Glucose kinetics and exercise tolerance in mice lacking the GLUT4 glucose transporter

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1 J Physiol (27) pp Glucose kinetics and exercise tolerance in mice lacking the GLUT4 glucose transporter Patrick T. Fueger 1, Candice Y. Li 1, Julio E. Ayala 1, Jane Shearer 1, Deanna P. Bracy 1,3, Maureen J. Charron 4, Jeffrey N. Rottman 2,3 and David H. Wasserman 1,3 1 Department of Molecular Physiology and Biophysics, 2 Department of Internal Medicine and 3 Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA 4 Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 461, USA The absence of GLUT4 severely impairs basal glucose uptake in vivo, but does not alter glucose homeostasis or circulating insulin. Glucose uptake in isolated contracting skeletal muscle (MGU) is also impaired by the absence of GLUT4, and onset of muscle fatigue is hastened. Whether the body can compensate and preserve glucose homeostasis during exercise, as it does in the basal state, is unknown. One aim was to test the effectiveness of glucoregulatory compensation for the absence of GLUT4 in vivo. The absence of GLUT4 was also used to further define the role of hexokinase (HK) II, which catalyses glucose phosphorylation after it is transported in the cell. HK II increases MGU during exercise, as well as exercise endurance. In the absence of GLUT4, HK II expression will not affect MGU. A second aim was to test whether, in the absence of GLUT4, HK II retains its ability to increase exercise endurance. Wild-type (), GLUT4 null (GLUT4 / ), and GLUT4 null overexpressing HK II (GLUT4 / HK Tg ) mice were studied using a catheterized mouse model that allows blood sampling and isotope infusions during treadmill exercise. The impaired capacity of working muscle to take up glucose in GLUT4 / is partially offset by an exaggerated increase in the glucagon : insulin ratio, increased liver glucose production, hyperglycaemia, and a greater capillary density in order to increase the delivery of glucose to the exercising muscle of GLUT4 /. Hearts of GLUT4 / also exhibited a compensatory increase in HK II expression and a paradoxical increase in glucose uptake. Exercise tolerance was reduced in GLUT4 / compared to. As expected, MGU in GLUT4 / HK Tg was the same as in GLUT4 /. However, HK II overexpression retained its ability to increase exercise endurance. In conclusion, unlike the basal state where glucose homeostasis is preserved, hyperglycaemia results during exercise in GLUT4 / due to a robust stimulation of liver glucose release in the face of severe impairments in MGU. Finally, studies in GLUT4 / HK Tg show that HK II improves exercise tolerance, independent of its effects on MGU. (Received 2 March 27; accepted after revision 9 May 27; first published online May 27) Corresponding author P. T. Fueger: Duke University Medical Center, Department of Pharmacology and Cancer Biology, 4321 Medical Park Drive, Suite 2, Durham, NC 2774, USA. patrick.fueger@duke.edu GLUT4 is the major glucose transporter isoform in skeletal and cardiac muscle, yet fasting blood glucose and insulin concentrations are normal in sedentary whole-body GLUT4 null mice (Katz et al. 1995). This is the case even though skeletal muscle GLUT1 expression is no greater than in wild-type littermates. Glucose uptake is normal in the isolated heart suggesting that GLUT1 is adequate to allow compensation for the deficit in GLUT4. Glucose uptake in isolated skeletal muscle from GLUT4 null mice has been shown to be both normal (Stenbit et al. 2) and impaired (Ryder et al. 1999). How these observations in isolated tissues translate to the whole animal is poorly defined. Little is known about compensatory mechanisms for the absence of GLUT4, particularly in vivo. The effectiveness of compensation is important to understand since Type 2 diabetes is characterized by altered glucose metabolism, including impairment in skeletal muscle glucose uptake (MGU). Exercise increases glucose fluxes and is effective in exposing deficits in glucoregulation. Since GLUT4 is the transporter recruited to the plasma membrane in contracting muscle (Douen et al. 199), it is reasonable to hypothesize that MGU is impaired in the absence of GLUT4. Skeletal muscle isolated from GLUT4 null mice is more fatigable, possibly due to lower glycogen stores (Gorselink et al. 22) and an inability to match glucose DOI:.1113/jphysiol

2 82 P. T. Fueger and others J Physiol uptake with the energy demand of contraction. It is notable that while the heart is enlarged in these mice, glucose uptake is not impaired in cardiac muscle (Stenbit et al. 2). It is important to recognize that control of glucose uptake is not determined solely by glucose transport, but rather is distributed between the ability to supply glucose to muscle, glucose transport, and the ability of a hexokinase (HK) isozyme (primarily HK II in rodent skeletal and cardiac muscles) to phosphorylate glucose (Wasserman & Halseth, 1998; Wasserman & Ayala, 25). Transport is the primary site of control in sedentary short-term fasted mice, while glucose phosphorylation is the primary site of control during exercise (Halseth et al. 1998, 1999; Fueger et al. 24b). HK II overexpression increases MGU during exercise, as well as exercise endurance (Fueger et al. 25). Metabolic control analysis predicts that in the absence of GLUT4, HK II expression will no longer be an effective MGU modifier. If, in fact, the absence of GLUT4 nullifies the effects of HK II overexpression on glucose uptake, the next question is whether or not HK II overexpression can still increase exercise tolerance in the absence of GLUT4. That is to say, is the ability of HK II overexpression to increase MGU required for it to increase endurance? The hypothesis was that HK II overexpression would lose its ability to enhance endurance capacity in mice lacking GLUT4 since it would not be able to enhance exercise-stimulated MGU. The aims of these studies were to test (a) the effectiveness of glucoregulatory compensation for the absence of GLUT4 in vivo, and (b) whether, in theabsence of GLUT4, HK II retains its ability to increase exercise endurance. These aims were addressed using isotopic methods and chronic catheterization to create a novel mouse exercise model. genomic DNA obtained from a tail biopsy. All mice were fed standard chow (Purina 51, Purina Mills, St Louis, MO, USA) ad libitum, were handled at least twice a week, and were studied at 4 months of age. Immunoblotting Total GLUT4 and HK II protein were determined in homogenates of gastrocnemius and cardiac muscles (Fueger et al. 25). A quantity of 2 μg of protein was resolved on 4 12% Bis-Tris SDS-PAGE gels, followed by electrophoretic transfer to polyvinylidine fluoride membranes. Membranes were blocked with 1X milk buffer (Chemicon; Temecula, CA, USA) for 15 min, probed with rabbit anti-glut4 (1 : ; Abcam) overnight at 4 C, and then incubated with anti-rabbit horseradish peroxidase (1 : 2 ; Pierce, Rockford, IL, USA) for 1 h at 23 C. Membranes were exposed to chemiluminescent substrate and imaged using the VersaDoc imaging system (Bio-Rad; Hercules, CA, USA). Membranes were stripped with a Re-Blot Western Blot Recycling Kit (Chemicon), probed with rabbit anti-hk II (1 : ; Chemicon), incubated with anti-rabbit horseradish peroxidase (1 : 2 ), and developed as before. To confirm equal protein loading and transfer, membranes were stripped and reprobed with monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1 : 4, Abcam) and then incubated with anti-mouse horseradish peroxidase (1 : 2 ). All antibodies were diluted in 1% polyvinylpyrrolidone in TBS (Haycock, 1993), and membranes were washed between antibody incubations three times with TBS for min. Densitometry was performed using Quantity One Analysis Software (Bio-Rad Laboratories; Hercules, CA, USA). Methods Mouse maintenance and genotyping All procedures performed were approved by the Vanderbilt University Animal Care and Use Committee. Male C57 BL/6 J mice with a heterozygous knockout of the GLUT4 gene selectively overexpressing HK II (GLUT4 +/ HK Tg ) in skeletal muscle were bred with female C57 BL/6J mice with a heterozygous knockout of the GLUT4 gene (GLUT4 +/ ) (Katz et al. 1995; Fueger et al. 24c,d). The HK II transgene contains human HK II cdna driven by the rat muscle creatine kinase promoter. Wild-type mice (), GLUT4 null mice (GLUT4 / ), and GLUT4 null HKII-overexpressing mice (GLUT4 / HK Tg )were studied. Mice were compared to littermates in each protocol. Littermates were separated by sex at 3 weeks of age and were maintained in microisolator cages. Genotyping was done by polymerase chain reactions of Echocardiography and blood pressure measurement At 16 weeks of age transthoracic echocardiograms were performed on resting, conscious mice using a 15 MHz transducer (Sonos 55 system, Agilent) as previously described (Exil et al. 23; Rottman et al. 23). In addition, systolic blood pressure was measured using tail cuff plethysmography (Weisberg et al. 25). Echocardiographic and blood pressure measurements were made in conscious mice two and three times, respectively, in order to account for acclimation. Measurement of heart and muscle glucose uptake in vivo Four-month-old mice were anaesthetized with sodium pentobarbital (i.p. injection, 7 mg (kg body weight) 1 ) and had carotid artery and jugular vein catheters surgically

3 J Physiol Exercise in GLUT4 null mice 83 implanted (Niswender et al. 1997; Halseth et al. 1999). Following an 5-day period in which body weight was restored (within % of pre-surgery body weight) mice were acclimated to treadmill running with a single min bout of exercise ( m min 1, % gradient), and experiments were performed on 5 h-fasted mice 2 days following the acclimation trial (Fueger et al. 23, 24a,b). Approximately 1 h prior to an experiment, mice were placed on a treadmill to acclimate them to the changed environment. At t = min, a baseline arterial blood sample (15 μl) was drawn for the measurement of blood glucose, haematocrit (Hct), and plasma insulin and non-esterified fatty acids (NEFAs). To prevent a fall in Hct, the remaining erythrocytes were washed once with.9% saline containing U ml 1 of heparin and reinfused. Mice received.1 U heparin. Mice either remained sedentary or ran for up to 3 min at 16.7 m min 1 with a % gradient (n = 9 11 for each experimental group and genotype). This work intensity is 8% of maximal oxygen consumption (Fernando et al. 1993). At t = 5 min, a 12 μci bolus of [2-3 H]deoxyglucose ([2-3 H]DG; Dupont, Boston, MA, USA) was administered in order to measure a tissue-specific glucose metabolic index (R g ). At t =, 15 and 2 min, 5 μl of arterial blood was sampled in order to determine blood glucose and plasma [2-3 H]DG. At t = 3 min, a 15 μl arterial sample was taken in order to determine blood glucose, Hct and plasma insulin, [2-3 H]DG and NEFAs, and mice were anaesthetized with an arterial infusion of sodium pentobarbital (7 mg (kg body weight) 1 ). Blood was centrifuged, and plasma was isolated and frozen at 2 C until analysis. Skeletal muscles and heart were excised, frozen in liquid nitrogen, and stored at 7 C. Mice were killed by the excision of the heart under anaesthesia. Immunoreactive insulin was assayed with a double antibody method (Morgan & Lazarow, 1965). NEFAs were measured spectrophotometrically (Wako NEFA C kit, Wako Chemicals Inc., Richmond, VA, USA). Following deproteinization with Ba(OH) 2 (.3 N) and ZnSO 4 (.3 N), [2-3 H]DG radioactivity of plasma was determined by liquid scintillation counting (Packard TRI-CARB 29TR, Packard, Meriden, CT, USA) with Ultima Gold (Packard) as scintillant. Muscle samples were homogenized in.5% perchloric acid. Homogenates were centrifuged and neutralized with KOH. Radioactivity in [2-3 H]DG and phosphorylated [2-3 H]DG ([2-3 H]DGP) was determined in one aliquot. A second aliquot was treated with Ba(OH) 2 and ZnSO 4 to remove [2-3 H]DGP and radioactivity was counted. [2-3 H]DGP is the difference between the two aliquots. [2-3 H]DGP was normalized to tissue weight. R g was calculated from these data as previously described (Kraegen et al. 1985; Fueger et al. 23, 24a). Glycogen was determined by the method of Chan & Exton (1976) in the gastrocnemius and superficial vastus lateralis (SVL) muscles, and heart. Soleus glycogen could not be determined since both solei were required to assay for [2-3 H]DG and [2-3 H]DGP. Capillary density Capillary density was assessed 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.3% 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 : 4, Santa Cruz Biotechnology) for 45 min. The Vectastain ABC Elite System (Vector Laboratories, Inc.) and DAB+ (DakoCytomation) were 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. Measurement of whole-body glucose kinetics A separate cohort of 4-month-old mice underwent catheterization and recovery as described above. Mice were acclimated to treadmill running with a single min bout of exercise (12 m min 1, % gradient), and experiments were performed on 3.5 h-fasted mice 2 days following the acclimation trial. The protocol consisted of a 9 min tracer equilibration period (t = 9 to min) followed by a 3 min experimental period (t = 3 min) during which mice ran on a treadmill at 12 m min 1.A3μCi bolus of [3-3 H]glucose purified by high-performance liquid chromatography was given at t = 9 min followed by a.5 μci min 1 infusion for the remainder of the experiment. At t = 15 and 5 min, blood samples were taken for assessment of basal glucose turnover. Blood samples were taken at t = 5,, 15, 2 and 3 min for the assessment of glucose turnover during exercise. Larger blood samples ( 2 μl) were taken at t = 15 and 3 min for the determination of circulating glucose, insulin, glucagon and corticosterone. Mice received saline-washed erythrocytes from donors throughout the experimental period (5 6 μl min 1 )topreventafallof > 5% haematocrit. Insulin, glucagon and corticosterone were determined as previously described (Morgan & Lazarow, 1965; Jacobson, 1999; Jacobson & Pacak, 25). [3-3 H]Glucose was assessed in plasma deproteinized with Ba(OH) 2 and ZnSO 4. Endogenous glucose production (R a ) and whole-body glucose disappearance (R d ) were determined using Steele s non-steady-state equations (Altszuler et al. 1956).

4 84 P. T. Fueger and others J Physiol Statistical analysis Dataare presentedasmeans± s.e.m. Differences between groups were determined by ANOVA followed by a Tukey s post hoc test. The significance level was set at P <.5. Results Descriptive characteristics of genetic models Total GLUT4 and HK II content in gastrocnemius and cardiac muscles are shown in Fig. 1. GLUT4 null mice had virtually undetectable levels of GLUT4. The HK II transgene increased gastrocnemius HK II content over sixfold. Interestingly, GLUT4 ablation increased total HK II content threefold in cardiac muscles. The HK II transgene did not further increase cardiac HK II content in mice lacking GLUT4. Ablating the GLUT4 gene resulted in a reduction in body weight (Table 1). Despite the absence of GLUT4, arterial blood glucose and plasma insulin concentrations were not altered. Skeletal muscle R g (Fig. 2) and arterial NEFA levels (Table 1) were reduced in GLUT4 / compared to. HK II overexpression was not able to increase resting skeletal muscle R g or arterial NEFA levels. GLUT4 / and GLUT4 / HK Tg had greater muscle capillary densities compared to (Fig. 3). A Genotype: HK Tg Genotype: HK Tg GLUT4 GLUT4 HKII HKII GAPDH GAPDH Gastrocnemius Cardiac B C GLUT4 protein content (Arbitrary units) HK II protein content (Arbitrary units) Gastrocnemius Gastrocnemius HK Tg Cardiac HK Tg Cardiac Figure 1. Total muscle GLUT4 and HK II content Immunoblotting was performed to measure total GLUT4 (A and B) and HK II (A and C) protein content in the gastrocnemius and cardiac muscles of wild-type mice (), GLUT4 knockout mice (GLUT4 / ), and GLUT4 knockout mice overexpressing HK II (GLUT4 / HK Tg ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as an internal control. Representative blots are shown in A. Data are means ± S.E.M. for 4 mice per group. P <.5 versus.

5 J Physiol Exercise in GLUT4 null mice 85 Table 1. Basal metabolic characteristics of 5 h-fasted C57BL/6J mice GLUT4 / GLUT4 / HK Tg Body weight (g) 25 ± 1 22 ± 1 22 ± 1 Glucose (mg dl 1 ) 165 ± 9 17 ± ± 8 Insulin (ng ml 1 ).66 ±..5 ±.9.55 ±. NEFA (mm) 1.5 ±.1.8 ±.1.6 ±.1 Data are means ± S.E.M. n = per group., wild-type mice; GLUT4 knockout mice (GLUT4 / ) and GLUT4 knockout mice overexpressing HK II (GLUT4 / HK Tg ); NEFA, non-esterified fatty acids. P <.5 versus. Mice lacking GLUT4 did not exhibit alterations in resting heart rate or systolic blood pressure (Table 2). However, marked increases in left ventricular mass, relative cardiac mass and absolute cardiac mass were evident. GLUT4 null mice had impaired cardiac function as evidenced by decreased fractional shortening. While HK II overexpression on the background of GLUT4 ablation improved several echocardiographic parameters and improved cardiac function, it did not restore the alterations in cardiac mass. MGU in GLUT4 null mice during exercise with and without HK II overexpression Ablating GLUT4 dramatically reduced exercise-stimulated R g in skeletal muscles (Fig. 2). Exercise led to hyperglycaemia in both GLUT4 / and GLUT4 / HK Tg within the first min and persisted throughout the trial (Fig. 4). GLUT4 / had a more rapid onset of fatigue than. HKII overexpression did not alter the aberrations in blood glucose or R g during exercise in mice lacking GLUT4. In light of this it was surprising that HK II overexpression significantly delayed fatigue associated with GLUT4 ablation. Glycogen content was decreased in gastrocnemius and SVL from both sedentary and exercised GLUT4 null mice (Fig. 5). GLUT4 / had increased skeletal muscle glycogen breakdown during exercise compared to and GLUT4 / HK Tg. Exercise decreased plasma insulin in all genotypes (16 ± 2, 11 ± 1 and 18 ± 3 μuml 1 in, GLUT4 / and GLUT4 / HK Tg, respectively). Plasma insulin was lowest in GLUT4 / compared to other genotypes. NEFAs were not altered by exercise in, GLUT4 / and GLUT4 / HK Tg (1.5 ±.2 to 1.6 ±.2,.7 ±.1 to.6 ±.1 and.6 ±.1 to.7 ±.1 mm for pre- and post-exercise samples, respectively). A Basal R g (μmol/g/min) HK Tg B 7 Soleus Gastrocnemius SVL Figure 2. GLUT4 knockout decreases muscle glucose metabolic index during rest and exercise Glucose metabolic index at rest (A), and exercise (B), were measured in the soleus, gastrocnemius and superficial vastus lateralis (SVL) muscles of mice, GLUT4 knockout mice (GLUT4 / ), and GLUT4 knockout mice overexpressing HK II (GLUT4 / HK Tg ). Data are means ± S.E.M. for 9 12 mice per group. P <.5 versus. Exercise-stimulated R g (μmol/g/min) Soleus Gastrocnemius SVL

6 86 P. T. Fueger and others J Physiol Table 2. Cardiovascular parameters of conscious C57BL/6J mice GLUT4 / GLUT4 / HK Tg HR (beats min 1 ) 681 ± ± ± 11 SBP (mmhg) 9 ± ± ± 3 Absolute heart mass (mg) 8 ± 5 15 ± ± Relative heart mass (mg g 1 ) 4.4 ± ± ±.3 LV mass (mg) 9 ± ± 6 ± 9 FS (%) 52 ± 2 46± 3 5 ± 3 IVSd (mm).9 ±.2.96 ±.3.91 ±.2 LVIDd (mm) 3.9 ± ± ±. LVPWd (mm).9 ± ±.5.97 ±.3 IVSs (mm) 1.66 ± ± ±.5 LVIDs (mm) 1.47 ± ± ±.14 LVPWs (mm) 1.32 ± ± ±.4 Data are means ± S.E.M., n = 8 per group. HR, heart rate; SBP, systolic blood pressure; LV mass, left ventricular mass; FS, fractional shortening, IVSd, interventricular septal thickness in diastole; LVIDd, LV end-diastolic dimension; LVPWd, LV posterior wall thickness in diastole; IVSs, interventricular septal thickness in systole; LVIDs, LV end-systolic dimension; LVPWs, LV posterior wall thickness in systole. P <.5 versus. As compensation for the loss of GLUT4, the hearts of GLUT4 null mice have a 5% increase in GLUT1 expression (Katz et al. 1995). Relative cardiac R g was not reduced in mice lacking GLUT4 during rest (23 ± 6, 26 ± 8 and 36 ± 9 μmol ( g) 1 min 1 in, GLUT4 / and GLUT4 / HK Tg, respectively) or exercise (4 ± 8, 59 ± 12 and 63 ± 16 μmol ( g) 1 min 1 in, GLUT4 / and GLUT4 / HK Tg, respectively). In fact, ablating GLUT4 increased absolute cardiac R g during rest and exercise (Fig. 6). Cardiac glycogen was not altered by GLUT4 ablation in resting mice but was greater in GLUT4 / compared to following exercise. Whole-body glucose kinetics during exercise in and GLUT4 / Since GLUT4 / become fatigued at a treadmill speed of 16.7 m min 1, a second group of and GLUT4 / were studied while running at 12 m min 1 in an experiment designed to measure whole-body glucose kinetics. Even at this slower speed, GLUT4 / hyperglycaemia developed (Fig. 7). During rest, basal glucose, insulin, glucagon, corticosterone, R a, R d and glucose clearance were not different between and GLUT4 /. Exercise led to a decrease in insulin concentration and an increase in glucagon concentration in both genotypes. However, the decrease in insulin concentration was more dramatic in GLUT4 / compared to despite marked hyperglycaemia. Exercise increased corticosterone in GLUT4 / but not. Glucose clearance rose twofold in. This rise was absent in GLUT4 /. R d was also increased twofold with exercise in. Despite the absence of an increase in glucose clearance with exercise, R d increased by 3% in GLUT4 / due to the development of hyperglycaemia. Exercise increased R a equally independent of the presence of GLUT4. Thus, the profound hyperglycaemia associated with exercising GLUT4 / is due to attenuation of the increase in glucose clearance in the presence of normal R a. Discussion GLUT4 null mice have normal circulating glucose and insulin compared to mice at rest. We tested the Capillary density (no/mm 2 ) Soleus Gastrocnemius SVL HK Tg Figure 3. GLUT4 knockout increases muscle capillary density Capillary density was measured in the soleus, gastrocnemius and SVL muscles of mice, GLUT4 knockout mice (GLUT4 / ) and GLUT4 knockout mice overexpressing HK II (GLUT4 / HK Tg ). Data are means ± S.E.M. for 9 12 mice per group. P <.5 versus.

7 J Physiol Exercise in GLUT4 null mice 87 hypothesis that the metabolic demands of physical exercise unmask robust alterations in the absence of GLUT4 that are silent in the sedentary state. Skeletal muscle R g was severely compromised during both rest and exercise in the absence of GLUT4 (Fig. 2). These results are consistent with the demonstration that isolated skeletal muscles from GLUT4 null mice are incapable of sufficiently increasing glucose uptake when exposed to hypoxia (Zierath et al. 1998). The absence of GLUT4 hastened fatigue when mice were challenged with exercise. This result is consistent with increased fatigability in isolated skeletal muscles of GLUT4 null mice (Gorselink et al. 22). While rodents tend to use less glycogen during exercise than humans (Baldwin et al. 1973; Reitman et al. 1973; Ivy, 1999; Pederson et al. 25), GLUT4 null mice underwent significant glycogenolysis in an effort to use more carbohydrate-based carbons. Glucose delivery is a key component to muscle glucose uptake, especially during exercise (Schultz et al. 1977; Halseth et al. 1998, 21; Wasserman & Halseth, 1998). Although skeletal muscle is less permeable to glucose in GLUT4 null mice, they have increased vascular glucose delivery during exercise due to increased blood glucose concentration and greater capillary density. Quite strikingly, blood glucose continued to increase beyond 3 mg dl 1. This level of glycaemia normally suppresses R a (Glinsmann et al. 1969; Sacca et al. 1978; Shulman et al. 198a,b; Ader et al. 1985; Bell et al. 1986). We speculate that skeletal muscle, which is starved for glucose carbons, sends a neural or humoral signal overriding the inhibitory effect of hyperglycaemia on the liver. Despite the abundance of glucose in the blood, the livers of GLUT4 null mice may get a signal reflecting systemic glucose deficit when a physiological challenge such as exercise is employed. It is possible that the signal from the muscle during this time when it is relatively intracellularly hypoglycaemic is either sent directly to the liver or to the pancreas in a direct or indirect manner whereby the response would be to increase the glucagon : insulin ratio. This exaggerated glucagon : insulin ratio might then override the normal response of hyperglycaemia to suppress hepatic glucose production. To ascertain the role of the liver in creating the exercise-induced hyperglycaemia in GLUT4 null mice, whole-body glucose kinetics were measured in and GLUT4 / during exercise. We demonstrate that mice exhibit an twofold increase in R a and R d during moderate exercise as rates increase from 2 to 4 mg kg 1 min 1. In addition, circulating insulin decreases, glucagon increases, and corticosterone does not appreciably change during exercise. In GLUT4 /, resting R a, R d, and clearance and exercise-stimulated R a are essentially the same as. Glucose clearance is completely unresponsive to exercise. R d increases solely due to the mass action effect driven by the increase in R a and hyperglycaemia. It was demonstrated decades ago that the exercise-induced increase in R a closely matches the exercise-induced increase in R d in dogs and then humans. It was subsequently shown that the increase in R a is driven by increased release of glucagon and decreased release of insulin from the pancreas. Here we show that pancreatic hormone and R a responses in mice can occur independent of feedback related to increased glucose uptake by working skeletal muscle. We previously showed that HK II overexpression enhanced both R g and exercise tolerance in mice with normal GLUT4 expression (Fueger et al. 25). The increase in R g was closely correlated to and proposed to be causal to the improved exercise tolerance. An interesting observation was that despite having no effect on glucose uptake or little impact on glycogen metabolism, HK II overexpression still improved exercise endurance in GLUT4 null mice by twofold. HK II A B Arterial blood glucose (mg/dl) Fatigue index (%) HK Tg 2 3 Time (min) HK Tg 2 3 Time (min) Figure 4. GLUT4 knockout alters arterial blood glucose during exercise and exercise performance Wild-type mice (), GLUT4 knockout mice (GLUT4 / ), and GLUT4 knockout mice overexpressing HK II (GLUT4 / HK Tg ) were chronically catheterized and allowed to recover from surgery for 7 days. Following a 5 h fast, mice were run on a treadmill for 3 min. Arterial blood concentration was measured during exercise (A). The fatigue index (B) represents the percentage of mice that were able to run for the indicated time points. Data are means ± S.E.M. for 9 12 mice per group. P <.5 versus.

8 88 P. T. Fueger and others J Physiol A Basal glycogen (mg/g muscle) HK Tg Gastrocnemius SVL Cardiac B 3 Post-exercise glycogen (mg/g muscle) 2 1 Gastrocnemius SVL Cardiac C 3 Glycogen breakdown (mg/g muscle/3 min) 2 1 Gastrocnemius SVL Cardiac Figure 5. Muscle glycogen following rest and exercise Muscle glycogen was measured after rest (A) or exercise (B) in 5 h-fasted, mice, GLUT4 knockout mice (GLUT4 / ) and GLUT4 knockout mice overexpressing HK II (GLUT4 / HK Tg ). Muscle glycogen breakdown (C) was calculated as the difference between rest and post-exercise glycogen masses. Data are means ± S.E.M. for 9 12 mice per group. P <.5 versus. forms a complex with voltage-dependent anion channels (VDACs) in the mitochondria. These channels are critical for mitochondrial function, adenine nuclear exchange with the cytosol, and maintenance of the oxidative state of the mitochondria. It may be that HK II overexpression increases the interaction of this enzyme with mitochondrial VDACs and thereby increases the efficiency of energy production from the small amount of glucose Absolute cardiac R g (nmol/min) HK Tg Sedentary Exercise Figure 6. Cardiac glucose uptake during rest and exercise is preserved in GLUT4 knockout mice Absolute cardiac glucose uptake was measured in mice, GLUT4 knockout mice (GLUT4 / ) and GLUT4 knockout mice overexpressing HK II (GLUT4 / HK Tg ) during rest and exercise. Data are means ± S.E.M. for 9 12 mice per group.

9 J Physiol Exercise in GLUT4 null mice 89 supplied to the muscle or by improving one of the exchange mechanisms with which VDACs are involved. In the heart, GLUT1 plays a significant role in mediating glucose transport. Thus, it is not surprising to find that A Exercise removing GLUT4 from the heart would have no effect upon cardiac glucose uptake during rest, especially given the fact that GLUT4 null mice display a 5% increase in cardiac GLUT1 (Katz et al. 1995). It might seem Arterial blood glucose (mg/dl) Glut4 -/ Time (min) B Insulin (ng/ml) E Ra (mg/kg/min) Glut4 -/- Glut4 -/- C Glucagon (pg/ml) Rest Exercise F Rd (mg/kg/min) Rest Exercise D Corticosterone (ng/ml) Glut4 -/- G Clearance (ml/min) Glut4 -/- Glut4 -/- Glut4 -/- Figure 7. Whole-body glucose kinetics during exercise in and GLUT4 knockout mice Wild-type mice () and GLUT4 knockout mice (GLUT4 / ) were chronically catheterized and allowed to recover from surgery for 7 days. Following a 3.5 h fast, [3-3 H]glucose was infused to measured whole-body glucose kinetics in vivo. Arterial blood was sampled to measure blood glucose (A), insulin (B), glucagon (C), corticosterone (D), and radioactivity for the determination of R a (E), R d (F) and clearance (G) during both rest and exercise. Data are means ± S.E.M. for 6 mice per group. P <.5 versus Rest; P <.5 versus.

10 8 P. T. Fueger and others J Physiol paradoxical that cardiac R g is actually increased during exercise in GLUT4 null mice. The severe hyperglycaemia forces glucose into the working heart. It is also likely that the threefold increase in cardiac HK II content created by GLUT4 ablation enhances exercise-stimulated R g.in addition, the lower NEFAs present in GLUT4 null mice limit their availability for metabolism by the heart and also reduce the inhibitory effect of fatty acids on glucose transport. Ablation of GLUT4 results in an unusual form of cardiac hypertrophy (Stenbit et al. 2; Weiss et al. 22). The stimulus for the increase in cardiac mass is possibly due to substrate utilization since blood pressure is not altered in GLUT4 null mice compared to their littermates. Since NEFAs are low and skeletal muscle is a poor consumer of glucose in GLUT4 null animals, the heart metabolizes more glucose. When the heart is compelled to utilize glucose, as is the case during treatment with inhibitors of fatty acid oxidation (Higgins et al. 1985; Rupp et al. 1992; Cabrero et al. 23), cardiac hypertrophy ensues. The altered heart mass and substrate utilization is also associated with a decrease in cardiac function, as evidenced by the decrease in fractional shortening at rest in GLUT4 null mice compared to mice. This cardiac insufficiency probably contributes to the observed exercise intolerance. Surprisingly, muscles from mice lacking GLUT4 still deposit glycogen, albeit to slightly lower levels. During exercise, GLUT4 null mice rapidly metabolize glycogen. Interestingly, the addition of increased glucose phosphorylation capacity on a GLUT4 null background spares muscle glycogen during exercise. In the hearts of GLUT4 null mice, where GLUT1 is increased compared to hearts (Katz et al. 1995), glycogen content is not altered and, in fact, cardiac glycogen is increased in GLUT4 / compared to. In conclusion, unlike the basal state where glucose homeostasis is preserved, hyperglycaemia results during exercise in GLUT4 null mice. This is due to increased liver glucose release in the face of severe skeletal muscle glucose intolerance. The stimulation of the liver in GLUT4 null mice is so robust that it is able to sustain the accelerated release of glucose in the presence of marked hyperglycaemia. Hyperglycaemia only partially compensates for the absence of GLUT4 in skeletal muscle but actually contributes to overcompensation in cardiac muscle. Finally, studies in mice lacking GLUT4 show that HK II improves exercise tolerance, independent of its effects on MGU. References Ader M, Pacini G, Yang YJ & Bergman RN (1985). Importance of glucose per se to intravenous glucose tolerance. 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12 812 P. T. Fueger and others J Physiol Acknowledgements We thank Wanda Snead of the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC) Hormone Assay Core for performing the insulin assays and Carlo Malabanan of the MMPC Metabolic Pathophysiology Core for excellent technical assistance. We thank Gemin Ni and ZhiZhang Wang of the MMPC Cardiovascular Pathophysiology Core for performance of echocardiography and blood pressure measurements. We greatly appreciate the assistance of Dr Lillian Nanney and Kelly Parman of the MMPC Immunohistochemistry Core for measurement of CD31. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R1 DK-5492 (to D.H.W.), R1 DK-5277 (to D.H.W.), R1 DK (to M.J.C.), and U24 DK (to D.H.W.).