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1 Department of Molecular Physiology & Biophysics
2 Mouse Metabolic Phenotyping Center
3 Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA
4 Department of Medicine
5 A. I. Virtanen Institute for Molecular Sciences and Department of Biotechnology and Molecular Medicine,University of Kuopio, Kuopio, Finland
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Abstract |
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3-fold HK overexpression (HKTg) were tested. Mice (n
= 12/group) completed both a maximal oxygen consumption 
test and an endurance capacity test (run at 75%
) on an enclosed treadmill equipped to measure gas exchange. Arterial and venous catheters were surgically implanted into separate groups of mice (n
= 9–11/group) in order to measure an index of muscle glucose uptake (Rg) during 30 min of treadmill exercise. Maximum work rate (0.95 ± 0.05, 1.00 ± 0.04 and 1.06 ± 0.07 kg m min–1), 
(137 ± 3, 141 ± 4 and 141 ± 5 ml kg–1 min–1) and maximal respiratory exchange ratio (1.04 ± 0.02, 1.00 ± 0.03 and 1.04 ± 0.04) were similar in HK+/–, WT and HKTg, respectively. Exercise endurance capacity (measured as time to exhaustion) increased as HK content increased (55 ± 11, 77 ± 5 and 98 ± 9 min) and this was related to Rg measured in mice during 30 min of exercise (13 ± 2, 24 ± 5 and 42 ± 5 µmol (100 g)–1 min–1). Muscle glycogen in sedentary HK+/– mice and HK+/– mice following 30 min of exercise were significantly lower than in HKTg and WT mice. However, the net exercise-induced muscle glycogen breakdown was equal in the three genotypes. In summary, HK protein content within the range studied (a) was not associated with a difference in the capacity to perform maximal intensity exercise, (b) was a powerful determinant of the ability to sustain moderate intensity exercise, as reducing HK content impaired endurance and increasing HK content enhanced endurance, and (c) although directly related to exercise endurance, was not a determinant of net muscle glycogen usage during exercise. In conclusion, adaptations that increase HK protein content and/or functional activity such as regular exercise contribute to increased muscular endurance.
(Received 11 February 2005;
accepted after revision 28 April 2005;
first published online 5 May 2005)
Corresponding author P. T. Fueger: Duke University Medical Center, Department of Pharmacology and Cancer Biology, 4321 Medical Park Drive, Suite 200, Durham, NC 27704, USA. Email: patrick.fueger{at}duke.edu
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Introduction |
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The aim of this study was to determine if alterations in exercise-stimulated MGU by manipulations to HKII content would affect exercise endurance capacity in the postabsorptive state and spare muscle glycogen. To this end, C57Bl/6J mice with (HK+/–) and without (WT) a partial deletion of HKII as well as mice overexpressing HKII (HKTg) were studied during 30 min of moderate-intensity exercise and a moderate-intensity exercise endurance test (defined as time to exhaustion). To distinguish a general physical impairment from one that is specific to endurance exercise, mice also underwent an incremental exercise test to exhaustion to determine the capacity for maximal intensity exercise.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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All procedures performed were approved by the Vanderbilt University Animal Care and Use Committee. Male Ukko1 (a mixture of BALB/c and DBA/2 strains) mice containing a partial deletion to the HKII gene (HK+/–) that results in a 50% reduction in HKII activity in heart and skeletal muscle, as well as in adipose tissue, but does not change HKI activity (Heikkinen et al. 1999) were backcrossed onto the C57BL/6J background for at least five generations. HK+/– mice on the C57BL/6J background were bred with mice containing a HKII transgene composed of the human HKII cDNA driven by the rat muscle creatine kinase promoter (Chang et al. 1996). This breeding strategy yielded C57BL/6J mice containing three different doses of HKII in skeletal muscle: HKII partial knockout, wild-type and HKII-overexpressing mice (HK+/–, WT and HKTg, respectively). At 3 weeks of age, littermates were separated by sex and maintained in microisolator cages. Genotyping was performed with the polymerase chain reaction on genomic DNA obtained and isolated from tail biopsies as previously described (Halseth et al. 1999; Heikkinen et al. 1999). Genotyping was performed after studies in mice tested for maximal and endurance exercise capacity so as to eliminate the possibility of investigator bias. Mice were fed standard chow diet ad libitum and studied at 4 months of age.
Surgical procedures
The surgical procedures utilized are the same as those previously described (Niswender et al. 1997; Halseth et al. 1999). Briefly, mice of either sex were anaesthetized with pentobarbital (I.P. injection, 70 mg (kg body weight)–1) and the left common carotid artery and the right jugular vein were catheterized for sampling and infusions, respectively. The free ends of catheters were tunnelled 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 o.d.), which were exteriorized and sealed with stainless steel plugs. Lines were kept patent by flushing each daily with 10–40 µl saline containing 200 U ml–1 of heparin and 5 mg ml–1 of ampicillin. Animals were housed individually after surgery and body weight was recorded daily. Following an 5-day period in which body weight was restored (within 10% of presurgery body weight) mice were acclimated to treadmill running with a single 10-min bout of exercise (15–16.7 m min–1, 0% grade). Experiments were performed 2 days following the treadmill acclimation trial.
Metabolic experiments
Experiments were performed on 5 h fasted mice as previously described (Fueger et al. 2003, 2004a,b). Approximately 1 h prior to an experiment, mice were placed on an enclosed treadmill in order to let them acclimate to the environment. At t
= 0 min, a baseline arterial blood sample (150 µl) was drawn for the measurement of blood glucose (HemoCue, Mission Viejo, CA, USA), haematocrit and plasma insulin and non-esterified fatty acids (NEFAs). To minimize a fall in haematocrit, the remaining erythrocytes were washed once with 0.9% saline containing 10 U ml–1 of heparin and reinfused. The mice either remained sedentary or ran on the treadmill for 30 min at 16.7 m min–1 with a 0% grade (n
= 8–10 for each experimental group and genotype). The selected work intensity is 80% of maximal oxygen consumption (Fernando et al. 1993). At t
= 5 min, a 12 µCi bolus of [2-3H]deoxyglucose ([2-3H]DG; New England Nuclear, Boston, MA, USA) was administered in order to provide an index of tissue-specific glucose uptake (Rg). At t
= 10, 15 and 20 min, 50 µl of arterial blood was sampled in order to determine blood glucose and plasma [2-3H]DG. At t
= 30 min, a 150 µl arterial blood sample was withdrawn in order to determine blood glucose, haematocrit and plasma insulin, [2-3H]DG and NEFAs and mice were anaesthetized with an arterial infusion of sodium pentobarbital (3 mg). The gastrocnemius muscles were excised, immediately frozen in liquid nitrogen and stored at –70°C until future tissue analysis. Mice were then killed by excising the heart under anaesthesia.
Maximal and endurance exercise testing
A separate group of animals (n
= 12 for each genotype) that had not undergone surgical catheterization were used to measure oxygen consumption 
during rest and exercise. Whole body 
and respiratory exchange ratio (RER) were measured with mice on an enclosed treadmill using an Oxymax Deluxe System (Columbus Instruments, Columbus, OH, USA) with an airflow rate of 1.0 l min–1.
For maximal 
testing, mice were placed on the enclosed treadmill and allowed to acclimate to their surroundings for 45 min. Resting 
was measured for 15 min. Mice then began running at 10 m min–1 and the speed was increased 4 m min–1 every 3 min until mice were no longer able to keep pace with the treadmill. Mice were encouraged to run as long as possible with the use of an electric grid placed at the end of the treadmill (1.5 mA, 200 ms pulses, 4 Hz). Mice were defined as exhausted if they remain on the shock grid for five continuous seconds. Maximal 
was achieved when 
no longer increased despite an increase in work rate.
One week later, exercise endurance was measured. Mice were placed in the enclosed treadmill and allowed to acclimate to their surroundings for 45 min as before. Resting 
was measured for 15 min. Mice were then run at 20 m min–1 until they were no longer able to keep pace with the treadmill. Exhaustion was determined as described above for the maximal exercise test. Total whole-body carbohydrate and fat oxidation were determined during rest, steady-state exercise and the minutes preceding exhaustion from gas exchange data based on established equations (Frayn, 1983). Gastrocnemius muscles were excised under anaesthesia as before in order to determine muscle glycogen following a 30 min recovery period and mice were killed as described above.
Echocardiography and blood pressure measurement
One week prior to completing maximal exercise testing transthoracic echocardiograms were performed on resting conscious mice using a 15-MHz transducer (Sonos 5500 system, Agilent) as previously described (Exil et al. 2003; Rottman et al. 2003). In addition, systolic blood pressure was measured in conscious mice at rest using tail cuff plethysmography (Weisberg et al. 2004).
Processing of plasma and muscle samples
Immunoreactive insulin was assayed with a double antibody method (Morgan & Lazarow, 1965). NEFAs were measured spectrophotometrically by an enzymatic colorimetric assay (Wako NEFA C kit, Wako Chemicals Inc., Richmond, VA, USA).
Following 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, USA) with Ultima Gold (Packard) as scintillant. Muscle samples were homogenized in 0.5% perchloric acid and neutralized with KOH. One aliquot was counted directly in order to determine total radioactivity ([2-3H]DG and [2-3H]DGP). A second aliquot was treated with Ba(OH)2 and ZnSO4 in order to remove [2-3H]DGP and any tracer incorporated into glycogen and then counted to determine [2-3H]DG radioactivity (Meszaros et al. 1987). [2-3H]DGP is the difference between the two aliquots. In all experiments the accumulation of [2-3H]DGP was normalized to tissue weight and bolus radioactivity. Rg was calculated as described by Kraegen et al. (1985). Muscle glycogen was determined by the method of Chan & Exton (1976) on the gastrocnemius muscle.
Total HKII protein content was determined on gastrocnemius muscles homogenized in a solution containing 10% glycerol, 20 mM sodium pyrophosphate, 150 mM NaCl, 50 mM Hepes (pH 7.5), 1% NP-40, 20 mM ß-glycerophosphate, 10 mM NaF, 2 mmol l–1 EDTA (pH 8.0), 2 mM phenylmethylsulphonyl fluoride, 1 mM CaCl2, 1 mM MgCl2, 10 µg ml–1 aprotinin, 10 µg ml–1 leupeptin, 2 mM Na2VO3 and 3 mM benzamide. After centrifugation (1 h at 4500 g) pellets were discarded and supernatants were retained for protein determination using a Pierce BCA protein assay kit (Rockford, IL, USA). Proteins (30 µg) were separated on a SDS-PAGE gel and then transferred to a PVDF membrane. Membranes were blocked, probed with rabbit anti-HKII (1: 1000; Chemicon International; Temecula, CA, USA) and then incubated with anti-rabbit horseradish peroxidase (1: 20000; Pierce, Rockford, IL, USA). In order to confirm equal protein loading and transfer, membranes were stripped and reprobed with monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1: 4000, Abcam) and then incubated with anti-mouse (1: 20000, Amersham). Densitometry was performed using ImageJ software (NIH).
Capillary density was determined in 5 µm sections of paraffin-embedded gastrocnemius muscles following immunohistochemical detection of CD-31 (platelet endothelial cell adhesion molecule-1, Pecam-1) in endothelial cells. Endogenous peroxidase was quenched with 0.03% hydrogen peroxide and samples were treated with diluted rabbit serum prior to primary antibody addition. Slides were incubated with goat anti-CD-31/Pecam-1 (1: 400, Santa Cruz Biotechnology) for 45 min. The Vectastain ABC Elite (Vector Laboratories, Inc.) System and DAB+ (DakoCytomation) was used to produce visible staining. Slides were lightly counterstained with Mayer's haematoxylin, dehydrated and coverslipped. For each muscle, capillaries in three visible fields were counted and averaged.
Statistical analysis
Data are presented as means ± S.E.M. Differences between groups were determined by ANOVA followed by Tukey's post hoc test. The level of significance was set at P < 0.05.
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Results |
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Total HKII protein content is shown in Fig. 1A and B. As expected, a partial HKII knockout decreased total HKII content by approximately 50%. The HKII transgene increased HKII protein content to approximately 3.5 times that observed in WT gastrocnemius muscles.
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relative to WT animals, but HKTg mice had increased 
compared to HK+/– mice at rest (Table 2).
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Exercise-stimulated Rg increased as HKII content increased (Fig. 1C). That is, relative to WT mice, HK+/– mice had a lower Rg during exercise and HKTg mice had a higher Rg. Interestingly, the reduction in exercise-stimulated Rg observed in HK+/– mice occurred despite elevated blood glucose throughout exercise (Fig. 2A), elevated insulin concentrations at the onset of exercise (Fig. 2B) and decreased NEFAs throughout exercise (Fig. 2C). Muscle glycogen in sedentary HK+/– mice and HK+/– mice following 30 min of exercise were significantly lower than during identical conditions in HKTg and WT mice (Fig. 3). However, the net exercise-induced muscle glycogen breakdown was equal in the three genotypes.
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Capacity for maximal intensity exercise were similar in HK+/–, WT and HKTg as mice obtained work rates of 0.95 ± 0.05, 1.00 ± 0.04 and 1.06 ± 0.07 kg m min–1, respectively. HK+/–, WT and HKTg had similar 
(Table 2) and RER at maximal exercise intensity (1.04 ± 0.02, 1.00 ± 0.03 and 1.04 ± 0.04, respectively).
Capacity for endurance exercise
Because all genotypes had comparable capacities for maximal intensity exercise, endurance testing was performed at similar absolute and relative exercise intensities (80 ± 3, 72 ± 4 and 70 ± 8%
for HK+/–, WT and HKTg, respectively). Compared to WT littermates, endurance capacity was reduced by 30% in HK+/– mice (Fig. 4). In contrast, HKTg mice had significantly improved endurance capacity compared to WT mice. During exercise, RER was not different between HK+/–, WT and HKTg mice (0.87 ± 0.01, 0.87 ± 0.03 and 0.84 ± 0.02, respectively). The dependence of exercise endurance on HKII content was qualitatively similar to that observed in exercise-stimulated Rg (Fig. 1B).
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Total carbohydrate and fat oxidation were not influenced by HKII content (Table 4). Exercise increased carbohydrate oxidation in all genotypes. Carbohydrate oxidation remained relatively constant throughout exercise even up to the minutes preceding exhaustion. Fat oxidation was consistent between rest, steady-state exercise and prior to exhaustion.
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Discussion |
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It was previously shown (Fueger et al. 2003) that a partial HKII knockout impaired MGU in oxidative muscles of mice on a background mixture of BALB/c and DBA/2 strains. The disrupted HKII gene was backcrossed onto the C57BL/6J background for at least five generations in order to study the effect of this genetic manipulation as well as HKII overexpression in the C57BL/6J mouse. We report here that a partial HKII knockout dramatically altered the normal physiological response to exercise in this strain. Exercise-induced hyperglycaemia and exercise intolerance resulted and were associated with impaired glucose uptake in the gastrocnemius muscle. In contrast, HKII overexpression in C57Bl/6J mice was beneficial to exercise endurance and this was associated with an increase in exercise-stimulated MGU.
In the present study, a partial disruption of HKII in C57BL/6J mice also created a metabolic phenotype under postabsorptive, sedentary conditions, characterized by mild hyperglycaemia and hyperinsulinaemia, suggesting glucose intolerance and insulin resistance. The net result of these changes is a slight paradoxical increase in Rg during rest. The lower NEFAs and muscle glycogen content in HK+/– mice may be an adaptation that blunts this monogenic form of insulin resistance. Interestingly, the heterozygous deletion of the HKII gene was not as detrimental in mice on a mixed background (Fueger et al. 2003). While exercise-stimulated MGU was reduced in oxidative muscles, these mice do not display exercise-induced hyperglycaemia.
Moderate exercise generally resulted in a decline in plasma insulin and NEFA concentrations in mice of all genotypes. Arterial blood glucose tended to rise at the onset of exercise and plateau by 15 min of the 30 min protocol. The rise in arterial blood glucose was most pronounced in mice with a partial HKII deletion. During exercise, the massive hyperaemia (Honig et al. 1980; Palm et al. 1983) and translocation of GLUT4 from the cytoplasm to sarcolemma (Ploug et al. 1992; Etgen et al. 1993; Brozinick et al. 1994) lead to a robust increase in MGU. The result of these two exercise-stimulated events is that glucose phosphorylation capacity is more important than glucose delivery or transport in determining MGU (Halseth et al. 1998, 1999; Fueger et al. 2003; Fueger et al. 2004b). Here we report an almost 10-fold increase in Rg in the exercising gastrocnemius of WT mice. Despite the relative hyperglycaemia and hyperinsulinaemia in HK+/– mice fed a chow diet, exercise was only able to increase Rg by three- to fourfold (compare Table 1 to Fig. 1C).
One might hypothesize that the impairment in MGU created by reducing HKII content would accelerate glycogen breakdown and lead to reduced endurance capacity. The same line of thought suggests that increasing MGU by increasing HKII content would preserve glycogen mass and increase endurance capacity. Indeed, people with McArdle's disease have an inability to mobilize glycogen (McArdle, 1951; Schmidt & Mahler, 1959) and fatigue more quickly. Supplementation with glucose or fatty acids can improve exercise tolerance in these patients (Lewis et al. 1985; Lewis & Haller, 1986; Mineo et al. 1990; Dorin et al. 1996; Yamauchi et al. 1996). Basal and postexercise muscle glycogen content is, in fact, reduced but not completely depleted in the more readily fatigued HK+/– mice. Muscle glycogen levels in WT and HKTg mice were similar in sedentary and exercised mice showing that glycogen is not spared by HKII overexpression. Moreover, the amount of gastrocnemius glycogen broken down by exercise was the same, regardless of genotype. These data suggest that muscle glycogen, within the range of HKII concentrations studied, does not limit endurance capacity in 5 h fasted mice. Along the same lines, Pederson et al. (2005) have recently demonstrated that mice without the muscle isoform of glycogen synthase do not have impaired exercise endurance. Therefore, at least in mice, it appears that it is the ability to sustain the influx of blood glucose, independent of muscle glycogen depletion, which limits exercise endurance.
The possibility exists that changes in the cardiovascular system are the cause of such changes in exercise endurance observed in the present study. This is unlikely to be the case as muscle capillary density, left ventricular mass and cardiac function and dimensions are similar in all genotypes examined in the present study. In addition to these results is the observation that 
and the maximal work rate achieved during the maximal exercise test were not altered by manipulations to HKII content. Similar capacities for maximum exercise performance, regardless of HKII expression, suggest that endurance exercise was performed at the same absolute and relative work intensities.
Despite the increased muscle glucose utilization in exercising HKTg mice, blood glucose was comparable to that seen in WT mice. It can be calculated that, if the gastrocnemius of mouse is representative of mouse muscle in general and if 60% of mouse weight is muscle, then whole body glucose utilization would increase by approximately 20 mg kg–1 min–1 during exercise compared to WT mice. Of course this may be a slight overestimation since some muscles may not have comparable energy requirements to the gastrocnemius during exercise. Since arterial blood glucose was the same in WT and HKTg, it can be calculated that endogenous glucose production must have been equally increased as well. Thus, despite the massive increase in glucose requirements, the livers of HKTg mice precisely accommodate the enhanced exercise-stimulated MGU, suggesting that liver glucose production is highly sensitive to muscle glucose utilization during exercise. This is consistent with studies in dogs and humans (Wasserman, 1995). However, the mechanism for this tight coupling process is not well understood. Although not measured in the present study, it is possible that lactate flux is greater in HKTg mice compared to WT mice. The increased lactate load to the liver could, in turn, be used for enhanced gluconeogenesis in order to support the increased glucose requirements of HKTg mice during exercise.
The observation here that simply manipulating one gene that encodes a metabolic enzyme can markedly affect exercise endurance is striking but obviously does not completely explain the physiological changes that occur with exercise training. For example, it is well-known that endurance-trained individuals actually rely less on carbohydrates during exercise due to their increased ability to oxidize fatty acids. Interestingly, while manipulating HKII in skeletal muscle altered exercise-stimulated MGU and exercise endurance capacity, it did not affect total whole body carbohydrate or fat oxidation. It is possible that substrate oxidation was changed by the genetic manipulations at the muscle level but these differences could not be detected in the whole animal due to the high metabolic rates in the mouse, even at rest. The adaptive training response in humans involves myriad changes in gene expression. It is indeed rare that the impact of a change in the expression of any single protein can be studied in humans (with the exception being rare polymorphisms). Thus, in vivo experiments in transgenic rodents (as employed here) are a useful method for studying organ-specific manipulations to selected proteins in the context of the whole animal.
In summary, manipulating muscle HKII content in the mouse provides a model system for assessing the impact of increased MGU without having to create marked changes in blood glucose or insulin. HK protein content within the range studied (a) was not associated with a difference in the capacity to perform maximal intensity exercise, (b) was a powerful determinant of the ability to sustain moderate intensity exercise, as reducing HK content impaired endurance and increasing HK content enhanced endurance, and (c) while directly related to exercise endurance time, was not a determinant of muscle glycogen usage. In conclusion, adaptations that increase HK protein content and/or functional activity such as regular exercise contribute to increased muscular endurance.
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P < 0.05 versus HK+/– and WT. C, an index of muscle glucose uptake (Rg) was determined in a separate group of mice that had catheters surgically implanted 1 week prior to an experiment into a jugular vein and carotid artery for infusions and arterial blood sampling, respectively. For an experiment, mice were fasted for 5 h, run on a motorized treadmill at 16.7 m min–1 for 30 min and given a 12 µCi bolus of [2-3H]deoxyglucose at 5 min into the exercise trial. Gastrocnemius Rg was calculated as has been previously described (
(ml kg–1 min–1) during rest and exercise in C57BL/6J mice with three levels of HKII
P < 0.05 versus WT and HK+/–.
