest period
between each stimulation frequency. The frequency
(optimum frequency) that produced the maximum
fused tetanic tension (P0) was used in all further
stimulations. The maximum tetanic tension was
recorded, and all measured forces were normalized
using an estimate of cross sectional area as previously
described.16
The fatigue characteristics of each muscle were
determined by stimulating the muscle with a 1 s
tetanic stimulation, every 5 s for 5 min. The tension
produced at time zero (t0) and every subsequent
minute for the 5 min period of stimulation was then
recorded and expressed as a decline in force from the
initial P0 measured at t0. A measure of recovery from
fatigue was also made by recording the tetanic tension
after a 5 min rest period (no stimulation) following the
fatigue protocol.
Muscle histochemistry
After completion of all isometric contractile experiments, muscles were removed from the force recording apparatus, and tendons and thread were carefully
trimmed from the muscles with a scalpel. Muscles
were then blotted dry on ®lter paper (Whatman no. 1)
and individually weighed on an analytical balance.
Muscles were snap frozen at L0 by placing them in
isopentane cooled in liquid nitrogen, then stored at
785 C. On the day of sectioning, muscles were
thawed to 720 C in a refrigerated cryostat microtome (Leica CM=1900, Leica Instruments, Nussloch,
Germany) and mounted in tissuetek OCT compound.
Serial 8 mm cross sections were cut from each muscle
and mounted on coverslips for staining. The standard
staining techniques for highlighting the citric acid
cycle enzyme, succinate dehydrogenase (SDH) and
myosin-ATPase phosphate deposition (metachromatic
dye-ATPase stain) were used to determine the ®bre
type composition and individual ®bre areas of each
muscle.17 ± 19
Sections were viewed on an upright microscope
(Leitz, Wetzlar, Germany) with digital images captured from each section using a black-and-white CCD
camera (WAT-902A, Watec, Japan). Images were
subsequently analysed using Apple Macintosh software NIH Image (version 1.57). Fibre type percentages for type I, IIA and IIB were determined from
each section according to their greyscale brightness as
International Journal of Obesity
previously described,15 and average ®bre cross sectional areas were also measured using this software.
Statistics
Unless otherwise stated, all data is presented as a
mean s.e.m. (standard error of the mean). A twoway analysis of variance (ANOVA) was used to detect
any differences between both the CON and Ob=Ob
groups, LEPTIN and PBS treated groups, and whether
the treatments had varying effects in the two animal
strains (interaction term). This was followed by a
Newman±Keuls post-hoc analysis to determine where
differences existed. In the force frequency and fatigue
relationships, statistical analysis was performed
at each stimulation frequency and time point,
respectively.
Results
Morphology
Throughout the treatment period there was an obvious
difference between the body mass of the CON and the
Ob=Ob groups, with the obese groups having approximately double the body mass of the lean groups
(Figure 1). This difference was not corrected by the
4 week period of leptin administration; however, with
leptin treatment there was a gradual loss of body mass
in the Ob=Ob-leptin group only, which became signi®cant (P < 0.05) at day 22 and continued until the
end of the study (Figure 1). All other groups showed
no evidence of a change in body mass with either PBS
or Leptin administration, while the Ob=Ob-leptin
group displayed a 10.3% overall reduction in body
mass from day 1 to the end of the 4 week treatment
period.
Figure 1 Body mass recorded throughout the treatment period.
Clear differences are evident from day 1 between the CON
groups and the Ob=Ob groups. In the Ob=Ob groups, leptintreated animals showed a loss of body mass which was signi®cant from day 22 until the end of the treatment period;
*P < 0.05.
Skeletal muscle characteristics in ob/ob mice
SA Warmington et al
Hind limb skeletal muscle mass of EDL and soleus
was 26% and 33% lower, respectively, in the Ob=Ob
groups compared with the CON groups (Table 1).
Similarly, optimum muscle length (L0) in the EDL and
soleus muscles was 6% and 13% lower, respectively,
in the Ob=Ob compared with the CON groups. However, in the EDL this was not signi®cant, achieving a
P value of 0.07 (Table 1). In contrast to the mass of
skeletal muscle, heart mass of the Ob=Ob groups was
21% greater than the CON groups (Table 1). Despite
the apparent effects of leptin on whole body mass in
the obese animals, leptin had no effect on the morphology of cardiac or skeletal muscle.
Isometric contractile properties
The isometric contractile properties for the EDL and
soleus muscles from both the CON and Ob=Ob groups
showed no evidence of an effect of leptin. The twitch
characteristics, maximum tetanic tension and Pt=P0
were all unaltered in these skeletal muscles following
4 weeks of leptin administration (Table 2).
The twitch characteristics were very similar in the
CON and Ob=Ob groups. In both the EDL and soleus
muscles there was no difference in speci®c twitch
force development following a single (1 Hz) stimulation (Pt) in the Ob=Ob compared with the CON
groups. However, TTP and 12RT were both 25%
greater in the EDL muscles, and 35% and 66%
greater, respectively, in the soleus muscles of the
Ob=Ob groups compared with the CON groups
(Table 2). In contrast to twitch tension, P0 in soleus,
but not in EDL, was signi®cantly reduced by 17% in
the Ob=Ob groups compared with CON groups, which
subsequently resulted in an elevation in the Pt=P0 ratio
in the soleus muscles from the Ob=Ob groups. The
Pt=P0 ratio for EDL was also signi®cantly elevated
despite no signi®cant change in the individual parameters of this ratio (Table 2).
1043
Force ± frequency relationship
At various sub-optimal stimulation frequencies in
both the EDL and soleus muscles, the CON groups
produced a lower percentage of P0 compared with the
Ob=Ob groups. For the EDL these were at the frequencies of 1, 10 and 30 Hz, while for soleus this
occurred at 1, 5, 10, 30 and 50 Hz (Figure 2). Further-
Table 1 Mean muscle mass and optimal length for CON and Ob=Ob groups. Mean s.e.m. for the
individual skeletal muscles EDL and soleus, and the heart from each experimental group. The top half
of the table shows the mass of the individual muscles, while the bottom section displays the optimal
length (L0) for each of the muscles. With leptin treatment having no effect on these parameters, the
Sig. column indicates differences between CON and Ob=Ob groups resulting from a two-way ANOVA
where **P < 0.01; ***P < 0.001; {P < 0.0001; and {P ˆ 0.07
CON
Mass (mg)
Heart
EDL
Soleus
L0 (mm)
EDL
Soleus
Ob=Ob
PBS
Leptin
Sig.
PBS
Leptin
146.3 3.0
8.9 0.3
6.7 0.8
152.9 12.0
9.3 0.7
6.3 0.4
**
{
***
192.6 11.9
7.1 0.5
4.5 0.4
170.7 8.5
6.3 0.5
4.3 0.3
13.1 0.4
12.1 0.3
13.4 0.4
12.0 0.4
{
***
12.3 0.3
10.7 0.3
12.6 0.4
10.2 0.2
Table 2 Isometric contractile properties for CON and Ob=Ob groups. Mean s.e.m. for the individual
skeletal muscles EDL and soleus. The upper half of the table shows the isometric contractile results
from the EDL muscles, while the bottom half are the results from soleus muscles. With no effects of
leptin on these contractile characteristics, the Sig. column indicates differences between the CON and
Ob=Ob groups resulting from a two-way ANOVA where *P < 0.05; **P < 0.01; ***P < 0.001; and
{P < 0.0001
CON
Ob=Ob
PBS
Leptin
EDL
Pt (kN=m2)
TTP (ms)
1
2RT (ms) 2
P0 (kN=m )
Pt=P0
27.5 3.5
36.1 3.8
48.6 9.4
153.8 7.6
17.6 1.6
30.6 2.3
40.0 2.3
52.0 4.3
158.7 8.3
19.4 1.4
Soleus
Pt (kN=m2)
TTP (ms)
1
2RT (ms) 2
P0 (kN=m )
Pt=P0
17.5 1.5
99.4 3.1
179.4 13.8
112.9 10.4
15.6 0.1
22.2 0.9
97.7 3.0
159.3 6.8
138.6 7.4
16.1 0.2
Sig.
PBS
Leptin
*
30.6 1.8
47.7 2.6
63.2 6.7
140.8 7.7
21.7 0.3
33.0 2.4
47.2 1.8
62.6 4.0
153.6 14.8
21.9 1.2
***
***
*
{
22.5 2.2
135.6 9.3
291.9 4.2
103.9 13.2
22.8 2.1
22.0 1.8
131.2 11.4
271.7 18.3
104.6 8.1
21.2 1.2
**
**
International Journal of Obesity
Skeletal muscle characteristics in ob/ob mice
SA Warmington et al
1044
Figure 2 The relationship of recorded force as a percentage of P0, to the frequency of stimulation in both the EDL (a) and soleus (b)
muscles. At various sub-optimal stimulation frequencies the CON groups produced a lower force (percentage of P0) than the Ob=Ob
groups in both the EDL and soleus, *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.
more, the optimal frequency for stimulation, although
dependant in each experiment on the individual
muscle, appears more likely to occur around 90 Hz
for EDL muscles from both the CON and Ob=Ob
groups. However, in soleus it appears that the optimal
stimulation frequency for maximal force production is
around 30 Hz in the Ob=Ob groups compared with
60 ± 70 Hz in the CON groups. Again, there was no
indication that leptin had an effect on the force
frequency relationship in either muscle group.
Stimulation to fatigue
There was no evidence of leptin action on force
production during stimulation to fatigue, or on the
recovery of force in either the EDL or soleus muscles
from both the CON and Ob=Ob groups (Figure 3).
However, in both the EDL (all recorded time points
during fatiguing stimulations) and soleus (at t ˆ 3, 4
and 5) muscles, the force decline during the fatigue
International Journal of Obesity
protocol in the CON groups was signi®cantly greater
to that of the Ob=Ob groups. This difference was not
present following the 5 min recovery period where the
EDL and soleus returned to around 56% and 100% of
P0, respectively.
Muscle histochemistry
In the EDL muscles leptin did not appear to have any
effect on either the ®bre type composition or the areas
of individual muscle ®bres in both the CON and
Ob=Ob groups. However, differences were observed
between groups in this muscle with a 13% elevation in
type IIA ®bre composition and a similar reduction
(12%) in the type IIB composition in the Ob=Ob
groups compared with the CON groups (Figure 4a).
Also, type IIA ®bre areas were 19% greater in the
Ob=Ob groups, with type I and type IIB areas 12%
and 21% lower, respectively (Figure 5a).
Skeletal muscle characteristics in ob/ob mice
SA Warmington et al
1045
Figure 3 Recorded force as a percentage of P0 at t ˆ 0 during stimulation to fatigue in both the EDL (a) and soleus (b) muscles. At all
recorded time points during stimulation in the EDL the CON groups were signi®cantly more fatigued than the Ob=Ob groups. In the
soleus similar differences were recorded between the CON and Ob=Ob groups at t ˆ 3, 4 and 5 min. All differences were abolished in
both muscle types during the 5 min recovery period from fatigue. **P < 0.01; ***P < 0.001; and ****P < 0.0001.
In contrast, in the soleus muscles, leptin produced a
reduction in the percentage of type IIA ®bres and an
elevation in the percentage of type IIB ®bres of the
soleus muscles from both the CON and Ob=Ob groups
(Figure 4b). However, as in the EDL muscles there
was a signi®cantly lower percentage of type IIB ®bres
in the soleus of the Ob=Ob groups compared with the
CON groups, with an increase in the type IIB ®bre
areas of the Ob=Ob groups (Figure 5b).
Discussion
Morphology
The initial morphological results of this study con®rm
a number of previous ®ndings. Firstly, leptin administration reduced body mass and the subsequent
established obesity in genetically obese (ob=ob)
mice, while in contrast leptin had no effect on their
lean (non-obese) counterpart mice (Figure 1).20 ± 22
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Skeletal muscle characteristics in ob/ob mice
SA Warmington et al
1046
Secondly, limb skeletal muscle displayed a reduced
mass in the ob=ob mice compared with the same
muscles from lean mice (Table 1)4 ± 9 and, despite
the presence of peripheral leptin receptors on skeletal
muscle,23 4 weeks of leptin administration did not
correct this reduction in skeletal muscle mass. The
reason for this reduced skeletal muscle mass is
unknown. However, it has been shown that passive
Figure 4 Percentage ®bre type (I, IIA, IIB) composition of the (a) EDL and (b) soleus muscles from the CON and Ob=Ob groups. Leptin
treatment (clear bars), signi®cantly reduced type IIA ({P < 0.05) and increased type IIB ({P < 0.01) percentages in the CON and Ob=Ob
groups of the soleus muscle only. However, the Ob=Ob groups showed a signi®cantly reduced percentage of type IIB ®bres in both
muscles (**P < 0.01), while the EDL only showed an increase in the type IIA percentage (**P < 0.01) in the Ob=Ob groups compared
with the CON groups.
International Journal of Obesity
Skeletal muscle characteristics in ob/ob mice
SA Warmington et al
tension exerted via long bone growth is essential for
full development of skeletal muscle,11 and that ob=ob
mice have reduced femur and tibia lengths. This may
subsequently result in a decrease in muscle length and
mass.8 In addition the present study found a signi®-
cantly reduced muscle length (L0) in the ob=ob animals when compared to the CON mice, adding to the
suggestion that a decrease in the local trophic action
of long bone growth is likely to be responsible for the
reduced skeletal muscle mass seen in this and other
1047
Figure 5 Fibre areas for type I, IIA, and IIB from the (a) EDL and (b) soleus muscles of the CON and Ob=Ob groups. There were no
effects of leptin treatment (clear bars) in either muscle type. However, in the EDL muscles the Ob=Ob groups had a reduced type I and
IIB area combined with an elevated type IIA ®bre area when compared with the CON groups, while in the soleus there was an elevated
type IIB area only in the Ob=Ob groups compared with the CON groups; **P < 0.01; ***P < 0.001; and ****P < 0.0001.
International Journal of Obesity
Skeletal muscle characteristics in ob/ob mice
SA Warmington et al
1048
studies.4 ± 9 However, it has also been reported that
ob=ob mice have an elevated level of circulating
corticosteroid hormones which limit protein synthesis and promote protein degradation in skeletal
muscle.4,10 Combined with an overall reduction in
DNA available for transcription, it appears that these
animals have a limited ability for muscle mass acretion.8 The possibility also exists that a decrease in the
level of physical activity in conjunction with obesity
may induce a reduction in skeletal muscle mass
through disuse muscle atrophy.
Although it may be expected that leptin administration could correct this muscle mass de®cit given the
increase in activity associated with leptin administration in these animals (data not shown; qualitative
observations), the lack of alteration in muscle mass
with leptin administration in the present study would
make this seem unlikely. However, alterations in
skeletal muscle mass and body mass may potentially
occur on a temporal scale such that a prolonged leptin
treatment period may provide even greater changes in
body mass towards normal, and the beginnings of any
changes to skeletal muscle mass. Of course this does
not eliminate the possibility that ultimately the skeletal muscle from ob=ob mice may be physiologically,
both morphologically and functionally, different from
that of the CON mice.
Isometric contractile parameters
Functionally, leptin also had no effect on the performance parameters of skeletal muscle measured in
this study. In both the EDL and soleus muscles
there was no effect of leptin on the isometric contractile parameters (Pt, TTP, 12RT and P0; Table 2),
force frequency relationship (Figure 2), or the fatigue
characteristics (Figure 3) of these two muscles. However, differences were observed in these parameters
when comparing the Ob=Ob and CON groups of mice.
Although twitch force was not different in muscles
from either the Ob=Ob or CON groups, both the TTP
and 12RT were prolonged in the EDL and soleus
muscles from the obese mice when compared to the
lean mice (Table 2). This is re¯ected in the ®bre type
data (Figure 4) where the Ob=Ob groups appear to
have a more slow type pro®le than the CON groups
with reductions in the type IIB composition in both
the EDL and soleus and an elevation in the type IIA
composition of the EDL only. This data may also
suggest a reduced Ca2‡ cycling ability of the sarcoplasmic reticulum (SR) in the Ob=Ob skeletal
muscle.24,25 Both Ca2‡ release from the SR to initiate
contraction and the reuptake of Ca2‡ into the SR
during relaxation are re¯ective of both TTP and
1
2RT, respectively, and thus appear to be impaired in
the Ob=Ob groups compared with the CON
groups.24,25 However, with no measure of the
force ± velocity relationship in these muscles it is
impossible to comment on whether the differences
in contraction and relaxation times are totally due to a
International Journal of Obesity
reduced SR function or are a result of variations in
cross bridge kinetics, myosin ATPase activity, or in
the light or heavy chains of myosin between the obese
and lean animals.26
Fatigue protocol
Fatigue resistance in the Ob=Ob groups was greater
than in the CON groups in both the EDL and soleus
muscles (Figure 3). This most likely re¯ects a difference in the metabolism of the skeletal muscle from the
two strains where by skeletal muscle from the Ob=Ob
groups appears to operate a more oxidative metabolism, compared to the CON groups that appear to
utilise a more glycolytic metabolism.27 Again this is
supported by the ®bre type proportions of the two
muscle types (Figure 4). In the EDL, the Ob=Ob
groups have a lower proportion of glycolytic type
IIB muscle ®bres in conjunction with a greater proportion of the more oxidative type IIA muscle ®bres
when compared with the CON groups. Similarly in the
soleus, the Ob=Ob groups have an overall lower
proportion of glycolytic type IIB ®bres compared to
the CON groups. Thus, the metabolism of both the
EDL and soleus muscles from the Ob=Ob groups
appears less glycolytic and thus, less fatigable. However, it is interesting to note that the CON groups
appear to have a faster rate of recovery from fatigue
compared with the Ob=Ob groups. In this stimulation
protocol, metabolic factors are responsible for
the induced fatigue rather than other conditions such
as impaired excitation contraction coupling, with
prolonged (days) reductions in force seen during
low-frequency fatigue, or alterations in t-system conduction as seen during high-frequency fatigue.28 Thus,
given that the CON groups reach a similar level of
force production after a 5 min rest period from a lower
percentage of P0 at the end of the fatiguing stimulations, this may be due to a faster rate of clearance of
metabolites during recovery in the CON compared to
Ob=Ob groups.
Histochemistry
In analysing the ®bre proportions present in the EDL
and soleus muscles it appears that the slower ®bre
pro®le in the Ob=Ob groups results in some degree of
compensatory hypertrophy of the faster ®bre types
present, evidenced by an increase in the type IIB ®bre
areas in the soleus and type IIA ®bre areas in the EDL
(Figure 5). However, this does not seem to be able to
offset the changes observed in the isometric contractile properties and certainly is not enough to in¯uence
the total mass of the skeletal muscle present. These
®ndings are in agreement with the previous data of
Almond and Enser,5 who also found a reduced proportion of type IIB ®bres in the biceps Brachii of
ob=ob mice, although in contrast no hypertrophy was
reported in this previous study. In fact, the study by
Almond and Enser,5 and another study by Stickland
Skeletal muscle characteristics in ob/ob mice
SA Warmington et al
et al,9 both found a decrease in the individual ®bre
areas of skeletal muscle from ob=ob mice which we
also observe in the type IIB and type I ®bres of the
EDL. Therefore, the reason suggested for the decrease
in muscle mass in these mice was a result of muscle
atrophy not a reduction in the total number of muscle
®bres present, and based on these cases and the
present study, skeletal muscle from obese mice
appears to lack the ability to hypertrophy.
Force frequency
The force recorded following stimulation at various
sub-optimal frequencies in the Ob=Ob groups is signi®cantly elevated compared with the CON groups for
both the EDL and soleus (Figure 2). This suggests
there is an imbalance in the SR Ca2‡ handling ability
in the obese skeletal muscle with the rate of SR Ca2‡
release being less reduced than the rate of reuptake of
Ca2‡ by the SR.29 This is supported by the results
observed for TTP and 12RT in both the EDL and soleus
muscles (Table 2). Particularly in soleus, there is a
66% prolongation of 12RT (reduction in reuptake of
Ca2‡) compared with a 35% prolongation in TTP
(reduction in release of Ca2‡), while in the EDL
they appear similar (25% prolongation for both TTP
and 12RT). A greater difference in the uptake of Ca2‡
by the SR (more reduced 12RT) than the release of
Ca2‡ by the SR (less reduced TTP) will result in a
greater force production as a percentage of P0 at suboptimal stimulation frequencies, than if the two were
balanced. Thus, in the soleus force ± frequency curve
there are differences observed between the Ob=Ob
and CON groups at more of the sub-optimal stimulation frequencies than in the EDL, with no difference
in the maximal tetanic force at optimal and supraoptimal stimulation frequencies. These changes are
again likely to be attributed to the altered ®bre type
pro®le of these two groups with the Ob=Ob being
somewhat slower than the CON groups.
However, the difference in the force frequency
curves may only partially be explained by differences
in the ability of the SR to cycle Ca2‡ in these muscles.
In conjunction with the greater Pt=P0 in the Ob=Ob
compared with CON groups (Table 2), this suggests
that in the obese animals there is a reduced elastic
component present in both the EDL and soleus
muscles compared with the lean mice.30,31 This
would also provide an avenue for greater force production at sub-optimal stimulation frequencies in the
Ob=Ob compared with the CON mice because during
force production, elastic components must be overcome before any measurable force output or muscle
shortening can occur. Thus, in the Ob=Ob groups
a reduced elastic component suggests a greater contribution of the tension generated during contraction
to muscle shortening or force production at suboptimal stimulation frequencies, and hence, a greater
Pt as a percentage of P0 when compared with the
CON groups. This decrease in elasticity of these obese
muscles is also supported by the reduced muscle
length (L0) observed in the Ob=Ob groups compared
with the CON groups (Table 1).
1049
Conclusion
Overall, the two skeletal muscles (EDL and soleus)
assessed in this study from genetically obese (ob=ob)
mice appear morphologically and functionally different from the same skeletal muscles obtained from lean
mice. It appears that altered ®bre proportions in these
obese muscles are responsible for the changes in
contraction and relaxation times observed, as well as
fatigability and, most likely, these ®bre type differences arise from the inactivity associated with the
present obesity. It is important to note that despite the
decreased absolute force production (data not shown)
as a result of the reduced muscle mass in these obese
mice, there was no alteration in normalized force
production with obesity. Interestingly, it also appears
that the skeletal muscle from ob=ob mice displays a
number of characteristics similar to that of aged
skeletal muscle. Brooks and Faulkner16 found that
in aged mouse skeletal muscle there was a similar
reduction in skeletal muscle mass, a prolongation of
TTP and 12RT, and an elevation in Pt=P0 with a left
shifted force frequency relationship when compared
to normal adult mouse skeletal muscle.
Leptin treatment in the obese mice used in this
study signi®cantly reduced body mass towards the end
of the treatment period, however, despite these effects
there was no evidence that leptin altered any functional or morphological characteristics associated with
the skeletal muscles investigated. There was an apparent move toward a more fast ®bre type pro®le in the
muscles from these animals when treated with leptin,
however, further investigation of leptin's effects on
skeletal muscle is warranted.
Acknowledgements
Dr Fred Andrews from the Department of Physiology,
Trinity College, supplied the recombinant murine
leptin used in this study. Professor Chris Bell provided
®nancial support through allocations from the Department of Physiology, Trinity College, small research
project fund.
References
1 Zhang Y, Proenca R, Maffei M, Barone M, Leopold L,
Friedman JM. Positional cloning of the mouse obese gene
and its human homologue. Nature 1994; 372: 425 ± 432.
2 Bray GA, York DA. Hypothalmic and genetic obesity in
experimental animals: an autocrine and endocrine hypothesis.
Physiol Rev 1979; 59: 719 ± 809.
3 JeÂquier E, Tappy L. Regulation of body weight in humans.
Physiol Rev 1999; 79: 451 ± 480.
4 Almond RE, Enser M. Effects of adrenalectomy on muscle
®bre growth and ®bre-type composition in obese-hyperglycaemic (ob=ob) and lean mice. Int J Obes 1989; 13: 791 ± 800.
International Journal of Obesity
Skeletal muscle characteristics in ob/ob mice
SA Warmington et al
1050
5 Almond RE, Enser M. A histochemical and morphological
study of skeletal muscle from obese hyperglycaemic ob=ob
mice. Diabetologia 1984; 27: 407 ± 413.
6 Walker HC, Romos DR. Effects of cimaterol, a beta-adrenergic agonist, on energy metabolism in ob=ob mice. Am J
Physiol 1988; 255: R952 ± R960.
7 Smith CK, Romsos DR. Cold acclimation of obese (ob=ob)
mice: effects on skeletal muscle and bone. Metabolism 1984;
33: 858 ± 863.
8 Shapira JF, Kircher I, Martin RJ. Indicies of skeletal muscle
growth in lean and obese Zucker rats. J Nutr 1980; 110:
1313 ± 1318.
9 Stickland NC, Batt RA, Crook AR, Sutton CM. Inability of
muscles in the obese mouse (ob=ob) to respond to changes in
body weight and activity. J Anat 1994; 184: 527 ± 533.
10 Goldberg AL, Tischler M, De Martino G, Grif®n G. Hormonal
regulation of protein degredation and synthesis in skeletal
muscle. Fed Proc 1980; 39: 31 ± 36.
11 Stewart DM. The role of tension in muscle growth. In: Gross
RJ (ed). Regulation of organ and tissue growth. Academic
Press: New York, 1972, pp 77 ± 100.
12 Douketis JD, Feightner JW, Attia J, Feldman WF. Periodic
health examination, 1999 update: 1. Detection, prevention and
treatment of obesity. Can Med Assa J 1999; 160: 513 ± 525.
13 Carek PJ, Dickerson LM. Current concepts in the pharmacological management of obesity. Drugs 1999; 57: 883 ± 904.
14 Stunkard AJ. Current views on obesity. Am J Med 1996; 100:
230 ± 236.
15 Hayes A, Lynch GS, Williams DA. The effects of endurance
exercise on dystrophic mdx mice. 1. Contractile and histochemical properties of intact muscles. Proc R Soc Lond [Biol]
1993; 253: 19 ± 25.
16 Brooks SV, Faulkner JA. Contractile properties of skeletal
muscles from young, adult and aged mice. J Physiol 1988;
404: 71 ± 82.
17 Pearse AGE (ed). Histochemistry: theoretical and applied, 3rd
edn, Vol. 1. Churchill: London, 1968.
18 Pullen AH. The distribution and relative size of ®bre types in
the extensor digitorum longus and soleus muscles of the adult
rat. J Anat 1977; 123: 467 ± 486.
19 Doriguzzi C, Mongini T, Palmucci L, Schiffer D. A new
method for myo®brillar Ca‡‡-ATPase reaction based on the
use of metachromatic dyes: its advantages in muscle ®bre
typing. Histochemistry 1983; 79: 289 ± 294.
International Journal of Obesity
20 Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT,
Rabinowitz D, Lalone RL, Burley SK, Friedman JM. Weight
reducing effects of the plasma protein encoded by the obese
gene. Science 1995; 269: 543 ± 546.
21 Pellymounter MA, Cullen MJ, Baker MB, Hecht R, Winters
D, Boone T, Collins F. Effects of the obese gene product on
body weight regulation in ob=ob mice. Science 1995; 269:
540 ± 543.
22 Rentsch J, Levens N, Chiesi M. Recombinant ob-gene product
reduces food intake in fasted mice. Biochem Biophys Res
Commun 1995; 214: 131 ± 136.
23 Houseknecht KL, Baile CA, Matteri RL, Spurlock ME.
The biology of leptin: a review. J Animal Sci 1998; 76:
1405 ± 1420.
24 Baker AJ, Longuemare MC, Brandes R, Weiner MW. Intracellular tetanic calcium signals are reduced in fatigue of whole
skeletal muscle. Am J Physiol 1993; 264: C577 ± C582.
25 Westerblad H, Allen DG. The role of sarcoplasmic reticulum
in relaxation of mouse muscle; effects of 2,5-di(tert-butyl)-1,4benzohydroquinone. J Physiol 1994; 474: 291 ± 301.
26 Westerblad H, LaÈnnergren J, Allen DG. Slowed relaxation in
fatigued skeletal muscle ®bres of Xenopus and mouse. Contribution of [Ca2‡]I and cross-bridges. J Gen Physiol 1997;
109: 385 ± 399.
27 Larsson L, Ansved T, Edstrom L, Gorza L, Schiaf®no S.
Effects of age on physiological, immunohistochemical and
biochemical properties of fast-twitch single motor units in the
rat. J Physiol 1991; 443: 257 ± 275.
28 Jones DA. High- and low-frequency fatigue revisited. Acta
Physiol Scand 1996; 156: 265 ± 270.
29 Kossler F, Kuchler G. Contractile properties of fast and slow
twitch muscles of the rat at temperatures between 6 and 42
degrees C. Biomed Biochim Acta 1987; 46: 815 ± 822.
30 Edman KA. The velocity of unloaded shortening and its
relation to sarcomere length and isometric force in vertebrate
muscle ®bres. J Physiol 1979; 291: 143 ± 159.
31 Walker SM. Lengthening contraction and interpretations of
active state tension in the isometric twitch response of skeletal
muscle. Am J Phys Med 1976; 55: 192 ± 204.