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Skeletal muscle in the diabetic mouseHistochemical and morphometric analysis.

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THE ANATOMICAL RECORD 225:41-45 (1989)
Skeletal Muscle in the Diabetic Mouse:
Histochemical and Morphometric Analysis
Medical Sciences Program (K.M.K.,J.D.F.,J.B.W.), Indiana University School of
Medicine, and School of Health, Physical Education, and Recreation (G.S.), Indiana
University, Bloomington, Indiana 47405
Despite the extensive literature concerning the neuropathy associated with diabetes, only limited information describes changes in the associated
muscle. The objective of this study was to evaluate the histochemical and morphometric characteristics of diabetic muscle in the C57BL/KsJ db-m strain of mouse.
The histochemical analysis of myofiber type for the diabetic mouse revealed that
the extensor digitorum longus muscle consisted of 53.1% type 2a, 46.0% type 2b,
and 0.9% type 1 myofibers, a significant shift from the percentages found in the
nondiabetic litter mates (44.4% type 2a, 55.6% type 2b, no type 1).Computerassisted morphometric analysis of myofiber size by fiber type indicated a significant difference in myofiber size for the type 2b fibers in muscles from diabetic mice.
Similarly, there was a shift in the fiber size distribution to include a greater
number of small type 2b myofibers when compared to controls. Skeletal muscle
from diabetic mice exhibited a significant change in the percentage of fiber types,
with an increase in the number of type 2a fibers, a fiber type grouping that implies
possible denervation and reinnervation, and a decrease in myofiber size. These
findings may explain why some diabetic patients complain of muscle weakness.
Striated muscle requires insulin for normal growth
and development (Cheek and Graystone, 1970; Trenkle, 1974; Young, 1974; Allen et al., 1979)) and muscle
wasting has been described in laboratory animals with
uncontrolled insulin-dependent diabetes (Olson and
Smith, 1981) and in patients with chronic disease conditions (Adams, 1975). Peripheral nerve damage is frequently associated with diabetes and has been recognized clinically for years (Woltman and Wilder, 1929;
Jordon, 1936; Rudles, 1945; Dyck et al., 1987).Animals
with experimentally induced diabetes have a wide variety of morphologic (Powell et al., 1977; Moore et al.,
1981; Mandlebaum et al., 1983; Westfall et al., 1983)
and metabolic alterations in peripheral nerves
(Baughman et al., 1981). In fact, the peripheral neuropathy described in the homozygote (db/db) mouse obtained from matings of the C57BWKsJ-db-m strain of
mouse, a genetic model of diabetes, is thought to be an
axonopathy resulting from a metabolic change in the
axon (Sima and Robertson, 1978; Robertson and Sima,
1980; Sharma et al., 1983). In addition, muscle often
exhibits depressed physiologic function that can be related to the histochemical type of myofiber, i.e., there is
greater depression of function in the fast twitch fibers
in muscles from diabetic animals (Paulus and Grossie,
Muscle and nerve are dependent on each other for
normal growth and development, and alteration of one
of these two tissues can result in pronounced changes
in the structure andlor function of the other (as reviewed by Grinnell, 1986). The diabetic state has been
shown to affect both of these tissues a t some time in the
0 1989 ALAN
pathogenesis of the disease. The C57BL/KsJ-db/db
mouse has been extensively studied because it displays
peripheral nerve abnormalities such as demyelination
and physiological alterations of nerve conduction
(Sima and Robertson, 1978; Robertson and Sima, 1980;
Sharma et al., 1983) that are similar to those observed
in animals with diabetes mellitus (Herberg and Coleman, 1977). However, little attention has been given to
the morphologic alterations in the muscle associated
with nerves exhibiting a peripheral neuropathy. The
muscle weakness seen in some diabetic patients may be
due to changes in glucose metabolism, to degenerative
changes within the peripheral nerve, or to denervationinduced changes within the muscle. Since there is a
diabetic neuropathy, there can be changes in the integrity of the muscle. A healthy nerve is necessary for the
determination of myofiber size and fiber type distribution (Grinnell, 1986). In myopathies where partial denervation occurs, myofibers can be reinnervated by
healthy neurons within the muscle. When this occurs,
the muscle loses its normal checkerboard arrangement
of fiber types and fiber type grouping occurs. The degree of fiber type grouping depends on the extent of
denervation-reinnervation of the muscle. Similarly,
Received January 14, 1988; accepted November 22,1988
Address reprint requests to Dr. Kathleen M. Klueber, Department
of Anatomical Sciences and Neurobiology, University of Louisville,
School of Medicine, Louisville, KY 40292.
denervation of a myofiber results in atrophy and a de- TABLE 1. Histochemical fiber type profile for diabetic
and control EDL muscles
crease in fiber size (Carpenter and Karpati, 1984).
Since the strain of mouse used in this study has a dock e 2a
Tvpe 2b
TVpe 1
umented neuropathy, a histochemical analysis of the Grour,
0.9% ? 0.4
46.0% * 0.9*
53.1% * 0.7*
muscle will give preliminary insights into the neuro- dbldbl
44.4% & 1.5
55.6% * 1.8
muscular relationships of the muscle and nerve. Thus, dbl+
the aim of this study was to evaluate the neuromuscu- ‘N = 8; 250 myofiberdmuscle.
lar and morphologic integrity of a diabetic muscle *P < .05; values expressed as mean t SEM.
through histochemical and morphometric analysis of
the fiber type profile.
dye (toluidine blue). Following this procedure, the type
1 myofibers stain turquoise and appear white in black
and white photos whereas the type 2a stain deep blue
The C57BLIKsJ-db-m strain of mouse used in this and photograph dark grey and the type 2b stain light
study was obtained from stock colonies at Jackson Lab- blue-violet and photograph light gray in black and
oratories (Bar Harbor, ME) and maintained as a breed- white photos. Intensity and color of the fiber type stain
ing colony within the animal care facilities in a 12 hr is the result of the phosphate content within the myolightldark cycle. This strain of mouse was chosen be- fiber after incubation in a basic (pH 9.4) medium. Because it is a genetic model of diabetes, with character- cause this procedure was new, comparison of serial secistics similar to type I1 diabetes. By using this model of tions reacted by standard myosin ATPase procedures
the disease, any possible myo- or neurotoxic effects of (Guth and Samaha, 1970) with those stained by the
the chemicals used to induce diabetes in other strains technique of Doriguzzi et al. (1983) was completed to
would be eliminated. Thus, only those changes in the confirm the fiber types. The percentage of fiber types
muscle and nerve resulting from a reduced insulin was determined from counts of all myofibers within the
environment would be noted. The homozygous cross section.
C57BLIKsJ diabetic mouse (dbldb; N = 8, age 100
Morphometric Analysis
days) is recognized by 3-4 weeks of age by the onset of
measured for each fiber type (N =
obesity (Herberg and Coleman, 1977), hindleg flexion,
photomicrographs ( x 450) of the
hyperglycemia and matted hair (Hanker et al., 1980).
Heterozygous nondiabetic carrier litter mates (db/+ ;N ATPase-treated sections on a Hipad tablet connected to
an Apple IIe computer programmed with Bioquant I1
= 8, age 100 days) served as controls and were selected
by their smaller size, lower blood glucose concentra- (R&M Biometrics, Nashville, TN) morphometric analtion, and smooth black hair. Animals used in this study ysis package. This software generated graphic reprewere housed and treated according to the NIB guide- sentation of the data and performed statistical analysis
of the data, including mean and standard error of the
lines for animal care.
The extensor digitorum longus (EDL) muscle was mean. Differences between the data from the diabetic
chosen for study for a number of reasons. The muscle is and nondiabetic carrier muscles were analyzed by a
nearly 100% fast in fiber type composition and fast Student’s t-test, which is part of the Bioquant software
muscles respond differently than slow muscles to dis- package. The level of significance was set at P < .05.
ease and trauma. Slow muscles are more adaptable to
change. Data in the lab from other studies of both conThe
were significantly
trol and other myopathies are available for comparison
with data from the diabetic muscle. Finally, the small higher for the dbldb mice (677 mgldl 53 SEM vs. 150
size of the EDL in the mouse lends itself well to a mgldl 9 SEM for dbl + ). The average body weight for
variety of other techniques, such as serial section elec- the db/db and the dbl + animals was 40.7 g 2.7 SEM
tron microscopy, that will enable correlation of histo- and 22.8 g 0.8 SEM, respectively. Although the diabetic animals were significantly heavier, the EDL muschemical data with morphological data.
cle from db/db mice was significantly smaller (4.5 mg 2
0.2 SEMI than that from db/+ mice (6.4 mg 2 0.2
Blood Glucose Determination
Blood samples (250 p1) were obtained a t the time of
Fiber type percentages for muscle from dbldb mice
sacrifice and blood glucose levels were determined with was significantly different from the percentages of fia YSI model 27 blood glucose analyzer (Yellow Springs ber types for the dbl + muscles (Table 1). Comparison of
Instruments, OH). Blood sugar concentrations above a normal histochemical profile for the EDL (Fig. l a )
300 mgldl were considered pathologic.
with that of the dbldb EDL (Fig. lb) indicates fiber type
rather than the normal “checkerboard” arHistochemical Analysis
The EDL muscle was removed from mice anestheAnalysis of myofiber size by fiber type (Table 2) retized with methoxyflurane (Metofane: Pitman-Moore, vealed a decrease in average fiber area for type 2a fiWashington Crossing, NJ) and weighed. The tissue bers of diabetic mouse (dbldb); however, this was not a
samples were frozen in isopentane (cooled over liquid significant shift in average area. Analysis of the fiber
nitrogen) and sectioned on a cryostat a t 30 pm.
size distributions (Fig. 2a) for the type 2a myofibers of
The frozen serial sections were reacted according to both groups indicated that dbldb myofibers have
the ATPase technique of Doriguzzi et al. (1983) for my- greater variability in size distribution than those from
ofibrillar ATPase. This technique uses metachromatic the control group but are similar in distribution. There
Fig. 1. a: Photomicrographof Ca + +-myosin ATPase histochemical
reaction of control (db/+) myofibers using the metachromatic stain
technique (Doriguzziet al., 1983). Fiber types are indicated as type 2a
and 2b. Note the lack oftype 1 fibers and the “checkerboard”arrangement of the myofibers. b: Photomicrograph of Ca+ +-myosin ATPase
histochemical reaction of diabetic (db/db)myofibers using the metachromatic stain technique (Doriguzzi et al., 1983). Fiber types are indicated as type 1, 2a, and 2b. Note slight fiber type groupings (arrowheads).
TABLE 2. Mean fiber area @m2)of individual fiber
t n e s for diabetic and control EDL muscles
the diabetic-like syndrome is indistinguishable between the two strains. Both are characterized by many
abnormalities, including hyperphagia, fasting hyperglycemia, and hyperinsulinemia (Herberg and Coleman, 1977; Almond and Enser, 1984). The differences
between the two strains are life span and changes in
the degree of hyperinsulinemia (Sprott, 1972; Herberg
and Coleman, 1977). At 4 months of age, the BL/Ks
db-m strain stops gaining weight, has a decrease in
insulin levels, and dies before 10 months of age. In
contrast, the ob/ob mice are hyperinsulinemic throughout life, continually gain weight, and have a 18-20
month life span (Sprott, 1972; Herberg and Coleman,
1977). The small muscle in the ob/ob muscle is thought
to be a result of retarded myofiber development early
in life. However, as the animal matures, there is no
catch-up growth for the affected myofibers, thus indicating a metabolic disturbance within the muscle (Almond and Enser, 1984). Whether this pattern is true of
the db/db muscle remains to be determined.
Concomitant with the small size of the diabetic EDL
are decreases in the size of the myofibers that comprise
the muscle. Only a slight change in the size of type 2a
fibers was seen in this study. Similar results were described in chemically induced diabetic muscle (Armstrong et al., 1975; Ianuzzo and Armstrong, 1976; Armstrong and Ianuzzo, 1977; Hegarty and Rosholt, 1981)
as well as the ob/ob muscle (Almond and Enser, 1984).
Type 2a
644 -+ 11
689 12
Type 2b
1206 15*
1759 t 23
Type 1
* 31
8; 225 fiberdmuscle for all fiber groups.
*P < .05; values expressed as mean ? SEM.
is also a significant decrease in size of the type 2b fibers
from diabetic mice (db/db) (Table 21, with a shift of the
size distribution toward smaller fibers for the diabetic
(db/db) type 2b myofibers (Fig. 2b).
Previous studies have examined diabetic muscle
from animals with chemically induced diabetes (Armstrong et al., 1975; Ianuzzo and Armstrong, 1976; Armstrong and Ianuzzo, 1977), but little information has
been reported on changes in muscle from animals with
the genetically induced disease. The significant increase in body weight is characteristic of the db/db
mouse (Herberg and Coleman, 1977), but the decrease
in muscle weight observed in this study is more similar
to that observed in the ob/ob mouse (Almond and
Enser, 1984). The diabetic mouse (BL/Ks db-m) is phenotypically similar to the obese mouse (BL/G-ob/ob)and
2A Fibers
AREA IN pm2 (BW = 100 pm2)
2B Fibers
AREA IN pm2 (BW =100pm2)
Fig. 2. a: Normalized histograms of type 2a myofiber size distributions for the db/db and db/+ muscles. Bin width = 100 pm2. Note
that the size distributions for type 2a fibers are comparable for both
groups. b: Normalized histogram of type 2b myofiber size distribution
for the db/db and db/ + muscles. Bin width = 100 pm2. Note the shift
toward smaller-sized myofibers in the dbidb muscles, indicating atrophy of the type 2b fibers in the diabetic state.
Type 2a fibers seem to be more adaptable than type 2b
fibers and are resistant t o changes in their environment, as shown by their regeneration to normal size
after transplantation (Thomas et al., 1984).
However, type 2b fibers are more susceptible than
type 2a fibers to changes in their environment. Studies
of myofiber size by fiber type in diabetic muscle indicate that type 2b fibers show a significant decrease in
size that becomes more pronounced with time (Armstrong et al., 1975; Ianuzzo and Armstrong, 1976; Armstrong and Ianuzzo, 1977; Niederle and Mayr, 1978;
Almond and Enser, 1984). The type 2b fiber is apparently incapable of regenerating to normal size after
transplantation (Thomas et al., 1984). Type 2b fibers
undergo atrophy after denervation at a faster rate than
type 2a fibers and show pronounced atrophy after 120
days (Niederle and Mayr, 1978). The reason type 2b
fibers show such great changes after trauma or disease
may be that the myofibers are more dependent than
type 2a fibers on neurotrophic influences (Engel and
Karpati, 1968). Why this differential response of the
two fast twitch fibers occurs in response to disease or
trauma remains to be determined. This shift in fiber
size for the type 2b fibers could result in diminished
physiologic function of the muscle, since the power produced by a muscle is related to the size of the myofibers
that compose it.
The alteration of myofiber pattern toward clustering
of similar fiber types is a sign of denervation followed
by reinnervation (Peyronnard and Charron, 1980). The
shift in percentages of myofiber types may indicate a
shift toward a more oxidative metabolism (Armstrong
et al., 1975; Ianuzzo and Armstrong, 1976) and a slowing of the muscle as a result of the presence of type 1
fibers (Rutschmann et al., 1984). Muscle from diabetic
(db/db) animals exhibits a prolonged contraction time
and a shift in oxidative capacity (Klueber, unpublished
data). Further analysis of the contractile properties
and the metabolic shifts of diabetic muscle are ongoing.
In a morphologic study of the db/db muscle, some motor
endplates have been shown to be in various states of
degeneration, effectively denervating the myofiber
(Feczko and Klueber, 1988). Since normal axons have
been shown to sprout and reinnervate denervated myofibers (Peyronnard and Charron, 1980), this may account for the slight fiber type grouping noted in the
db/db muscle. Similar shifts in fiber type percentages
have been noted in muscle from animals with chemically induced diabetes (Armstrong et al., 1975) and in
the ob/ob muscle (Almond and Enser, 1984).
Postulated reasons for this shift in percentages of
fiber types include a dedifferentiation of the myofibers
(Romanul and Hogan, 19651, as evidenced by changes
in isomyosins (Rutschmann et al., 1984) or a selective
neuropathy of motor neurons specific to fast twitch
muscle fibers (Armstrong et al., 1975). The latter may
be pertinent to this study because there is a neuropathy associated with this model of diabetes (Sima and
Robertson, 1978; Sharma et al., 1983). In addition,
there are signs of denervation within the EDL muscle
of the C57BL/KsJ db-m mouse, as observed by Feczko
and Klueber (1988).The changes in fiber type arrangements noted in this study suggest denervation followed
by reinnervation. Because the fiber type of a myofiber
is determined in part by its innervation (Engel and
Karpati, 1968; as reviewed by Grinnell, 1986; as reviewed by Gauthier, 19861, changes within the neuron
supplying the myofiber will result in changes within
the myofiber. In the diabetic mouse, the partial denervation may lead to nerve sprouting and reorganization
of the myofiber types, as described by Peyronnard and
Charron (1980). The increase in type 1myofibers over
that seen in the controls in this study may be the result
of nerve sprouting and reinnveration from surrounding
muscles. The changes noted in the histochemical analysis indicate a need t o examine both the myosin
isozyme patterns as well as the neuromuscular relationships of the diabetic mouse.
The decrease in myofiber size in diabetic muscle may
impair its physiologic function, because muscle
strength is dependent on both size and number of myofibers that comprise the muscle. How these morphologic changes affect the function of diabetic muscle remains to be determined. These morphologic alterations
in the diabetic muscle could account a t least partially
for the muscle weakness that is often observed in
poorly controlled diabetic patients.
J.D.F. was a summer research fellow supported by
the Indiana Affiliate of the American Diabetes Association. This research was supported in part by grants
from the American Medical Association Education and
Research Foundation and the American Diabetes Association. The competent secretarial assistance of Ms.
Deborah Pryor, Ms. Beverly Hankins, Ms. Rita Crouch,
and Ms. Diane Richardson is gratefully acknowledged.
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skeletal, muscle, morphometric, mousehistochemical, analysis, diabetic
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