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(The FASEB Journal. 2001;15:958-969.)
© 2001 FASEB

Metabolic adaptations in skeletal muscle overexpressing GLUT4: effects on muscle and physical activity

TSU-SHUEN TSAO*, JING LI*, KENNETH S. CHANG{dagger}, ANTINE E. STENBIT*, DANA GALUSKA{ddagger}, JUDY E. ANDERSON§, JULEEN R. ZIERATH{ddagger}, ROGER J. MCCARTER{dagger} and MAUREEN J. CHARRON*,1

1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA;
{dagger} Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229 USA;
{ddagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To understand the long-term metabolic and functional consequences of increased GLUT4 content, intracellular substrate utilization was investigated in isolated muscles of transgenic mice overexpressing GLUT4 selectively in fast-twitch skeletal muscles. Rates of glycolysis, glycogen synthesis, glucose oxidation, and free fatty acid (FFA) oxidation as well as glycogen content were assessed in isolated EDL (fast-twitch) and soleus (slow-twitch) muscles from female and male MLC-GLUT4 transgenic and control mice. In male MLC-GLUT4 EDL, increased glucose influx predominantly led to increased glycolysis. In contrast, in female MLC-GLUT4 EDL increased glycogen synthesis was observed. In both sexes, GLUT4 overexpression resulted in decreased exogenous FFA oxidation rates. The decreased rate of FFA oxidation in male MLC-GLUT4 EDL was associated with increased lipid content in liver, but not in muscle or at the whole body level. To determine how changes in substrate metabolism and insulin action may influence energy balance in an environment that encouraged physical activity, we measured voluntary training activity, body weight, and food consumption of MLC-GLUT4 and control mice in cages equipped with training wheels. We observed a small decrease in body weight of MLC-GLUT4 mice that was paradoxically accompanied by a 45% increase in food consumption. The results were explained by a marked fourfold increase in voluntary wheel exercise. The changes in substrate metabolism and physical activity in MLC-GLUT4 mice were not associated with dramatic changes in skeletal muscle morphology. Collectively, results of this study demonstrate the feasibility of altering muscle substrate utilization by overexpression of GLUT4. The results also suggest that as a potential treatment for type II diabetes mellitus, increased skeletal muscle GLUT4 expression may provide benefits in addition to improvement of insulin action.—Tsao, T.-S., Li, J., Chang, K. S., Stenbit, A. E., Galuska, D., Anderson, J. E., Zierath, J. R., McCarter, R. J., Charron, M. J. Metabolic adaptations in skeletal muscle overexpressing GLUT4: effects on muscle and physical activity.


Key Words: free fatty acids • EDL • glycogen synthesis • oleate oxidation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
UNDER INSULIN-STIMULATED CONDITIONS, skeletal muscle is quantitatively the most important tissue responsible for whole body glucose uptake in humans (1 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
MLC-GLUT4 and control mice used in this study are described in our first report (19) . 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 {chi}2 statistics. Changes in proportions of fiber types are considered significant if {chi}2 P value is less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Glycolysis rates under basal and insulin-stimulated conditions
Isolated soleus and EDL were incubated for a total of 105 min under basal or insulin-stimulated conditions. Glycolysis was measured by release of 3H2O from 5-3H-glucose. No statistically significant differences in glycolysis rates were observed in either isolated soleus or EDL between the female MLC-GLUT4 and sex-matched control groups under basal and insulin-stimulated conditions (Fig. 1A , 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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  		Figure 1. Glycolysis and glycogen synthesis rates in isolated solei (A) and EDL (B) muscles from female control and MLC-GLUT4 mice. Solei and EDL muscles were isolated and incubated as described in Materials and Methods. Glycolysis was measured as µmol water released into incubation medium/g wt weight/h at 5 mM glucose in the absence (basal, closed bars) or presence (insulin, open bars) of 20 nM porcine insulin. Specific activity of 3H2O (cpm/µl) was obtained from scintillation counting of incubation medium after radiolabeled glucose was separated by ion-exchange chromatography. Glycogen synthesis was measured as µmol glucose incorporated into glycogen x g wt weight-1 x h-1 at 5 mM glucose in the absence (basal, closed bars) or presence (insulin, open bars) of 20 nM porcine insulin. Values determined using 5-[3H]glucose and U-[14C]glucose were nearly identical and were averaged to give final values. Values are mean ± SE for 5 to 12 muscles from each group. Statistical significance was assessed by ANOVA with Fisher’s PLSD for post hoc analyses and is noted where *P < 0.03.



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  		Figure 2. Glycolysis and glycogen synthesis rates in isolated solei (A) and EDL (B) muscles from male control and MLC-GLUT4 mice. Solei and EDL muscles were isolated and incubated as described in Materials and Methods. Glycolysis was measured as µmol water released into incubation medium x g wt weight-1 x h-1 at 5 mM glucose in the absence (basal, closed bars) or presence (insulin, open bars) of 20 nM porcine insulin. Specific activity of 3H2O (cpm/µl) was obtained from scintillation counting of incubation medium after radiolabeled glucose was separated by ion-exchange chromatography. Glycogen synthesis was measured as µmol glucose incorporated into glycogen x g wt weight-1 x h-1 at 5 mM glucose in the absence (basal, closed bars) or presence (insulin, open bars) of 20 nM porcine insulin. Values determined using 5-[3H]glucose and U-[14C]glucose were nearly identical and were averaged to give final values. Values are mean ± SE for 5 to 12 muscles from each group. Statistical significance was assessed by ANOVA with Fisher’s PLSD for post hoc analyses and is noted where *P < 0.03.

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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  		Figure 3. Glycogen content in isolated solei (A, C) and EDL (B, D) muscles from female (A, B) and male (C, D) control and MLC-GLUT4 mice. Solei and EDL muscles were isolated and incubated as described in Materials and Methods. Glycogen was purified and converted into glucose as described in Materials and Methods after incubation at 5 mM glucose in the absence (basal, closed bars) or presence (insulin, open) of 20 nM porcine insulin. Glycogen content is expressed as µg glucose in the form of glycogen x mg wt weight-1. Values are mean ± SE for 6 to 12 muscles from each group. Comparisons were made against control groups using unpaired, two-tailed Student’s t test. Statistical significance was assessed by ANOVA with Fisher’s PLSD for post hoc analyses and is indicated where *P < 0.05 and {dagger} denotes P < 0.005.

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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  		Figure 4. Oleate oxidation rate in isolated solei and EDL muscles from female (A) and male (B) control (closed bars) and MLC-GLUT4 (open bars) mice. Solei and EDL muscles were isolated and oxidation was measured for 45 min in the presence of 0.5 mM oleate and 5 mM glucose as described in Materials and Methods. Results are expressed as µmol oleate x g wet weight-1 x 45 min-1. Values are mean ± SE for muscles from 8 to 12 muscles from each group. Comparisons were made against control groups using unpaired, two-tailed Student’s t test. Statistical significance is indicated where *P < 0.03 and {dagger} denotes P < 0.0003.

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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  		Figure 5. Liver (A) and skeletal muscle (B) lipid content in male MLC-GLUT4 and control mice. Tissues were dissolved in 1M KOH at 40°C to release the glycerol moiety of lipids. Total glycerol levels were determined by as detailed in Materials and Methods. Results obtained from MLC-GLUT4 tissues were normalized against controls with the control glycerol levels set at 100%. Values are mean ± SE for tissues from 15 MLC-GLUT4 and 16 control mice. Comparisons were made against the control groups using unpaired, two-tailed Student’s t test. Statistical significance is indicated where *P < 0.005.

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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  		Figure 6. Voluntary wheel exercise (A) and daily cage activity (B) of male MLC-GLUT4 and control mice. Average wheel distance traveled by MLC-GLUT4 and control mice was measured over a 24-h period during a span of 5 wk as described in Materials and Methods. Results are expressed as meters/24 h Average daily cage activity of MLC-GLUT4 and control mice was measured as accumulative horizontal and vertical distances traveled in free cages without exercise wheels. Results are expressed as centimeters/24 h Values are mean ± SE from 7 MLC-GLUT4 and 7 control mice. Statistical significance is indicated where *P < 0.05.



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  		Figure 7. Hourly free activity of male MLC-GLUT4 (bolded lines) and control (plain lines) mice. Hourly horizontal and vertical distances traveled in rodent cages were measured over a 24-h day/light cycle. Results are expressed as centimeters/h. Values are mean ± SE from 7 MLC-GLUT4 and 7 control mice. Difference in overall cage activity was not statistically significant between the two groups.



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  		Figure 8. Food consumption (A) and body weight (B) of MLC-GLUT4 and control mice in cages with exercise wheels. Food consumption was measured over 3-day periods for a span of 3 wk when animals had free access to exercise wheels. Results are expressed as grams of food/24 h Average body weight in grams was measured weekly for 4 wk during the 5-wk wheel exercise period. Values are mean ± SE from 7 MLC-GLUT4 and 7 control mice. Statistical significance is indicated where *P < 0.05.

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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  		Table 1. Individual muscle weights from female and male MLC-GLUT4 transgenic and control micea

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 ({chi}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 ({chi}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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  		Table 2. Skeletal muscle fiber composition of soleus, EDL, and gastrocnemius from male MLC-GLUT4 transgenic and control micea


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  		Table 3. Mean fiber area and capillary density of soleus, EDL, and gastrocnemius from male MLC-GLUT4 and control micea


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A greater understanding of the metabolic and functional consequences of increased GLUT4 content has become increasingly important as strategies to increase muscle GLUT4 expression have been proposed as treatment for type II diabetes mellitus (19 , 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
 
We thank F. Bone for expert technical support. We also thank E. B. Katz and L. Rezende for valuable discussions throughout this study. This work was supported by grants from the National Institutes of Health (DK47425, HL58119, and AG14674), American Heart Association, American Diabetes Association, and Albert Einstein College of Medicine Cancer Center (5P30CA13330) (to M.J.C.), Novo-Nordisk Foundation, Swedish Diabetes Association, Marcus and Amalia Wallenbergs Foundation, Foundation for Strategic Research, Swedish Medical Research Council (11823 and 12211) (to J.R.Z.), and Muscular Dystrophy Association and Heart and Stroke Foundation of Canada (to J.E.A.). M.J.C. is a recipient of an Irma T. Hirschl Career Scientist Award. A.E.S. and T.-S.T. were supported by NIH (5T32GM07491 and 5T32HL07675). This work is submitted in partial fulfillment of the requirements for the Ph.D. degree for the Albert Einstein College of Medicine (T.-S.T.) and in partial fulfillment of the requirements for the M.S. degree of the University of Texas Health Science Center at San Antonio (K.S.C.).

Received for publication June 16, 2000. Revision received September 27, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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