Distributed control of glucose uptake by working muscles of conscious mice: roles of transport and phosphorylation

Patrick T. Fueger,1 Deanna P. Bracy,1,2 Carlo M. Malabanan,2 R. Richard Pencek,1 and David H. Wasserman1,2

1Department of Molecular Physiology and Biophysics, 2Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Submitted 8 July 2003 ; accepted in final form 9 September 2003


    ABSTRACT
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Muscle glucose uptake (MGU) is determined by glucose delivery, transport, and phosphorylation. C57Bl/6J mice overexpressing GLUT4, hexokinase II (HK II), or both were used to determine the barriers to MGU. A carotid artery and jugular vein were catheterized for arterial blood sampling and venous infusions. Experiments were conducted in conscious mice ~7 days after surgery. 2-Deoxy-[3H]glucose was administered during rest or treadmill exercise to calculate glucose concentration-dependent (Rg) and -independent (Kg) indexes of MGU. Compared with wild-type controls, GLUT4-overexpressing mice had lowered fasting glycemia (165 ± 6 vs. 115 ± 6 mg/dl) and increased Rg by 230 and 166% in the gastrocnemius and superficial vastus lateralis (SVL) muscles under sedentary conditions. GLUT4 overexpression was not able to augment exercise-stimulated Rg or Kg. Whereas HK II overexpression had no effect on fasting glycemia (170 ± 6 mg/dl) or sedentary Rg, it increased exercise-stimulated Rg by 82, 60, and 169% in soleus, gastrocnemius, and SVL muscles, respectively. Combined GLUT4 and HK II overexpression lowered fasting glycemia (106 ± 6 mg/dl), increased nonesterified fatty acids, and increased sedentary Rg. Combined GLUT4 and HK II overexpression did not enhance exercise-stimulated Rg compared with HK II-overexpressing mice because of the reduced glucose concentration. GLUT4 combined with HK II overexpression resulted in a marked increase in exercise-stimulated Kg. In conclusion, control of MGU shifts from membrane transport at rest to phosphorylation during exercise. Glucose transport is not normally a significant barrier during exercise. However, when the phosphorylation barrier is lowered by HK II overexpression, glucose transport becomes a key site of control for regulating MGU during exercise.

delivery; glucose transporter 4; hexokinase; exercise; 2-deoxyglucose


THE CONTROL OF skeletal muscle glucose uptake (MGU) is distributed over the following three serial steps: delivery of glucose from the blood to the sarcolemma, transport across the sarcolemmal membrane, and intracellular phosphorylation by hexokinase (HK; see Ref. 42). Both blood flow and capillary recruitment are important determinants of glucose delivery. Glucose transport is mediated by the facilitated diffusion of glucose through a GLUT family member. Under basal and sedentary conditions, GLUT1 facilitates much of the transport into skeletal muscle (10, 30, 38); however, after stimulation by contractions (3, 8, 37), GLUT4 translocates from an intracellular pool to the cell surface, thereby increasing the permeability of the sarcolemma to glucose. Whether HK activity or localization and hence the functional rate of glucose phosphorylation to glucose 6-phosphate (G-6-P) changes during acute stimulation by exercise remains to be clearly determined. While one step may exert dominance in controlling MGU under one condition, the primary control step may shift with physiological stimuli (11-14, 34).

During sedentary conditions, glucose transport is the predominant rate-controlling step of MGU in the fasted state (38). However, during acute exercise when glucose delivery is increased because of an elevation in muscle perfusion and membrane transport is increased as a result of translocation of GLUT4 to the sarcolemma, the rate of glucose phosphorylation by HK II becomes an important rate-determining step (11, 12, 22). Reducing the phosphorylation barrier by selectively overexpressing HK II in skeletal muscle of FVB/NJ mice has been shown to increase exercise-stimulated MGU while having no apparent effect on sedentary MGU (12) after an overnight fast designed to reduce G-6-P levels. In contrast, GLUT4 overexpression in skeletal muscle increases sedentary MGU (15, 41) and lowers fasting glycemia (2, 15, 25, 41). It is unknown whether GLUT4 overexpression alone augments exercise-stimulated MGU in vivo. To test this, we examined the effect of GLUT4 overexpression on exercise-stimulated MGU in the conscious animal. Glucose phosphorylation is a major control step for MGU during exercise. Therefore, we also tested the hypothesis that GLUT4 overexpression would be particularly effective in enhancing MGU if the resistance to glucose phosphorylation was reduced by HK II overexpression.


    RESEARCH DESIGN AND METHODS
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mouse maintenance and genotyping. All procedures performed were approved by the Vanderbilt University Animal Care and Use Committee. Male FVB/NJ mice that selectively overexpress HK II in skeletal muscle using a transgene containing the human HK II cDNA driven by the rat muscle creatine kinase promoter that yields a three-, five-, and sevenfold increase in HK activity in the soleus, gastrocnemius, and SVL muscles, respectively, were obtained from Dr. David E. Moller (6) and backcrossed on the C57Bl/6J background for at least five generations. Female mice carrying the HK II transgene (HKTg) were subsequently bred with male C57Bl/6J mice transgenic for the hGLUT4-11.5 construct (GLUT4Tg) that yields a two-, three-, and threefold increase in GLUT4 protein content in the soleus, gastrocnemius, and SVL muscles, respectively, (35) to obtain mice of the following four genotypes: wild type (WT), GLUT4Tg, HKTg, and HKTg + GLUT4Tg. After a 3-wk weaning period, littermates were separated by gender and maintained in microisolator cages. Genotyping for the HK II and hGLUT4-11.5 transgenes was performed with the PCR on genomic DNA obtained from a tail biopsy and isolated by a DNeasy Tissue Kit (Qiagen, Valencia, CA), as previously described (12, 35). Mice were fed standard chow ad libitum and were studied at ~4 mo of age.

Surgical procedures. The surgical procedures used are similar to those described previously (12, 33, 40). Mice were anesthetized with pentobarbital sodium (70 mg/kg body wt). The left common carotid artery was catheterized for sampling of arterial blood with a two-part catheter consisting of PE-10 (inserted in the artery) and Silastic (0.025 in. OD) tubing. The right jugular vein was catheterized for infusions with a Silastic catheter (0.025 in. OD). The free ends of catheters were tunneled under the skin to the back of the neck, where they were attached via stainless steel connectors to lines made of Micro-Renathane (0.033 in. OD), which were exteriorized and sealed with stainless steel plugs. Lines were flushed daily with 10-50 µl saline containing 200 U/ml heparin and 5 mg/ml ampicillin. Considering heparin's half-life, the amount that reaches the circulation would not be expected to create sustained increases in lipolysis (28). Animals were housed individually after surgery, and body weight was recorded daily. After an ~5-day period in which body weight was restored (within 10% of presurgery body wt), mice were acclimated to treadmill running with a single 10-min bout of exercise (0.5-0.6 miles/h, 0% grade). Experiments were performed 2 days after the treadmill acclimation trial.

Sedentary and exercise experiments. On the day of the experiment, conscious mice were placed in a 1-liter plastic container lined with bedding and fasted for 5 h. Approximately 1 h before an experiment, Micro-Renathane (0.033 in. OD) tubing (22 cm long) was connected to the catheter leads, and mice were placed on an enclosed treadmill. At time (t) = 0 min, a baseline arterial blood sample (150 µl) was drawn for the measurement of arterial blood glucose (HemoCue, Mission Viejo, CA), hematocrit, and plasma insulin and nonesterified fatty acids (NEFA). The remaining red blood cells were washed one time with 0.9% saline containing 10 U/ml heparin and reinfused. The mice either remained sedentary (n = 9 WT, 8 GLUT4Tg, 12 HKTg, and 10 HKTg + GLUT4Tg) or ran (n = 9 WT, 8 GLUT4Tg, 10 HKTg, and 10 HKTg + GLUT4Tg) on the treadmill (0.6 mph, 0% grade) for 30 min. Exercise was standardized so that the same amount of absolute work was performed rather than matching relative work intensities so as to create similar energy requirements. It has, however, previously been reported that transgenic overexpression of GLUT4 does not significantly alter oxygen consumption during treadmill exercise at a similar absolute work rate (2). The selected work intensity is designed to increase oxygen consumption to ~80% of maximal oxygen consumption (9). This was confirmed in a small subset of animals of each genotype during a maximal oxygen consumption test. At t = 5 min, a 12-µCi bolus of 2-deoxy[3H]glucose ([2-3H]DG; New England Nuclear, Boston, MA) was administered to provide an index of tissue-specific glucose uptake. At t = 10, 15, and 20 min, ~50 µl of arterial blood were sampled to determine blood glucose and plasma [2-3H]DG. Blood glucose and hormones were not clamped, and concentrations reflect the native physiological responses. At t = 30 min, a 150-µl arterial blood sample was withdrawn, and mice were anesthetized with an arterial infusion of pentobarbital sodium. The soleus (~44% type I, ~51% type IIA, and ~5% type IID fibers), gastrocnemius (~6% type IIA, ~11% type IID, and ~83% type IIB fibers), and superficial vastus lateralis (SVL; ~3% type IIA, ~10 type IID, and ~87% type IIB fibers) muscles (1) were excised, immediately freeze-clamped in liquid nitrogen, and stored at -70°C until future tissue analysis.

Processing of plasma and muscle samples. Immunoreactive insulin was assayed with a double-antibody method (32). NEFA were measured spectrophotometrically by an enzymatic colorimetric assay (Wako NEFA C kit; Wako Chemicals, Richmond, VA). After deproteinization with Ba(OH)2 (0.3 N) and ZnSO4 (0.3 N), [2-3H]DG radioactivity of plasma was determined by liquid scintillation counting (Packard TRI-CARB 2900TR; Packard, Meriden, CT) with Ultima Gold (Packard) as scintillant. Muscle samples were weighed and homogenized in 0.5% perchloric acid. Homogenates were centrifuged and neutralized with KOH. One aliquot was counted directly to determine [2-3H]DG and [2-3H]DG-6-phosphate ([2-3H]DGP) radioactivity. A second aliquot was treated with Ba(OH)2 and ZnSO4 to remove [2-3H]DGP and any tracer incorporated into glycogen and then counted to determine [2-3H]DG radioactivity. [2-3H]DGP is the difference between the two aliquots. In all experiments, the accumulation of [2-3H]DGP was normalized to tissue weight. Clearance of [2-3H]DG (Kg) and the metabolic index (Rg), which serves as an index of tissue-specific glucose uptake, were calculated as previously described (24)

where [2-3H]DGPmuscle is the [2-3H]DGP radioactivity in the muscle in dpm/g, AUC [2-3H]DGplasma is the area under the plasma [2-3H]DG disappearance curve in dpm·ml-1·min-1, and Glucoseplasma is the average blood glucose in millimolar during the experimental period. Plasma [2-3H]DG data for t = 5 min (i.e., time of tracer administration) have been extrapolated using a binomial equation based on the tracer disappearance curve from t = 10 to 30 min.

Muscle glycogen was determined by the method of Chan and Exton (5) on the contralateral gastrocnemius and SVL muscles. Glycogen breakdown in response to exercise is calculated as the average glycogen concentration for sedentary mice within a genotype minus the individual postexercise glycogen concentrations. After deproteinization with 0.5% perchloric acid, tissue glucose and G-6-P were measured enzymatically (26) and expressed as micromole per gram wet weight.

Statistical analysis. Data are presented as means ± SE. Differences between groups were determined by ANOVA. The significance level was set at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Baseline characterization of mice. Baseline characteristics of the mice used in these protocols are shown in Table 1. Body weight was higher in the GLUT4Tg and HKTg + GLUT4Tg (28 ± 1 and 28 ± 1 g) mice compared with WT and HKTg (26 ± 1 and 25 ± 1 g, P < 0.001) mice because of the greater percentage of males in these groups. If body weight is compared by gender, there are no differences between any groups (data not shown). As has been reported previously, GLUT4 overexpression, either alone (2, 10, 19, 20, 25, 29, 38, 41) or in combination with HK II overexpression (27), reduced glycemia. Similar to previous reports in FVB/NJ mice (6, 12), HK II overexpression in C57Bl/6J mice by itself had no effect on fasting glycemia relative to WT controls. Plasma insulin concentrations were not significantly different between groups. Plasma NEFAs were significantly increased in HKTg + GLUT4Tg mice compared with all other groups.


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Table 1. Baseline characteristics in 5-h-fasted C57B1/6J mice

 

Effect of GLUT4 overexpression during rest and exercise. Blood glucose was constant relative to baseline throughout both the sedentary and exercise period in WT and GLUT4Tg mice so that the reduction in blood glucose observed when GLUT4 was overexpressed persisted throughout the study (Fig.1). These data are consistent with fasting blood glucose values obtained in previous investigations (2, 10, 19, 20, 25, 29, 38, 41) and reflect the accelerated basal glucose uptake in GLUT4-overexpressing mice resulting from the two- to threefold increase in GLUT4 protein content in skeletal muscle.



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Fig. 1. Arterial blood glucose during a 30-min sedentary or exercise condition in chronically catheterized, conscious mice. Arterial blood was obtained and measured for blood glucose as described in RESEARCH DESIGN AND METHODS. Data are means ± SE for 8-12 mice/group. A: wild type (WT; {circ}, {bullet}) or mice overexpressing GLUT4 (GLUT4Tg; {square}, {blacksquare}) were fasted for 5 h and subjected to 30 min of rest ({circ}, {square}) or treadmill running at 0.6 mph ({bullet}, {blacksquare}). *P < 0.05 for GLUT4Tg vs. WT at all time points measured in both conditions. B: mice overexpressing HK II (HKTg; {circ}, {bullet}) or both HKTg + GLUT4Tg ({square}, {blacksquare}) were fasted for 5 h and subjected to 30 min of rest ({circ}, {square}) or treadmill running at 0.6 mph ({bullet}, {blacksquare}). *P < 0.05 for HKTg + GLUT4Tg vs. HKTg at all time points measured in both conditions.

 

The disappearance curves of the radioactive glucose analog, [2-3H]DG, from the plasma for the WT and GLUT4Tg mice are shown in Fig. 2. Compared with the WT mice during the sedentary condition, a downward shift in a disappearance curve is indicative of increased clearance of the tracer. In both sedentary and exercise conditions, GLUT4Tg mice had a significantly greater rate of disappearance of [2-3H]DG from the plasma compared with WT mice, as evidenced by the lower curves. Exercise elicited only a slight increase in [2-3H]DG disappearance in WT mice and yielded no change in [2-3H]DG disappearance in GLUT4Tg mice.



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Fig. 2. Disappearance of 2-deoxy-[3H]glucose ([2-3H]DG) from the plasma during a 30-min sedentary or exercise condition in chronically catheterized, conscious mice. Arterial blood was obtained and measured for plasma [2-3H]DG as described in RESEARCH DESIGN AND METHODS. Data are means ± SE for 8-12 mice/group. A: WT mice ({circ}, {bullet}) or mice overexpressing GLUT4 ({square}, {blacksquare}) were fasted for 5 h and subjected to 30 min of rest ({circ}, {square}) or treadmill running at 0.6 mph ({bullet}, {blacksquare}). *P < 0.05 for GLUT4Tg vs. WT at all time points measured in both conditions; {dagger}P < 0.05 for exercise vs. sedentary within the WT genotype at 20 and 30 min. B: mice overexpressing hexokinase (HK) II (HKTg; {circ}, {bullet}) or HKTg + GLUT4Tg ({square}, {blacksquare}) were fasted for 5 h and subjected to 30 min of rest ({circ}, {square}) or treadmill running at 0.6 mph ({bullet}, {blacksquare}). *P < 0.05 for HKTg + GLUT4Tg vs. HKTg at all time points measured in both conditions. {dagger}P < 0.05 for exercise vs. sedentary within a genotype at all time points.

 

Kg and Rg values for the soleus, gastrocnemius, and SVL from WT and GLUT4Tg mice during the sedentary and exercise conditions are shown in the left half of Figs. 3 and 4, respectively. Consistent with previous results in isolated skeletal muscle (15, 41), sedentary GLUT4Tg mice had greater Kg and Rg values in the gastrocnemius and SVL compared with WT mice in vivo. Exercise increased both Kg and Rg in all muscles of WT and GLUT4Tg mice with the exception of the soleus of GLUT4Tg mice. The soleus is tonically active; therefore, a marked increase in Kg or Rg with exercise was not expected in WT mice. It was notable that GLUT4 overexpression did not augment the exercise-induced increase in Rg in any of the muscles studied in relation to WT mice. One might speculate that reduced muscle glucose delivery resultant from lower plasma glucose associated with GLUT4Tg mice limited the ability of the transgene to have an effect during exercise. This, however, was not the case, since Kg, which is largely glucose concentration independent, was also not augmented by GLUT4 overexpression in exercising mice.



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Fig. 3. Sedentary and exercise-stimulated glucose clearance in intact skeletal muscles from chronically catheterized, conscious mice. WT or mice overexpressing GLUT4 (GLUT4Tg), HK II (HKTg), or both (HKTg + GLUT4Tg) were chronically catheterized and allowed to recover from surgery for ~7 days. After a 5-h fast and a bolus injection of 12 µCi [2-3H]DG during a sedentary (open bars) or exercise (closed bars) condition, soleus (A), gastrocnemius (B), and superficial vastus lateralis (SVL; C) Kg was calculated as described in RESEARCH DESIGN AND METHODS. Data are means ± SE for 8-12 mice/group. *P < 0.05 for exercise vs. sedentary within a genotype; {dagger}P < 0.05 for GLUT4Tg and HKTg + GLUT4Tg vs. WT and HKTg within the sedentary condition; {ddagger}P < 0.05 for HKTg and HKTg + GLUT4Tg vs. WT and GLUT4Tg within the exercise condition; §P < 0.05 for HKTg + GLUT4Tg vs. WT, GLUT4Tg, and HKTg within the exercise condition.

 


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Fig. 4. Sedentary and exercise-stimulated glucose uptake in intact skeletal muscles from chronically catheterized, conscious mice. WT or mice overexpressing GLUT4 (GLUT4Tg), HK II (HKTg), or both (HKTg + GLUT4Tg) were chronically catheterized and allowed to recover from surgery for ~7 days. After a 5-h fast and a bolus injection of 12 µCi [2-3H]DG during a sedentary (open bars) or exercise (closed bars) condition, soleus (A), gastrocnemius (B), and SVL (C) Rg, an index of tissue-specific glucose uptake, was calculated as described in RESEARCH DESIGN AND METHODS. Data are means ± SE for 8-12 mice/group. *P < 0.05 for exercise vs. sedentary within a genotype; {dagger}P < 0.05 for GLUT4Tg and HKTg + GLUT4Tg vs. WT and HKTg within the sedentary condition; {ddagger}P < 0.05 for HKTg and HKTg + GLUT4Tg vs. WT and GLUT4Tg within the exercise condition.

 

GLUT4 overexpression did not appreciably increase muscle glycogen content (Table 2) or muscle glucose and G-6-P concentrations (data not shown) in 5-h fasted, sedentary mice. Exercise led to a reduction in muscle glycogen in both WT and GLUT4Tg mice compared with their sedentary counterparts and no notable changes in muscle glucose or G-6-P. Glycogen breakdown in response to exercise was significantly increased in GLUT4-overexpressing mice compared with WT mice (Fig. 5).


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Table 2. Muscle glycogen

 


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Fig. 5. Glycogen breakdown during 30 min of exercise. WT or mice overexpressing GLUT4 (GLUT4Tg), HK II (HKTg), or both (HKTg + GLUT4Tg) were chronically catheterized and allowed to recover from surgery for ~7 days. Mice ran on a treadmill for 30 min at 0.6 mph. Gastrocnemius (A) and SVL (B) glycogen breakdown (expressed as µmol glucosyl units·100 g tissue-1·min-1) was calculated as the average muscle glycogen within a genotype minus the individual muscle glycogen of exercised mice. Data are means ± SE for 8-12 mice/group. *P < 0.05 for GLUT4Tg vs. WT and HKTg; {dagger}P < 0.05 for HKTg + GLUT4Tg vs. HKTg.

 

Effect of GLUT4 overexpression in conjunction with HK II overexpression during rest and exercise. Blood glucose was constant relative to baseline throughout both the sedentary and exercise period in HKTg and HKTg + GLUT4Tg mice so that the reduction in blood glucose observed when GLUT4 was overexpressed on the background of HK II overexpression persisted throughout the study (Fig. 1). HK II overexpression by itself had no effect on fasting glycemia relative to WT controls. With respect to blood glucose, HK II overexpression had no effect during sedentary or exercise conditions compared with WT mice despite three- to sevenfold increases in HK activity in skeletal muscle, a major site for glucose utilization. HK II overexpression combined with GLUT4 overexpression did not further reduce blood glucose compared with GLUT4 overexpression alone. HKTg + GLUT4Tg mice were able to maintain glycemia constant during both sedentary and exercise conditions, even with dramatic increases in glucose transport and phosphorylation capacities.

The disappearance curves of the radioactive glucose analog, [2-3H]DG, from the plasma for the HKTg and HKTg + GLUT4Tg mice are shown in Fig. 2. During both the sedentary and exercise conditions, HKTg + GLUT4Tg mice had increased clearance of the glucose tracer compared with HKTg mice. Unlike the response seen in WT and GLUT4Tg mice, exercise led to an increase in the disappearance of [2-3H]DG in HKTg and HKTg + GLUT4Tg mice, as seen by the large downward shift in the [2-3H]DG disappearance curves during exercise compared with during rest.

GLUT4 overexpression in conjunction with HK II overexpression led to an ~2.5-fold increase in sedentary Kg and a 2-fold increase in sedentary Rg in the soleus, gastrocnemius, and SVL muscles compared with HKTg mice (right half of Figs. 3 and 4, respectively). Exercise significantly increased Kg and Rg compared with the sedentary condition in all muscles studied of both genotypes. GLUT4 overexpression on the background of HK II overexpression did not further augment Rg during exercise. However, GLUT4 overexpression in combination with HK II overexpression led to a synergistic increase in exercise-stimulated Kg compared with HK II or GLUT4 overexpression alone. It should be noted that the effect of HK II overexpression alone on exercising Rg in C57Bl/6J mice is qualitatively similar to the response reported previously by this laboratory in FVB/NJ mice (12). However, the response seen in the 5-h-fasted C57Bl/6J mice here was larger than the response in 5-h-fasted FVB/NJ mice and more closely resembled the response in 18-h-fasted FVB/NJ mice.

Exercise led to a decrement in muscle glycogen of ~0.6 and 1.4 mg/g tissue wt in the HKTg and HKTg + GLUT4Tg mice, respectively (Table 2). Glycogen breakdown in response to exercise was increased significantly in the SVL of HKTg + GLUT4Tg mice compared with HKTg mice (Fig. 5). In comparison with HK II overexpression alone, GLUT4 overexpression combined with HK II overexpression had no effect on muscle G-6-P (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The aim of the present study was to define the barriers to MGU during exercise in vivo using transgenic mice. MGU can be separated into the following three sequential steps: delivery of glucose from the blood to the sarcolemma, transport across the sarcolemma, and intracellular phosphorylation by HK. It has been proposed that regulation of MGU can occur at all three of these steps and that they all may become barriers to MGU in healthy and diseased states (42). For example, during sedentary conditions, glucose transport is the predominant site of resistance to MGU (38), whereas glucose phosphorylation is not a barrier (12). However, during exercise, the control of glucose uptake shifts to phosphorylation (11, 12). The data presented here confirm these previous findings. Moreover, we show that membrane transport is not a primary barrier to MGU during moderate exercise in the intact mouse, since overexpression of GLUT4 has little effect on Rg or the glucose concentration-independent Kg. In fact, the incremental response with exercise is actually somewhat reduced. Only when the barrier to glucose phosphorylation is lowered by overexpressing HK II does glucose transport become a barrier to exercise-stimulated MGU.

Several studies have determined the effect of GLUT4 overexpression on MGU in isolated muscles (15, 25, 29, 38) or hindlimb preparations (4). These studies have been extremely informative but do not take into account glucose delivery and other physiological adaptations to exercise. The results of this study are novel in that they are the first actual measurements of muscle glucose utilization during exercise in GLUT4-overexpressing mice either with or without HK II overexpression. These measurements were obtainable because of the unique catheterization technique employed. The addition of arterial catheterization permits the use of [2-3H]DG to determine Kg and Rg, indexes of tissue-specific glucose uptake, in the conscious mouse during a physiological condition such as exercise. The former variable is glucose concentration independent and is, therefore, useful for comparing genotypes with differences in glycemia, as is the case here. This catheterization technique provides a better index of the physiological regulation of MGU than do isolated muscle or anesthetized animal preparations. In conjunction with the transgenic manipulations employed here, we are able to determine the functional barriers to MGU during moderate exercise in the intact animal.

The general premise in the design of this study is that, if a step is a significant barrier of a metabolic pathway, transgenic overexpression of the enzyme responsible for that step will remove or at least reduce this step as a barrier, thereby increasing the flux rate through the given pathway. Previous studies have shown that transgenic overexpression of GLUT4 with the hGLUT4-11.5 construct increases the GLUT4 protein content approximately two- to threefold in skeletal muscle, yielding an ~50% increase in basal glucose uptake of isolated soleus, epitrochlearis, and extensor digitorum longus skeletal muscles (15, 41). Although the massive increase in total cellular GLUT4 protein content likely overloads the translocation machinery, the GLUT4 protein content in the plasma membrane is still markedly increased in GLUT4Tg compared with WT mice during both basal and stimulatory conditions (4). The data from the sedentary mice presented here show that basal Rg is increased by 230 and 166% in the gastrocnemius and SVL muscles, respectively. The more dramatic response documented here as opposed to isolated muscles or hindlimb preparations is likely because of the fact that measurements are obtained from the conscious mouse. These results are even more impressive given the fact that GLUT4 overexpression leads to a marked reduction in muscle glucose delivery (i.e., reduced blood glucose). Previous work (11, 12, 16) has demonstrated that glucose phosphorylation is not a major barrier to glucose uptake in sedentary muscles of normal chow-fed animals. The data presented here support those findings, since HK II overexpression did not increase sedentary Rg. Even when basal glucose transport is increased at least twofold by GLUT4 overexpression, phosphorylation did not become a major barrier to MGU, since the addition of HK II overexpression did not further augment sedentary Rg. These data are consistent with those from GLUT1-overexpressing mice (16). Thus it is evident that glucose transport exerts most of the control of MGU under sedentary, postabsorptive conditions. The contribution that glucose delivery makes in controlling sedentary MGU remains to be determined but, in light of the effects of GLUT4 overexpression, is likely to be minor.

With muscle contractions, GLUT4 translocates from an intracellular pool to the sarcolemma (3, 8, 37), increasing the myocyte's permeability to glucose. The effectiveness of exercise-induced translocation is evident by the finding that GLUT4 overexpression alone was not able to further augment exercise-stimulated MGU. The finding that HK II overexpression increases exercise-stimulated Rg compared with the response observed in WT mice (see Fig. 4) is consistent with previous results in FVB/NJ mice (12) and supports the concept that the control of MGU shifts to glucose phosphorylation during exercise in vivo (11, 12). Additional support for this concept was generated by studies using multiple radioisotopic glucose analogs to calculate glucose concentration gradients from the arterial blood to the cytoplasm of the myocyte in the rat (11). Studies performed by Katz et al. (21, 22) in exercising humans have provided similar evidence regarding the role of glucose phosphorylation in the control of exercise-stimulated MGU, and they have purported that the initial increases in G-6-P cause the cell to rely more heavily on glycogen for energy rather than glucose. As the glycogen pool diminishes, the G-6-P levels will fall, thereby lowering the G-6-P inhibition of HK II and permitting more glucose entry. Another reason why the control shifts to glucose phosphorylation is that exercise also lowers the glucose delivery barrier by increasing blood flow and capillary recruitment and thus muscle perfusion (17, 36). As the barriers to both glucose delivery and transport are lowered with acute exercise, the primary control of MGU shifts to that of glucose phosphorylation. This is verified by the current study, since HK II overexpression alone increases exercising Rg by 82, 60, and 169% in soleus, gastrocnemius, and SVL muscles, respectively. Thus, in a muscle with normal glucose transport capacity, glucose phosphorylation by HK is the primary barrier to MGU during exercise in vivo.

Transgenic overexpression of GLUT4 in conjunction with HK II increased exercise-stimulated Kg, but not Rg, compared with HK II-overexpressing muscle alone. Thus, when MGU is considered after normalization of arterial glucose concentrations (i.e., Kg), glucose transport becomes a barrier to MGU when the barrier to glucose phosphorylation is reduced. The fact that Rg is not enhanced in HKTg + GLUT4Tg compared with HKTg mice may be resultant from reduced arterial glucose concentrations and suggests that glucose delivery becomes a chief barrier to MGU during exercise in muscles in which the barriers to glucose transport and phosphorylation have been lowered by transgenic overexpression. This hypothesis is supported by the decrease in free muscle glucose in response to exercise of HKTg + GLUT4Tg mice that was significant in the soleus muscle (data not shown).

The lower arterial plasma glucose concentration in mice overexpressing GLUT4 may limit the exercising Rg since MGU is dependent on blood glucose concentration (31). However, when comparing exercise responses of genotypes with similar arterial blood glucose concentrations (i.e., WT vs. HKTg and GLUT4Tg vs. HKTg + GLUT4Tg), the increase in Rg during exercise attributed to HK II overexpression is identical. That is, despite differences in arterial blood glucose concentrations, HK II overexpression enhanced exercised-stimulated Rg in all muscles studied. Furthermore, when Kg is considered to eliminate the confounding effect of arterial blood glucose concentration, an effect of GLUT4 overexpression is exposed only on the background of HK II overexpression (see Fig. 3). In fact, the combined transgenes have a synergistic effect on Kg during exercise. Thus GLUT4 overexpression only becomes an important site of control when the barrier to glucose phosphorylation is lowered by HK II overexression.

The effectiveness of muscle contractions to lower the resistance to membrane glucose transport is even more remarkable given that, in the WT and HKTg mice, the plasma insulin concentration significantly fell by ~6 µU/ml by the end of exercise (data not shown). There was no appreciable difference in the insulin concentrations at the beginning and end of exercise in the GLUT4Tg and HKTg + GLUT4Tg mice. Similarly, the elevation in resting Rg and Kg of GLUT4Tg and HKTg + GLUT4Tg mice occurs even though insulin concentration is not greater than in WT or HKTg mice.

It is also noteworthy that the liver retains an exquisite ability to maintain blood glucose during exercise despite dramatic increases in MGU seen in some of the transgenic mice. For example, blood glucose concentration did not fall during exercise in HKTg mice despite marked increases in MGU compared with sedentary HKTg mice. This suggests that endogenous glucose production is increased proportionately. Future studies will be needed to determine the mechanism for such an increase in endogenous glucose production resulting from the transgenic manipulations employed here as well as alterations in fatty acid metabolism.

GLUT4Tg and HKTg + GLUT4Tg mice had increased glycogen breakdown associated with exercise compared with WT and HKTg mice, respectively. Increased glycogen breakdown has been reported previously in GLUT4-overexpressing mice during exercise (2). The GLUT4Tg and HKTg + GLUT4Tg mice had comparable glycogen breakdowns during exercise, and thus HK II overexpression was not able to attenuate the accelerated glycogen breakdown created by GLUT4 overexpression. The accelerated glycogen breakdown may have been the result of a heightened counterregulatory response during exercise to prevent a decline in the already reduced blood glucose concentrations of these mice overexpressing GLUT4. Counterregulatory hormones, such as catecholamines and glucagon, were not measured in the present study. One can speculate that elevated catecholamines might increase muscle glycogenolysis and inhibit glucose transport in the contracting muscle (18, 23, 43, 44). One might ascribe the finding that GLUT4 overexpression did not augment exercise-stimulated MGU to the notion that these mice relied more heavily on glycogen for energy; thus, there was no need to further increase MGU. This is not likely the case, since HKTg + GLUT4Tg mice exhibited an equal exercise-stimulated MGU compared with HKTg mice and had a comparable glycogen breakdown compared with GLUT4Tg mice.

As expected, there are several key differences between muscles of different fiber types. For example, the soleus muscle, being more metabolically active and subjected to tonic contractions, has a significantly greater Rg during the sedentary condition in all genotypes. This high rate of MGU is maintained during exercise. Basal MGU is similar between the gastrocnemius and SVL, which is consistent with their similar fiber type composition (1). The exercise-stimulated Rg in the gastrocnemius is likely the result of different muscle recruitment patterns with treadmill running. Muscle glucose concentration follows a similar pattern, since the soleus muscle contains a higher concentration than that seen in the other muscles in all genotypes and conditions studied. Conversely, the G-6-P levels are greater in the gastrocnemius and SVL muscles compared with the soleus muscle in all genotypes and conditions studied. Importantly, the transgenic manipulations employed here do not alter any of these general muscle fiber type trends; thus, MGU per se does not likely determine the set point of muscle glucose and G-6-P.

Other investigators have studied the regulation of MGU during both basal and stimulated states, such as by insulin, and isolated muscle contractions using similar transgenic mice (i.e., GLUT1-, GLUT4-, and HK II-overexpressing mice). The data presented here in regard to the basal (i.e., sedentary) state are in agreement with previously published reports using GLUT4 (7, 15, 38) and HK II (6, 12, 16, 27) transgenic mice. That is, GLUT4 but not HK II overexpression increases basal MGU. During insulin stimulation, it has been reported that GLUT4 overexpression increases whole body glucose disposal in vivo (7, 39) and MGU in vitro (7, 15). Hansen et al. (15) go on to demonstrate that GLUT4 overexpression augments postexercise MGU. We and others have shown that, although HK II overexpression enhances insulin-stimulated MGU in vivo (12, 27), it has minimal, if any, effects on whole body glucose disposal (12, 16). The effectiveness of HK II overexpression, however, has varied in isolated muscle preparations. Chang et al. (6) initially reported that HK II overexpression increased insulin-stimulated [2-3H]DG uptake in extensor digitorum longus, soleus, and epitrochlearis muscles in vitro. In contrast, Hansen et al. (16) demonstrated that maximally insulin-stimulated [2-3H]DG uptake was similar in isolated extensor digitorum longus muscles from WT and HKTg mice. The current study is novel in that it demonstrates that HK II but not GLUT4 overexpression enhances MGU during exercise in intact, conscious C57Bl/6J mice.

In summary, isotopic methods were combined with transgenic manipulations to determine the barriers to MGU in the conscious mouse under physiological conditions. Although it is clear that increasing GLUT4 content in skeletal muscle has marked effects on MGU in the resting state, it was unknown whether GLUT4 overexpression would augment exercise-stimulated MGU. Previous work demonstrated that, during exercise, HK II exhibited control of MGU in FVB/NJ mice and led to the hypothesis that glucose transport was not limiting under this condition. The corollary to this is that GLUT4 overexpression would have no effect on exercise-stimulated MGU. This was, in fact, the case even after normalizing for reduced arterial glucose concentrations in GLUT4-overexpressing mice. It was our hypothesis that, if HK II activity were increased, glucose transport would then become a site of resistance to MGU during exercise. GLUT4 combined with HK II overexpression enhanced muscle Kg during exercise. In conclusion, the control of MGU shifts from membrane transport at rest to phosphorylation during exercise in conscious C57Bl/6J mice. Glucose transport is only a significant control point for exercise-stimulated MGU when the phosphorylation barrier is lowered by overexpression of HK II.


    GRANTS
 TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01 DK-54902 and U24-DK-59637.


    ACKNOWLEDGMENTS
 
We thank Wanda Snead of the Vanderbilt Hormone Assay Core for performing the insulin assays.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. T. Fueger, Dept. of Molecular Physiology and Biophysics, Vanderbilt Univ. School of Medicine, Nashville, TN 37232-0615 (E-mail: patrick.fueger{at}vanderbilt.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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