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1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA;
Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229 USA;
Department of Clinical Physiology, Karolinska Hospital, S-171 76, Stockholm, Sweden; and
Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, MB R3E 0W3, Canada
1Correspondence: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA. E-mail: charron{at}aecom.yu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: free fatty acids • EDL • glycogen synthesis • oleate oxidation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
and rodents
(4)
. Glucose transport is the rate-limiting step for
skeletal muscle glucose utilization in certain, but not all,
physiological states (5
6
7
8
9)
. The predominant glucose
transporter isoform expressed in skeletal muscle is glucose transporter
4 (GLUT4) (10
11
12
13
14)
. Under basal conditions, GLUT4 resides
in intracellular vesicles and is translocated to the plasma membrane
upon stimulation by insulin (15
16
17)
. GLUT4 is important
in both the regulation of glucose uptake in skeletal muscle and the
maintenance of whole body glucose homeostasis (18
19
20
21
22
23
24
25
26
27)
.
A primary lesion in type II diabetes mellitus is impaired
insulin-stimulated glucose uptake in skeletal muscle (28)
.
Numerous groups using transgenic mice have demonstrated that whole body
glucose utilization can be increased by overexpression of GLUT4 either
selectively in skeletal muscle (19)
or in tissues where
GLUT4 is normally expressed (skeletal muscle, heart, and adipose
tissue) (22
23
24)
. Thus, strategies aimed to increase
skeletal muscle GLUT4 expression have been proposed for the treatment
of type II diabetes mellitus (19
, 21
22
23
24
25
, 29)
.
Consequently, understanding the long-term metabolic and functional
consequences of increased GLUT4 content in skeletal muscle is important
in order to establish whether these strategies may be applied to humans
with type II diabetes. Increasing glucose transport by overexpressing
glucose transporters provides an interesting and unique metabolic
scenario. How does a cell respond metabolically to
increased glucose influx? It is unknown whether increased
glucose transport will result in increased glycolysis and/or glycogen
synthesis. Furthermore, it is unclear whether increased glucose influx
may affect utilization of free fatty acids (FFA), the other major fuel
substrate for skeletal muscle (30)
.
In addition to effects of GLUT4 overexpression on muscle substrate
metabolism, we revisited the question whether long-term changes in
insulin action and substrate utilization will affect an animal’s body
weight. This question is highly relevant since previous studies have
associated degree of insulin sensitivity with weight gain (31
, 32)
. It is necessary to investigate whether increased muscle
GLUT4 expression, as a possible therapeutic strategy for type II
diabetes, may result in undesirable weight gain. Several groups,
including us, have previously reported no apparent weight changes in
transgenic mice overexpressing GLUT4 in skeletal muscle (19
, 24
, 29
, 33
, 34)
. However, physical activity of these mice, which can
account for a large portion of daily energy expenditure, may have been
discouraged by the limited confines of the housing cages. As a result,
effects of insulin action or altered substrate utilization on weight
maintenance may have been masked in these previous studies.
These issues were studied in transgenic mice overexpressing GLUT4
selectively in fast-twitch skeletal muscle (MLC-GLUT4 mice)
(19)
. Promoter and enhancer elements from myosin light
chain 1/3 locus were used to drive targeted overexpression (2.5-fold)
of GLUT4 in muscles containing high proportions of fast-twitch fibers.
Glucose uptake was increased in MLC-GLUT4 muscles rich in fast-twitch
fibers, but not in muscles rich in slow-twitch fibers
(19)
. Since fast-twitch fibers comprise the predominant
fiber type of skeletal muscle (35
, 36)
, MLC-GLUT4 mice
also exhibit improved whole body glucose homeostasis and patterns of
altered lipid metabolism (19)
. With GLUT4 overexpression,
glucose transport should become less rate limiting, thereby shifting
the control of glucose utilization to downstream regulatory steps.
Thus, MLC-GLUT4 mice provide an excellent model to study the long-term
metabolic and functional consequences of increased GLUT4 content in
skeletal muscle. Here we report that increased glucose influx after
GLUT4 overexpression is preferentially targeted to glycolysis in male
MLC-GLUT4 extensor digitorum longus (EDL) muscle. In contrast,
increased glycogen synthesis was observed instead in female MLC-GLUT4
EDL. The long-term increase in glucose metabolism led to decreased
oxidation of FFA in transgenic EDL. Furthermore, decreased skeletal
muscle FFA oxidation was associated with increased liver, but not whole
body, lipid content. Last, the increased skeletal muscle GLUT4
expression was associated with elevated voluntary wheel exercise that
led to decreased body weight and increased food consumption.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Animals were fed ad
libitum and maintained in a murine hepatitis virus-free barrier
facility on a 12-h light and dark cycle. All protocols have been
approved by the Animal Care and Use Committee of the Albert Einstein
College of Medicine in accordance with the Public Health Service Animal
Welfare Policy.
Muscle incubation procedure for glucose metabolism assessment
Mice between 10 and 14 wk old were used in this study. After
cervical dislocation, soleus and EDL muscles were rapidly isolated from
hind limbs. The distal tendon of each muscle was tied to a piece of
suture to facilitate transfer among different media. Incubation was
carried out at 30°C in Krebs-Henseleit bicarbonate buffer
supplemented with 5 mM HEPES, pH 7.4, 0.1% bovine serum albumin
(fraction V, RIA grade, Sigma, St. Louis, Mo.), and 5 mM glucose. All
media were oxygenated (95% O2; 5%
CO2) before incubation and kept oxygenated as
indicated below with constant circulation. The gas mixture was hydrated
throughout the experiment by bubbling through a gas washer (Kontes
Inc., Vineland, N.J.). Isolated soleus and EDL muscles were first
rinsed for 15 min in 1 ml of oxygenated incubation media. The muscles
were then transferred to a second set of incubation media (1.5 ml)
containing 0.1 mCi/ml U-14C-glucose and 1 mCi/ml
5-3H-glucose (Amersham, Arlington Heights, Ill.)
and incubated with or without 20 nM porcine insulin (Eli Lilly,
Indianapolis, Ind.) for 45 min. Four blanks with no muscle samples were
incubated and processed exactly the same as the muscle samples. Vials
were sealed with a screw top lid containing a rubber septum, from which
a center well containing a piece of Whatman paper was suspended. After
oxygen circulation was stopped at the end of the 45 min period, muscles
were incubated under closed conditions for an additional hour.
Immediately, 200 µl of Solvable (Packard Instruments, Meriden, Conn.)
was injected onto the Whatman paper. Muscle vials were placed in an ice
water bath for 3 min. Muscles and 500 µl of incubation media were
removed and vials were quickly resealed.
Glycolysis, glycogen synthesis, and glucose oxidation in isolated
muscles
Glycolysis was assessed by the formation of
3H2O from
5-[3H]-glucose supplemented in the media as
described previously (37)
with minor modifications. Anion
exchange columns were prepared by packing 1 ml pipette tips first with
washed glass beads and then with Dowex 1 x 2 anion exchange resin
(Bio-Rad. Lab., Richmond, Calif.) (38)
. The rate of
glycolysis was expressed as nmol glucose/g wet muscle weight/h.
Glycogen synthesis was determined by accumulation of either
3H or 14C in glycogen. For
glycogen synthesis, muscles were removed from the incubation media and
dissolved in 1 N NaOH at 55°C. Glycogen purification from isolated
muscles using Whatman papers and glycogen breakdown has been described
previously (39)
. Glycogen synthesis was expressed as nmol
glucose/g wet muscle weight/h. Calculations using either
3H or 14C liquid
scintillation counting yielded similar results. Glucose oxidation was
determined by the release of
14CO2 from the media as
described previously (40)
and expressed as nmol glucose/g
wet muscle weight/h. ScintiSafe 30% (Fisher Scientific Inc.,
Pittsburgh, Pa.) was used in all liquid scintillation counting
procedures.
Glycogen content in isolated muscles
To determine glycogen content in muscles, glycogen precipitated
in Whatman papers was eluted in 0.2 M sodium acetate and hydrolyzed
with 0.1 mg/ml amyloglucosidase (Boehringer Mannheim, Indianapolis,
Ind.) as described previously (39
, 41)
. Amount of released
glucose was determined using Trinder glucose oxidase reagent (Sigma).
Glycogen content was expressed as µg glucose/g wet muscle weight.
Oleate oxidation in isolated skeletal muscle
To measure oleate oxidation, muscles from 10- to 14-wk-old mice
were incubated in the media as described for glucose metabolism
experiments, with a further addition of 0.5 mM oleate and 4% bovine
serum albumin (Sigma). After the 15 min rinse period, the muscles were
transferred to another set of media and incubated at 30°C in the
presence of 0.8 µCi/ml [1-14C] oleic acid (60
mCi/mmol, Amersham). After an initial incubation period of 15 min with
constant oxygenation, gas circulation was removed to close the system
to the outside environment. The muscles were incubated for 45 min at
30°C under closed conditions. At the end of this period, release of
14CO2 from the media was
determined as described previously (40)
. The rate of
oleate oxidation was expressed as nmol/g muscle wet weight/45 min.
Whole body and tissue lipid content determination
MLC-GLUT4 and control mice 
12 months old were killed by
cervical dislocation after a short fast (6 h). Carcasses were weighed
after bladder contents were emptied and the intestines were cleared of
waste products. Carcasses were digested with alcoholic KOH (1M KOH in
66% ethanol solution) at 60°C for 2 days with complete
saponification of fat (42)
. The amount of glycerol present
in the supernatant after MgCl2 precipitation was
assessed using a kit from Sigma (Sigma Diagnostics). To assess
triglyceride content in liver and muscle, small pieces of tissue were
dissected from mice 10–12 months old and dissolved in 1M KOH at
40°C. Glycerol content was measured in supernatant after
MgCl2 precipitation using a kit (Sigma
Diagnostics).
Voluntary wheel exercise, cage activity, body weight, and food
consumption
Male mice 3 months of age were housed individually in cages
containing stainless steel, freely rotating exercise wheels. The wheel
had a 14 cm diameter and a 1 cm space between each rung of the wheel.
Number of wheel rotations was recorded via a magnet attached to each
wheel and an electromagnetic counter attached to the outside of the
cage. The total distance run was calculated by multiplying the number
of wheel rotations by the circumference of the wheel. Results were
expressed as meters/day. Wheel running activity was measured for 5 wk
on a weekly basis. During and after the 5-wk voluntary exercise period,
mouse body weight, food consumption, and cage activity over a 24 h
period were recorded. Mice were placed individually in cages with free
access to food and water. Each cage was then placed into the Digiscan
animal activity monitor (Omnitech, Columbus, Ohio). The monitor
recorded horizontal and vertical movement in the cage by interruption
of intersecting beams of infrared light traversing the cage in two
different horizontal planes. As infrared beams were broken by a moving
mouse, total distance moved was calculated using software programs and
recorded by a computer.
Weights of individual skeletal muscles
Whole muscles (tibialis anterior, gastrocnemius, EDL, and
soleus) were dissected carefully from anesthetized male and female
transgenic and control mice. The muscles were weighed after they were
cleaned of visible fat deposits and connective tissues. Muscles
dissected from right and left hind limbs were pooled.
Fiber typing and capillary density analyses
Individual skeletal muscles from male mice were rapidly excised
from anesthetized mice and trimmed to remove visible connective tissue
and fat. Muscles were mounted in embedding medium (Tissue-Tek II O.T.C.
Compound, Lab-Tek Products, Naperville, Ill.), frozen in isopentane
cooled to its freezing point with liquid nitrogen, and stored at
-20°C until analysis. Serial transverse sections (10 µm) were cut
with a microtome at -20°C and tissue slices were stained for
myofibrillar adenosine triphosphatase activity at different pH levels
(4.5, 6.6, 10.3). On the basis of the myofibrillar adenosine
triphosphatase staining characteristics, the fibers were typed as I
(slow-twitch) and type II (fast-twitch) and into subgroups IIa, IIb,
and IIc (43)
. Capillary supply and fiber areas were
determined using sections stained by the amylase-periodic acid-Schiff
method (44)
. Male mice 24 wk of age were used in these
analyses.
Statistical analysis
Data are presented as mean ± SE of multiple
determinations. Statistical analysis was performed by two-tailed,
unpaired Student’s t test or ANOVA as indicated. Results of
muscle fiber composition measurements were also analyzed by
2 statistics. Changes in proportions of fiber
types are considered significant if 2
P value is less than 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, B
). In MLC-GLUT4 transgene-negative soleus, basal and
insulin-stimulated glycolysis rates were identical in male MLC-GLUT4
and sex-matched control mice (Fig. 2A
). In contrast, basal and insulin-stimulated
glycolysis were increased by 72% and 70% (P<0.03),
respectively, in transgene-positive EDL from male MLC-GLUT4 mice when
compared with sex-matched controls (Fig. 2B
).
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Glycogen synthesis rates under basal and insulin-stimulated
conditions
In transgene-negative soleus, both female and male MLC-GLUT4 mice
exhibited the same glycogen synthesis rates as controls under basal and
insulin-stimulated conditions (Fig. 1A
, Fig. 2A
).
In transgene-positive EDL, basal glycogen synthesis rates in female and
male MLC-GLUT4 mice were also identical to those of sex-matched
controls (Fig. 1B
and Fig. 2B
). Under
insulin-stimulated conditions, female MLC-GLUT4 glycogen synthesis was
increased by 53% over the sex-matched control group
(P<0.03, Fig. 1B
). In contrast, we did not
observe significant increases in insulin-stimulated glycogen synthesis
in male MLC-GLUT4 EDL (Fig. 2B
).
Glycogen content under basal and insulin-stimulated conditions
Similar to our results for glycolysis and glycogen synthesis, no
difference in glycogen content was observed in transgene-negative
soleus between MLC-GLUT4 transgenic and control groups in either sex
under basal and insulin-stimulated conditions (Fig. 3A
C
). In female MLC-GLUT4 EDL, glycogen content
was increased 83% (P<0.0005) over sex-matched control EDL
under insulin-stimulated conditions (Fig. 3B
) in accordance
with increases in glycogen synthesis rate (Fig. 1B
). Under
basal conditions, no statistically significant difference in glycogen
content was detected between female MLC-GLUT4 and control EDL (Fig. 3C
). However, in contrast to the lack of significant
increases in glycogen synthesis rates, male EDL glycogen contents under
basal and insulin-stimulated conditions were increased in MLC-GLUT4
mice by 56% (P<0.05) and 69% (P<0.003),
respectively, over sex-matched control groups (Fig. 3D
).
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Oxidation rates of glucose and oleate
In female soleus and EDL, glucose oxidation rates between
MLC-GLUT4 transgenic and control groups were similar under basal and
insulin-stimulated conditions (data not shown). Similarly, in male
soleus and EDL muscle, no differences in basal or insulin-stimulated
glucose oxidation rates were observed between MLC-GLUT4 and control
mice (data not shown).
The rate of FFA uptake and oxidation was measured in isolated soleus
and EDL muscles from female and male age-matched MLC-GLUT4 and control
mice. The rate of FFA oxidation in isolated muscles was measured using
0.5 mM oleate and in the presence of 5 mM glucose. Since in each group
studied insulin treatment did not alter oleate oxidation (data not
shown), only results obtained under basal conditions are presented. In
the transgene-positive EDL of female and male MLC-GLUT4 mice, oleate
oxidation rates were decreased by 31% (P<0.0003) and 26%
(P<0.03), respectively, when compared with controls
(Fig. 4A
, B
). In the soleus of MLC-GLUT4 mice, where
GLUT4 expression levels remained normal, oleate oxidation rates were
the same as for control groups (Fig. 4A
, B
).
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Whole body, liver, and muscle triglyceride content
To determine whether decreased in vitro FFA oxidation
rates observed in fast-twitch EDL muscles of MLC-GLUT4 mice can
lead to regional or whole body changes in lipid storage, we assessed
whole body, liver, and skeletal muscle triglyceride content in
MLC-GLUT4 mice. Total body lipid levels were similar between MLC-GLUT4
and control mice of either sex (data not shown). However, at the
regional level, livers from male MLC-GLUT4 mice exhibited an
threefold increase in triglyceride content compared with controls
(Fig. 5A
). In contrast, no significant difference in triglyceride
content was observed in hind-limb skeletal muscles between MLC-GLUT4
and control mice (Fig. 5B
).
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Voluntary exercise, cage activity, body weight, and food
consumption
Over the 5-wk period in which mice were allowed free access to the
exercising wheel, male MLC-GLUT4 mice voluntarily ran fourfold more
average distance on the wheel per day than the controls (Fig. 6A
A). However, in the absence of an exercising device that
encouraged physical activity, MLC-GLUT4 mice only tended to be more
active in their cages over a 24-h period (Fig. 6B
). A
breakdown of hourly horizontal and vertical movements in free cages
during a 24-h period is shown in Fig. 7
. Associated with the increased physical activity, average food
consumption by MLC-GLUT4 mice was increased by 45% over controls
(Fig. 8A
). Despite increased appetite, body weight of male MLC-GLUT4
mice was moderately decreased when compared with controls (Fig. 8B
).
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Weights of individual skeletal muscles
To assess possible functional consequences after alterations in
glucose and FFA utilization patterns in MLC-GLUT4 skeletal muscle,
muscle weights, fiber composition, fiber area, and capillary density
were measured in several individual muscles. Individual weights of
tibialis anterior, gastrocnemius, EDL, and soleus muscle were
determined for female and male MLC-GLUT4 and control mice (Table 1
). Small but significant decreases in muscle weights were observed in
tibialis anterior, gastrocnemius, and EDL of female MLC-GLUT4 mice
compared with sex-matched controls (Table 1)
. Small but significant
decreases in muscle weights were also observed in tibialis anterior and
gastrocnemius of male MLC-GLUT4 mice (Table 1)
. No differences between
MLC-GLUT4 and control groups were detected for female soleus, male
soleus, and male EDL muscle weights (Table 1)
.
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Fiber typing analysis of control and MLC-GLUT4 muscles
Fiber composition was determined in soleus, EDL, and gastrocnemius
muscle from male MLC-GLUT4 and control mice (Table 2
). The proportions of type I, type IIa, type IIb, and type IIc fibers
were similar between MLC-GLUT4 and control soleus (Table 2)
. In
gastrocnemius muscle from MLC-GLUT4 vs. control mice, the proportion of
type IIa fibers was 11% greater, with a corresponding 28% decrease in
the proportion of type IIb fibers (2
P value < 0.02, Table 2
). Changes in fiber type
proportions, in particular those of type I fibers, were also observed
in EDL muscles between transgenic and control groups
(2 P value < 0.05, Table 2
).
No differences in mean fiber area were observed between MLC-GLUT4 and
control groups in the predominantly fast-twitch EDL and gastrocnemius
muscles (Table 3
). MLC-GLUT4 soleus exhibited a 33% decrease (P<0.03) in
mean fiber area when compared with controls (Table 3)
. Soleus,
gastrocnemius, and EDL muscle of MLC-GLUT4 mice also displayed small
decreases in number of capillaries per fiber (Table 3)
. However, with
the exception of EDL (P=0.06 in unpaired, two-tailed
Student’s t test), these small differences were no longer
evident when capillary density was normalized to muscle area (Table 3)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 21
22
23
24
25
, 29)
. Here we examine the mechanism by which increased glucose
influx alters intracellular glucose handling and whether these changes
in glucose metabolism in turn may influence the function of individual
skeletal muscles and whole body activity. We addressed these questions
using a genetic model of stable GLUT4 overexpression in fast-twitch
skeletal muscles of transgenic mice (19)
. In MLC-GLUT4
transgenic mice, GLUT4 is expressed at high levels in skeletal muscles
that predominantly contain high levels of fast-twitch type II fibers
such as gastrocnemius and EDL muscles (19
, 20)
. In
contrast, the MLC-GLUT4 transgene is expressed to a negligible extent
in skeletal muscle that contains a high proportion of slow-twitch type
I fibers such as the soleus muscle, which is similar to only a small
number of skeletal muscles (19
, 20)
. In isolated soleus
and EDL muscle, we showed that there were modest differences in
intracellular glucose utilization between male and female MLC-GLUT4
mice as a result of increased glucose influx. Irrespective of how
glucose was metabolized, however, GLUT4 overexpression in skeletal
muscle led to a decrease in oxidation rate of exogenous FFA. The
decreased rate of FFA oxidation was associated with increased levels of
lipid content in liver, but not whole body lipid content. Surprisingly,
we observed a small decrease in the body weight of MLC-GLUT4 mice that
is associated with a fourfold increase in voluntary exercise wheel
running. The propensity of MLC-GLUT4 mice for increased physical
activity was not, however, associated with marked changes in skeletal
muscle morphology.
After glucose transport and phosphorylation, glucose utilization in
skeletal muscle diverges into two major pathways: glycogen synthesis
and glycolysis. However, regulation of the relative proportion of
glucose entering each pathway under various metabolic states is poorly
understood. With increased GLUT4 expression, it is unknown whether the
increased glucose uptake will lead to elevated glycogen synthesis rates
or glycolytic flux. In the present study, we found that the primary
fate of the increased glucose influx under basal and insulin-stimulated
conditions in male MLC-GLUT4 EDL was through the glycolysis pathway. In
comparison, there was only a tendency toward increased glycogen
synthesis in male MLC-GLUT4 EDL. Nevertheless, male MLC-GLUT4 EDL
contained more glycogen then controls under both basal and
insulin-stimulated conditions. This suggests that the lack of
statistically significant increase in male MLC-GLUT4 EDL glycogen
synthesis rate most likely is caused by the limited sensitivity of the
assay system. In contrast, female MLC-GLUT4 EDL exhibited a marked
increase in insulin-stimulated glycogen synthesis compared to controls.
Although basal and insulin-stimulated glycolysis tended to increase in
female MLC-GLUT4 EDL over controls, the magnitude of increases was
small when compared to those observed between male MLC-GLUT4 and
control EDL. These results suggest possible differences in the
regulation of intracellular glucose metabolism between female and male
mice. The molecular mechanisms underlying these differences are
currently unknown. Differences in the regulation of glycogen synthase
may be responsible. It has been demonstrated before that development of
insulin resistance after removal of female sex hormones in
oophorectomized rats is associated with impaired GLUT4 translocation as
well as decreased glycogen synthase expression (45)
.
Further reductions in glycogen synthase expression and insulin action
were observed when these oophorectomized rats were treated with
testosterone (45)
. These results strongly suggest that the
presence of estrogen can promote glycogen synthase expression and
glycogen synthesis while testosterone exerts the opposite effects.
Although speculative, the observed differences in glucose utilization
between male and female mice may contribute to the gender-specific
differences in the severity of abnormal glucose homeostasis in certain
animal models of type II diabetes (46)
.
Although basal and insulin-stimulated glycolysis was increased in male
MLC-GLUT4 EDL vs. controls, no difference in glucose oxidation rate was
observed. This should lead to an increase in lactate production in
fast-twitch MLC-GLUT4 skeletal muscle. Indeed, we and others have
previously reported that overexpression of GLUT4 in skeletal muscle is
associated with increased serum lactate levels (19
, 20
, 22
, 24)
. Taken together, these results may suggest that increased
flux through the Cori-cycle may occur in male MLC-GLUT4 mice. Should
this be confirmed in future studies, a therapeutic regimen involving
elevated muscle GLUT4 content should also be coupled with strategies
designed to shuttle increased glucose influx toward glycogen synthesis
rather than nonoxidative glycolysis. This would be necessary in order
to prevent an up-regulation of hepatic glucose output, which has been
shown to be a major contributor in the pathogenesis of type II diabetes
mellitus (28)
. In addition, it has been proposed that a
defect in muscle glycogen synthesis and glycogen recycling play a major
role in impaired glucose utilization in type II diabetes mellitus
(47)
.
The increase in glycogen synthesis was accompanied by increased
glycogen content in female MLC-GLUT4 EDL muscle measured under
insulin-stimulated conditions (Fig. 3B
). However, male
MLC-GLUT4 EDL exhibited elevated glycogen content under basal and
insulin-stimulated conditions, despite the lack of dramatic increases
in glycogen synthesis rates. This raises the possibility that the
increased glucose influx and glycolysis may have a glycogen sparing
effect by decreasing glycogen breakdown.
In both female and male MLC-GLUT4 mice, oleate oxidation rate was
decreased in transgene-expressing EDL muscle compared to controls. In
contrast, oleate oxidation rate in transgene-negative soleus muscle of
MLC-GLUT4 mice remained unchanged compared to controls. These results
suggest that a decrease in the FFA oxidation rate in EDL muscle from
MLC-GLUT4 mice may be directly related to an increase in glucose
utilization. Whether this occurs by direct substrate competition or
from an adaptation to the long-term elevation in GLUT4 content and
glucose utilization remains to be determined. Recently, it was
demonstrated in rodents and humans that suppression of fatty acid
oxidation after increased glucose utilization is correlated with
increased muscle malonyl-CoA levels (48
, 49)
. It is likely
that increased glucose utilization in MLC-GLUT4 EDL led to increased
levels of cytosolic citrate, which in turn can activate acetyl-CoA
carboxylase, resulting in increased malonyl-CoA concentration. Since
malonyl-CoA is an inhibitor of carnitine palmitoyltransferase I, an
increase in MLC-GLUT4 muscle malonyl-CoA levels may reduce fatty acid
oxidation. Paradoxically, this scenario parallels that seen in type II
diabetic patients with hyperglycemia and impaired FFA utilization
(50
, 51)
.
Decreased oleate oxidation rates may indicate decreased fatty acid
uptake in fast-twitch MLC-GLUT4 muscles, which in turn may lead to
increased triglyceride content in tissues that supply lipids to
skeletal muscle. Indeed, MLC-GLUT4 liver contained significantly more
triglyceride than the controls (Fig. 5A
). We have not
observed any difference in fat pad size between MLC-GLUT4 and control
mice (data not shown). However, increased muscle glucose utilization
may have resulted in a small decrease in insulin-stimulated glucose
uptake and subsequent triglyceride synthesis in MLC-GLUT4 adipocytes,
masking the effects of reduced muscle fatty acid oxidation
(52)
. Despite the decrease in fatty acid oxidation,
hind-limb triglyceride level in MLC-GLUT4 mice remained unchanged (Fig. 5B
). This lack of difference may reflect an offsetting
combination of decreased fatty acid uptake and decreased endogenous
triglyceride oxidation. Clearly, further studies are necessary to
determine whether the increased triglyceride content is associated
pathological changes in the liver of MLC-GLUT4 mice.
It is well documented that decreased glucose utilization rate is
associated with obesity in humans as well as animals (53
, 54)
. In conjunction, it has been hypothesized that insulin
resistance develops as an adaptive response to prevent further weight
gain (31
, 32)
. If this hypothesis is true, it is
reasonable to propose that animals with enhanced insulin action may be
predisposed to become obese. Yet studies of mice exhibiting increased
insulin action by GLUT4 overexpression have reported no apparent
increase in body weight under conditions of normal or elevated feeding
(19
, 24
, 29
, 33
, 34)
. However, the effects of improved
insulin action on body weight may have been masked in these previous
studies. This could have been caused by lack of physical activity
imposed as a result of limited living space. To promote physical
activity, in the present study mice were placed in cages that contained
training wheels. By encouraging physical activity, the metabolic
characteristics of insulin resistance (increased lipid utilization and
decreased carbohydrate utilization) may play a larger role in
determining total daily energy expenditure, and consequently body
weight. Unexpectedly, we showed in the present study that MLC-GLUT4
mice exhibited slightly decreased body weight compared with the
controls. This result is contrary to our original hypothesis. However,
we also observed a surprising increase in voluntary exercise activity
in MLC-GLUT4 mice. The decreased body weight in MLC-GLUT4 mice is most
readily explained by the increased energy expenditure that accompanies
the dramatic fourfold increase in exercise wheel running distance. This
is supported by the observation that despite decreased body weight,
MLC-GLUT4 mice ingested 45% more food when placed in cages with
exercise wheels. In addition, the lack of dramatic changes in MLC-GLUT4
skeletal muscle morphology cannot account for the observed changes in
wheel running distance.
It is unclear at the present why MLC-GLUT4 mice undergo increased
voluntary exercise. It is possible that somehow overexpression of
muscle GLUT4 altered behavioral patterns of mice, making them
hyperactive. However, in the absence of training wheels, MLC-GLUT4 mice
only tended to be more active in experiments designed to measure free
movement around cages. The lack of statistical significance may be
attributed to the fact that the animals were placed in cages without
any device that encouraged physical activity. One explanation for
increased voluntary exercise is that increased glucose uptake and/or
increased glycogen content in MLC-GLUT4 skeletal muscle may supply more
substrate toward energy production and thereby provide greater
tolerance for fatigue during exercise. During exercise, muscle GLUT4 is
mobilized to the cell surface from intracellular vesicles
(55)
. In addition, glucose transport has been shown to be
a rate-limiting step in exogenous glucose utilization during exercise
(56)
. In mammals that differ in maximal aerobic
capacities, fat oxidation supplies most of the required fuel during low
intensity exercise (57
, 58)
. During high-intensity
exercise, most of the fuel energy is provided by carbohydrate oxidation
(57
, 58)
. It is possible that the increased availability
of glucose and/or glycogen content for fuel oxidation may allow
MLC-GLUT4 mice to undergo the same or higher intensity exercise for a
longer period of time than controls. This conjecture is supported by a
recent study showing that UCP3 mRNA levels are up-regulated in skeletal
muscle of mice overexpressing GLUT4 (59)
, suggesting a
state of energy surplus in that GLUT4-overexpressing tissue.
Previous studies have demonstrated a clear link between skeletal muscle
fiber composition, muscle glucose transport, and whole body glucose
utilization under insulin-stimulated conditions in humans (60
, 61)
, as well as in rodents (62
, 63)
. A reduced
population of type I (slow-twitch oxidative) fibers and/or an increased
population of type IIb (fast-twitch glycolytic) fibers is associated
with impaired insulin-stimulated glucose disposal in first-degree
relatives of type II diabetic patients (64)
and in some
(63)
, but not all (65)
, type II diabetic
patients. However, it is unknown whether alterations in muscle fiber
composition precede development of insulin resistance or are
consequences of changes in muscle insulin sensitivity, functional
demand, or inactivity. In the present study we found a small increase
in type IIa as well as a small decrease in type IIb fibers in
gastrocnemius muscle of MLC-GLUT4 mice compared with controls. This
suggests that muscle fiber composition may be modified by changes in
muscle glucose metabolism. Indeed, expression levels of GLUT4 in
individual muscles are positively correlated with the amount of type I
and type IIa fibers (10
, 66)
. However, other studies have
clearly demonstrated that changes in fiber type composition are not
necessarily associated with changes in muscle glucose transport or
GLUT4 content in people with complete cervical cord lesions
(67)
or type II diabetes (65)
. When all
studies are taken into account, changes in glucose metabolism appear to
have a direct but limited effect on muscle fiber composition, since
there are other adaptive changes dependent on muscle energy demands
that also affect fiber type distribution.
In summary, we have examined the metabolic and functional consequences of a long-term increase in skeletal muscle GLUT4 content in transgenic mice selectively overexpressing GLUT4 in fast-twitch skeletal muscle. Whereas increased glucose influx into male MLC-GLUT4 EDL predominantly underwent glycolysis, increased glycogen synthesis was seen in female MLC-GLUT4 EDL. Increased glucose utilization in EDL muscle from MLC-GLUT4 mice was also associated with decreased exogenous FFA oxidation in muscle and in male mice an increased accumulation of lipid in liver. Furthermore, elevated skeletal muscle GLUT4 expression resulted in increased voluntary wheel exercise accompanied by increased food consumption and decreased body weight. The findings reported in this study illustrate the need to investigate further the plasticity of muscle and whole body metabolism in adaptation to genetic manipulation.
|
ACKNOWLEDGMENTS |
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Received for publication June 16, 2000.
Revision received September 27, 2000.
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REFERENCES |
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