Control of muscle glucose uptake: test of the rate-limiting step paradigm in conscious, unrestrained mice

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1 J Physiol (2005) pp Control of muscle glucose uptake: test of the rate-limiting step paradigm in conscious, unrestrained mice Patrick T.Fueger 1,Jane Shearer 1,Deanna P. Bracy 1,2,KellyA.Posey 1,R.RichardPencek 1, Owen P. McGuinness 1,2 and David H. Wasserman 1,2 1 Department of Molecular Physiology & Biophysics and 2 Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, Nashville, TN, USA The aim of this study was to test whether in fact glucose transport is rate-limiting in control of muscle glucose uptake (MGU) under physiological hyperinsulinaemic conditions in the conscious, unrestrained mouse. C57Bl/6J mice overexpressing GLUT4 (GLUT4 Tg ), hexokinase II (HK Tg ), or both (GLUT4 Tg + HK Tg ), were compared to wild-type (WT) littermates. Catheters were implanted into a carotid artery and jugular vein for sampling and infusions at 4 month of age. After a 5-day recovery period, conscious mice underwent one of two protocols (n = 8 14/group) after a 5-h fast. Saline or insulin (4 mu kg 1 min 1 ) was infused for 120 min. All mice received a bolus of 2-deoxy[ 3 H]glucose (2-3 HDG) at 95 min. Glucose was clamped at 165 mg dl 1 during insulin infusion and insulin levels reached 80 µuml 1. The rate of disappearance of 2-3 HDG from the blood provided an index of whole body glucose clearance. Gastrocnemius, superficial vastus lateralis and soleus muscles were excised at 120 min to determine 2-3 HDG-6-phosphate levels and calculate an index of MGU (R g ). Results show that whole body and tissue-specific indices of glucose utilization were: (1) augmented by GLUT4 overexpression, but not HKII overexpression, in the basal state; (2) enhanced by HKII overexpression in the presence of physiological hyperinsulinaemia; and (3) largely unaffected by GLUT4 overexpression during insulin clamps whether alone or combined with HKII overexpression. Therefore, while glucose transport is the primary barrier to MGU under basal conditions, glucose phosphorylation becomes a more important barrier during physiological hyperinsulinaemia in all muscles. The control of MGU is distributed rather than confined to a single rate-limiting step such as glucose transport as glucose transport and phosphorylation can both become barriers to skeletal muscle glucose influx. (Received 28 September 2004; accepted after revision 30 November 2004; first published online 2 December 2004) 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. patrick.fueger@duke.edu The regulatory mechanisms that control insulinstimulated muscle glucose uptake (MGU) in vivo and the processes involved are poorly understood. Glucose transport is commonly asserted to be the rate-limiting step for MGU (Wallberg-Henriksson & Zierath, 2001; Koistinen & Zierath, 2002; Petersen & Shulman, 2002). Evidence for the GLUT4 rate-limiting step paradigm in vivo is largely derived from studies using transgenic mice overexpressing GLUT4 (Marshall & Mueckler, 1994; Treadway et al. 1994; Tsao et al. 1996; Marshall et al. 1999) that show they have a greater glucose disposal rate during an insulin clamp than their wild-type controls. The difficulty with interpreting previous mouse clamp studies is that they were conducted under a variety of experimental conditions. Depending on the specific report, studies were done in the presence of one or more of the following conditions: suprapharmacological insulin levels, anaesthesia, after a protracted fast ( 18 h), and with excessive handling to obtain mixed blood from the cut tail rather than arterial blood. Moreover, isotopic methods used in these studies were not those needed to specifically assess MGU directly. One study showed that GLUT4 overexpression did not improve insulin-stimulated glucose disappearance in mice fasted for 3 4 h (Ren et al. 1995). What made these distinct from other previous studies and worth further examination was that conscious mice, physiological insulin levels and short-term fasts were used, and whole body glucose fluxes were measured. There were several aspects of that study, however, that complicated interpretation. Blood sampling was performed by bleeding the cut tail, requiring that mice were handled. This DOI: /jphysiol

2 926 P. T. Fueger and others J Physiol procedure may have introduced an element of stress into the studies as handling of rodents markedly increase circulating catecholamines (Benthem & Taborsky, 1998). If so, this could potentially influence results, given the role of adrenaline (epinephrine) in inhibiting muscle glucose transport (Han & Bonen, 1998). Due to difficulty in obtaining blood from the tail, insulin levels were not measured concurrently with glucose fluxes, but were measured in a separate set of experiments in the previous studies. The aim of the present study was to further explore the unresolved issue as to whether GLUT4 overexpression augments the increase in glucose utilization associated with physiological hyperinsulinaemia. Several critical modifications were made to previous in vivo studies of GLUT4 overexpressing mice. In order to more specifically examine skeletal muscle, the glucose analogue, 2-deoxyglucose, was used to measure a tissue-specific index of glucose utilization (R g ). Measurements were made 5h after food removal to insure that mice were postabsorptive, but not glycogen-depleted and catabolic as is the case for mice fasted overnight. Finally, to eliminate the stress associated with mouse handling during the study, catheters were surgically implanted into a carotid artery and jugular vein at least 5 days before study. GLUT4 overexpression was studied in C57Bl/6J mice in isolation or in combination with hexokinase II (HKII)overexpression. The hypothesis was that GLUT4 overexpression would not increase R g during an insulin clamp at a physiological dose. However, it was further hypothesized that GLUT4 overexpression would increase R g when the resistance to glucose phosphorylation was lowered by concomitant HKII overexpression. Methods Mouse maintenance and genotyping All procedures performed were approved by the Vanderbilt University Animal Care and Use Committee. Male C57Bl/6J mice transgenic for the hglut construct (GLUT4 Tg ) were bred with female C57Bl/6J mice carrying a transgene containing the human HKII cdna driven by the rat muscle creatine kinase promoter (HK Tg ) in order to obtain mice of four genotypes: wild-type (WT), GLUT4 Tg,HK Tg and double transgenic (GLUT4 Tg + HK Tg ). Littermates were separated by gender following a 3-week weaning period and were maintained in microisolator cages. Genotyping for the HKII and hglut transgenes was performed as previously described (Olson et al. 1993; Halseth et al. 1999). All mice were fed standard chow ad libitum, handled twice per week, and studied at 4month of age. Immunoblotting GLUT4 and HKII protein content was determined on representative gastrocnemius and superficial vastus lateralis (SVL) muscles. Muscles were homogenized in a solution containing 10% glycerol, 20 mm sodium pyrophosphate, 150 mm NaCl, 50 mm Hepes (ph 7.5), 1% Nonidet P 40 (NP-40), 20 mm β-glycerophosphate, 10 mm NaF, 2 mm EDTA (ph 8.0), 2mmphenylmethylsulphonyl fluoride, 1 mm CaCl 2,1mm MgCl 2,10µgml 1 aprotinin, 10 µgml 1 leupeptin, 2 mm Na 2 VO 3 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 SDS-PAGE gel and then transferred to apolyvinyl idene fluoride (PVDF) membrane. Equal protein loading was confirmed by briefly staining membranes with 0.1% Ponceau S in 5% acetic acid and destaining with deionized water. Membranes were blocked, probed with rabbit anti-glut4 (1 : 1000; Alpha Diagnostic International; San Antonio, TX, USA) and anti-hkii antibodies (1 : 1000; Chemicon International; Temecula, CA, USA), and then incubated with goat anti-rabbit-horseradish peroxidase (1 : 20000; Pierce, Rockford, IL, USA). Densitometry was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Surgical procedures The surgical procedures utilized for implanting chronic catheters have been previously described (Niswender et al. 1997; Halseth et al. 1999). Briefly, mice were anaesthetized with pentobarbitol (70 mg (kg body weight) 1 ). The left common carotid artery and right jugular vein were catheterized for arterial blood 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 (o.d., 0.033), which were exteriorized and sealed with stainless steel plugs. Following surgery, animals were housed individually and body weight was recorded daily. Lines were kept patent by flushing daily with µl saline containing 200 U ml 1 heparin and 5mgml 1 ampicillin. In vivo metabolic experiments Mice were allowed to recover from surgery for at least 5days. Mice were only studied when body weight was restored to within 10% of presurgery body weight. For metabolic studies, conscious, unrestrained mice were placed in an 1-l plastic container lined with bedding at h and fasted for 5 h. A 5-h fast was used as mice are postabsorptive but have not lost a significant amount of

3 J Physiol Control of insulin-stimulated muscle glucose uptake in vivo 927 weight, as is the case for 18-h fasted mice who lose 15% of their body weight (authors unpublished observation). Approximately 1 h prior to an experiment (12.00 h), Micro-Renathane (o.d., 0.033) tubing was connected to the catheter leads and infusion syringes for infusions and sampling. From this point until the end of the experiment the container was covered with a Plexiglass lid custom-designed to permit catheter access. Mice were not handled and were allowed to move freely in the container in order to eliminate stress. Following this 1-h acclimation period (13.00 h), a 150-µl baseline arterial blood sample was drawn for the measurement of arterial blood glucose level (HemoCue, Mission Viejo, CA, USA), haematocrit and plasma insulin and non-esterified fatty acid (NEFA) levels. The remaining red blood cells were washed with 0.9% saline containing 10 U ml 1 heparin and re-infused. An infusion of saline (n = 11, 8, 11 and 8 for WT, GLUT4 Tg,HK Tg and GLUT4 Tg + HK Tg,respectively)or 4mUkg 1 min 1 of insulin (n = 14, 9, 11 and 8 for WT, GLUT4 Tg,HK Tg and GLUT4 Tg + HK Tg,respectively), was infused at a flow rate of µl min 1 starting at t = 90 min (13.00 h). Euglycaemia was maintained during insulin infusions by measuring arterial blood glucose ( 5 µl whole blood) every 10 min and infusing 50% dextrose as necessary. Mice received saline-washed red blood cells from a donor mouse as needed to minimize reductions in haematocrit (> 5%). A 150-µl arterial blood sample was obtained at t = 0 min. At t = 5 min, a 12 µci bolus of 2-deoxy[ 3 H]glucose ([2-3 H]DG) was administered. At t = 7, 10, 15, 20 and 30 min, arterial blood was sampled in order to determine arterial blood glucose and plasma [2-3 H]DG concentrations. At t = 30 min (15.00 h), a final 150-µl arterial blood sample was withdrawn and mice were anaesthetized with an infusion of sodium pentobarbital. The gastrocnemius ( 6% type IIA, 11% type IID, 83% type IIB fibres), SVL ( 3% type IIA, 10% type IID, 87% type IIB fibres) and soleus ( 44% type I, 51% type IIA, 5% type IID fibres) muscles (Delp & Duan, 1996) were excised, immediately frozen in liquid nitrogen, and stored at 70 C until future tissue analysis. Following the removal of muscles, mice were killed during anaesthesia by rapidly excising the heart. Assays for plasma and muscle samples Immunoreactive insulin was assayed with a double antibody method (Morgan & Lazarow, 1965). NEFA levels were measured spectrophotometrically by an enzymatic colorimetric assay (Wako NEFA C kit, Wako Chemicals Inc., Richmond, VA, USA). [2-3 H]DG radioactivity of deproteinized plasma samples and both [2-3 H]DG and [2-3 H]DG-G-phosphate ([2-3 H]DGP) radioactivity levels of frozen muscle samples were determined by liquid scintillation counting (Packard TRI-CARB 2900TR, Packard, Meriden, CT, USA) as previously described (Fueger et al. 2003, 2004a,b). Muscle glycogen was determined by the method of Chan & Exton (1976) on the contralateral gastrocnemius and SVL muscles. Tissue glucose and glucose-6-phosphate (G6P) levels were measured enzymatically (Lloyd et al. 1978) following deproteinization with 0.5% perchloric acid and were expressed as mmol (l tissue water) 1. Calculations Tissue-specific clearance of [2-3 H]DG (K g ) and R g,were calculated as previously described (Kraegen et al. 1985): K g = [2-3 H]DGP muscle AUC[2-3 H]DG plasma R g = K g Glucose plasma where [2-3 H]DGP muscle is the [2-3 H]DGP radioactivity in the muscle in d.p.m. g 1,AUC[2-3 H]DG plasma is the area under the plasma [2-3 H]DG disappearance curve calculated by the trapezoid method in d.p.m. ml 1 min 1, and Glucose plasma is the average blood glucose concentration (in mm) during the experimental period. The rate of disappearance of [2-3 H]DG from arterial blood is an index of whole body glucose clearance. Data are presented as means ± s.e.m. Differences between groups were determined by ANOVA followed by Tukey s post hoc tests. The significance level was set at P < Results Characterization of mice GLUT4 overexpression increased total GLUT4 content approximately four- and five-fold in the gastrocnemius and SVL, respectively (Fig. 1). HKII overexpression increased total HKII content approximately three- and four-fold in the gastrocnemius and SVL, respectively. It is interesting that GLUT4 overexpression alone led to a 70% reduction in total HKII content in both muscles; however, this result was not present in muscles expressing both transgenes. Baseline characteristics are reported in Table 1. GLUT4 overexpression, either alone or in combination with HKII overexpression reduced fasting glycaemia and insulin concentration relative to WT mice. HKII overexpression had no effect on fasting glycaemia yet resulted in a mild hypo-insulinaemia. NEFA levels and body weight were not altered by any of the transgenic manipulations in the present study.

4 928 P. T. Fueger and others J Physiol Basal glucose metabolism Arterial blood glucose concentrations during the final 30 min of the saline-infusion are shown in (Fig. 2A). GLUT4 overexpression, either alone or in combination with HKII overexpression, increased the disappearance of [2-3 H]DG from the plasma (Fig. 3A and B) and increased basal K g and R g in the gastrocnemius and SVL compared to WT mice (Table 2). GLUT4 overexpression alone increased basal K g of the soleus, but because of the fall in blood glucose concentration, no effect on R g was observed. HKII overexpression had no effect on the disappearance of [2-3 H]DG from the plasma. As has been previously reported (Chang et al. 1996; Hansen et al. 2000), HKII overexpression also had no effect on basal K g or R g in the gastrocnemius or SVL but did lead to a small increase in R g in soleus. HKII overexpression in combination with GLUT4 overexpression did not lead to afurther enhancement in basal K g or R g of any of the muscles studied, nor an increased disappearance of [2-3 H]DG from the plasma compared to WT. These data are in agreement with fasting blood glucose concentrations and basal glucose metabolism data obtained in previous investigations (Liu et al. 1993; Ren et al. 1993; Gulve et al. 1994; Marshall & Mueckler, 1994; Treadway et al. 1994; Ikemoto et al. 1995a,b; Bao & Garvey, 1997) and clearly demonstrate the accelerated basal glucose uptake in GLUT4 overexpressing mice. None of the transgenic manipulations resulted in alterations in muscle glycogen (Table 3) or G6P (data not shown) in saline-infused mice. Muscle glucose concentration was lower in the soleus of GLUT4 Tg mice and all three muscles studied of the GLUT4 Tg + HK Tg mice compared to WT mice (data not shown). Insulin-stimulated glucose metabolism Arterial blood glucose concentration was clamped at 165 mg dl 1 in order to match concentrations in the 5-h fasted WT mice (Fig. 2A). No differences in arterial blood glucose or plasma insulin concentrations were observed (Figs 2B and 4, respectively). The glucose infusion rate (GIR) reflects the sum of the effect of insulin on liver and kidney glucose production and whole body glucose utilization. The GIRs required to maintain euglycaemia were 57.6 ± 3.1, 66.5 ± 4.4, 66.9 ± 8.1 and 71.9 ± 4.5 mg kg 1 min 1 in WT, GLUT4 Tg, HK Tg and GLUT4 Tg + HK Tg, respectively. GLUT4 overexpression, either alone or in combination with HKII overexpression increased GIR compared to WT mice. While HKII overexpression alone led to a similar GIR compared to GLUT4 overexpression alone, this increase was not significant compared to WT mice (P = 0.12). Stimulation by insulin significantly increased the disappearance of [2-3 H]DG from the plasma in all genotypes (Fig. 3D). Consistent with the work of Ren et al. Figure 1. Total GLUT4 and HKII content from skeletal muscles Immunoblotting was performed to measure total GLUT4 (A C) and HKII (D F) protein content in the gastrocnemius and SVL muscles of wild-type (WT) mice and their littermates overexpressing GLUT4 (GLUT4 Tg ), HKII (HK Tg )or both (GLUT4 Tg + HK Tg ). Representative blots are for GLUT4 (A) and HKII (D) are shown. Densitometry data are means ± S.E.M. for 3 4 mice per group. P < 0.05 versus WT and HK Tg ; P < 0.05 versus WT; P < 0.05 versus WT and GLUT4 Tg.

5 J Physiol Control of insulin-stimulated muscle glucose uptake in vivo 929 Table 1. Baseline characteristics in C57Bl/6J mice fasted for 5 h WT GLUT4 Tg HK Tg GLUT4 Tg + HK Tg n (male/female) 25 (14/11) 18 (12/6) 22 (9/13) 17 (9/8) Body weight (g) 27 ± 1 28± 1 25 ± 1 26± 1 Glucose (mg dl 1 ) 174 ± ± ± ± 6 Insulin (µu ml 1 ) 24± 2 15 ± 2 19 ± 2 14 ± 2 NEFA (mm) 1.5 ± ± ± ± 0.2 Data are means ± S.E.M. NEFA, non-esterified fatty acids; P < 0.05 versus WT and HK Tg mice; P < 0.05 versus WT mice. (1995), GLUT4 overexpression did not further augment the disappearance of [2-3 H]DG from the plasma (Fig. 3C and D)nor the insulin-stimulated K g in the gastrocnemius, SVL or soleus (Figs 5A and B, and 6A,respectively). GLUT4 overexpression increased insulin-stimulated R g in SVL but not in gastrocnemius or soleus muscles (Fig. 5D and C, and Fig. 6B, respectively). In contrast, HKII overexpression enhanced the disappearance of [2-3 H]DG from the plasma and the insulin-stimulated K g and R g in all three muscles studied. GLUT4 overexpression in combination with HKII overexpression did not further enhance the disappearance of [2-3 H]DG from the plasma or insulin-stimulated K g and R g compared to HKII overexpression alone. An increase in muscle glycogen content was only seen in the SVL of insulin-clamped GLUT4 Tg and HK Tg mice (Table 3). There were no differences in glycogen between genotypes in insulin-clamped mice. Consistent with increased transport and phosphorylation capacities, muscle glucose concentration was actually reduced in gastrocnemius and soleus (Table 4). Muscle G6P concentration was elevated in the soleus of GLUT4 Tg + HK Tg mice which also had the highest rate of glucose influx compared to all other genotypes (Table 4). Discussion The aim of the present study was to test the paradigm where GLUT4 is the single rate-limiting step for MGU during physiological hyperinsulinaemia in conscious, unstressed mice. In order to more specifically examine skeletal muscle, the glucose analogue, [2-3 H]DG, was used to measure indices of whole body and muscle-specific glucose utilization. As has been seen in numerous previous studies (Marshall & Mueckler, 1994; Treadway et al. 1994; Hansen et al. 1995, 2000; Tsao et al. 1996) glucose transport was shown here to be the primary control site for MGU in the basal state. However in the present study, GLUT4 overexpression did not increase an index of whole body glucose clearance (i.e. rate of blood [2-3 H]DG disappearance). Moreover, GLUT4 overexpression also did not increase R g in soleus and gastrocnemius muscles during insulin clamp at a physiological dose. GLUT4 overexpression only increased insulin-stimulated R g in SVL. HKII overexpression, on the other hand, caused marked increments in indices of whole body glucose clearance and tissue-specific glucose utilization in all muscles studied during physiological hyperinsulinaemia. Together these data show that glucose phosphorylation, more so than transport, limits the rate of MGU during physiological hyperinsulinaemia in conscious mice fasted for 5 h. Experimental details are crucial for interpreting in vivo metabolic studies. In this work, confounding factors due to stress and anaesthesia on whole body and muscle glucose metabolism were eliminated using the chronically catheterized, conscious mouse model in which arterial samples were obtained and venous infusions were made without handling the animal (Niswender et al. 1997; Halseth et al. 1999). Samples were taken after a 5-h fast, and the insulin infusion was selected to achieve insulin concentrations in the upper physiological range Figure 2. Arterial blood glucose cocncentration from chronically catheterized, conscious mice during the final 30 min of a 120-min saline infusion or hyperinsulinaemic euglycaemic clamp Wild-type (WT, ) and mice overexpressing GLUT4 (GLUT4 Tg, ), HKII (HK Tg, ) orboth (GLUT4 Tg + HK Tg, ) were fasted for 5 h and subjected to a 120-min saline infusion (A) or hyperinsulinaemic euglycaemic clamp (B). Data are means ± S.E.M. for 8 14 mice per group. P < 0.05 for GLUT4 Tg and GLUT4 Tg + HK Tg versus WT and HK Tg at all time points measured.

6 930 P. T. Fueger and others J Physiol ( 80 µuml 1 ). The present findings regarding GLUT4 overexpression are consistent with the earlier results of Ren et al. (1995). These investigators found that the greater GIR in conscious GLUT4 overexpressing mice was due entirely to suppression of endogenous glucose production during insulin clamp at a physiological dose (2.5 mu kg 1 min 1 ). Their work clearly demonstrates the differential effects a transgene may have depending on the duration of fast. Our laboratory has also shown that the effect of the HKII transgene employed here becomes magnified as the duration of the fast is increased from 5 to 18 h and is likely to be due to the effect of G6P inhibition of HKII (Halseth et al. 1999). A new finding of these studies was that overexpression of GLUT4 alone results in a 70% reduction in muscle HKII protein. Considering that HKII catalyses a vital step in MGU, one could speculate that GLUT4 overexpression was offset by the reduction in HKII. To test whether this was a possibility, GLUT4 overexpressing mice were bred with mice overexpressing the HKII transgene. This created HKII levels that were equal to and could thereby be compared to those of mice overexpressing HKII alone. It was shown that combined GLUT4 and HKII overexpression did not augment insulin-stimulated MGU beyond that seen with HKII overexpression alone. Thus even when the bottle neck at glucose phosphorylation is widened by HKII overexpression, MGU is not rate-limited by glucose transport. It is noteworthy that the HKII transgene containing the creatine kinase promoter is not affected by GLUT4 overexpression. However, the endogenous HKII promoter is sensitive to GLUT4 overexpression, and the lower HKII protein content may be due to the lower circulating insulin concentrations measured in GLUT4 Tg mice, given the role of insulin in modulating HKII gene expression (Postic et al. 1993; Printz et al. 1993). GIR during an insulin clamp is determined by the magnitude of the suppression of endogenous glucose production and the stimulation of whole body glucose utilization (mainly muscle). GLUT4 overexpression alone and in combination with HKII overexpression augmented GIR by 15 and 25%, respectively, during physiological hyperinsulinaemia. As indices of whole body and muscle glucose utilization were not increased it would appear that, as first shown by Ren et al. (1995), the primary means by Figure 3. Disappearance of [2-3 H]DG from the plasma in chronically catheterized, conscious mice during the final 25 min of a 120-min saline infusion or hyperinsulinaemic euglycaemic clamp Wild-type (WT, ) and mice overexpressing GLUT4 (GLUT4 Tg, ), HKII (HK Tg, )orboth (GLUT4 Tg + HK Tg, ) were fasted for 5 h and subjected to a 120-min saline infusion (A, B) or hyperinsulinaemic euglycaemic clamp (C and D). The area under the curve (AUC) for the disappearance of [2-3 H]DG from the plasma has also been calculated (B and D). Data are means ± S.E.M. for 8 14 mice per group. P < 0.05 versus WT; P < 0.05 versus HK Tg ; P < 0.05 versus GLUT4 Tg ; P < 0.05 versus saline-infused.

7 J Physiol Control of insulin-stimulated muscle glucose uptake in vivo 931 Table 2. Basal muscle glucose uptake during the last 30 min of a 120-min saline infusion from C57BL/6J mice fasted for 5 h WT GLUT4 Tg HK Tg GLUT4 Tg + HK Tg K g (ml (100 g) 1 min 1 ) Gastrocnemius 0.12 ± ± ± ± 0.07 SVL 0.13 ± ± ± ± 0.06 Soleus 0.83 ± ± ± ± 0.25 R g (µmol (100 g) 1 min 1 ) Gastrocnemius 1.19 ± ± ± ± 0.46 SVL 1.28 ± ± ± ± 0.44 Soleus 9.19 ± ± ± ± 1.79 Data are means ± S.E.M. in 8 14 mice per group. P < 0.05 versus WT and HK Tg mice; P < 0.05 versus WT mice; P < 0.05 versus GLUT4 Tg + HK Tg mice. Table 3. Muscle glycogen (mg glucosyl units (g muscle) 1 ) following a 120-min saline infusion or hyperinsulinaemic euglycaemic clamp from C57BL/6J mice fasted for 5 h WT GLUT4 Tg HK Tg GLUT4 Tg + HK Tg Saline Gastrocnemius 3.6 ± ± ± ± 0.8 SVL 3.6 ± ± ± ± 1.0 Insulin Gastrocnemius 4.0 ± ± ± ± 0.5 SVL 4.3 ± ± ± ± 0.5 Data are means ± S.E.M. in8 14 mice per group. P < 0.05 versus saline-infused mice. which GLUT4 overexpression increases GIR is by added suppression of endogenous glucose production. Although not the focus of the present study, the hepatic effects of GLUT4 overexpression are interesting in themselves and worthy of further investigation. The difference between GIR in WT and HK Tg mice during the insulin clamp was 9.2 mg kg 1 min 1.Although the difference in GIR was small and not statistically significant, it was actually very close to the rates projected from muscle R g.ifitisassumed that skeletal muscle accounts for 90% of insulin-stimulated glucose uptake in WT and HK Tg, then one would predict that R g of a representative muscle would increase by 8.3 mg kg 1 min 1. This is very close to the result of the present study that showed that the HKII transgene increases insulin-stimulated R g by 7.8 mg kg 1 min 1 (4.3 µmol (100 g) 1 min 1 )inthemixed fibre type gastrocnemius. Thus a result that is marked when assessing MGU specifically, was essentially undetectable when using GIR to assess the effect of the transgene. This emphasizes the added sensitivity obtained using isotopic methods specifically for evaluating muscle glucose metabolism. Previous studies using modelling approaches in rats (Furler et al. 1991; Youn et al. 1995; Petersen et al. 2003) and humans (Bonadonna et al. 1993, 1996; Kelley et al. 1999; Williams et al. 2001, 2003) have suggested that glucose phosphorylation becomes an important site for controlling MGU during hyperinsulinaemia. This study utilized an independent approach (i.e. transgenic manipulation in conscious mice) and provides further support for that hypothesis in that overexpression of HKII, but not GLUT4, was able to augment insulin-stimulated indices of whole body glucose clearance and tissue-specific glucose utilization in all muscles studied. The results presented here are consistent with previous work from our laboratory utilizing FVB/NJ mice overexpressing HKII (Halseth et al. 1999; Fueger et al. 2004a). Therefore, when muscle is stimulated by insulin, the control of glucose influx shifts from primarily glucose transport to predominantly glucose phosphorylation. On the other hand, results of other experiments using GLUT4 overexpressing mice that concluded transport is the rate-limiting step for insulin-stimulated glucose uptake differ from those of the present study and that of Ren et al. (1995). Experiments herein and by Ren et al. (1995) both used conscious mice and physiological insulin doses. We postulate that results that differ do so due to technical differences which generally make the preparations less physiological. These include the use of anaesthesia (Marshall & Mueckler, 1994; Lombardi et al. 1997; Marshall et al. 1999), in vitro preparations (Deems et al. 1994; Hansen et al. 1995; Tsao et al. 1996; Lombardi et al. 1997; Marshall et al. 1999), suprapharmacological insulin doses (Marshall & Mueckler, 1994; Ren et al. 1995; Marshall et al. 1999) and prolonged fasting (Ren et al. 1995). With regard to the latter two, it is noteworthy that, when Ren et al. (1995) used a prolonged fast that results

8 932 P. T. Fueger and others J Physiol Table 4. Muscle glucose and glucose 6-phosphate (mm) following a 120-min hyperinsulinaemic euglycaemic clamp from C57BL/6J mice fasted for 5 h WT GLUT4 Tg HK Tg GLUT4 Tg + HK Tg Glucose Soleus 1.03 ± ± ± ± 0.19 Gastrocnemius 0.55 ± ± ± ± 0.08 SVL 0.37 ± ± ± ± 0.13 Glucose-6-phosphate Soleus 0.48 ± ± ± ± 0.12 Gastrocnemius 0.89 ± ± ± ± 0.25 SVL 1.06 ± ± ± ± 0.15 Data are means ± S.E.M. in8 14 mice per group. P < 0.05 versus WT mice; P < 0.05 versus HK Tg mice; P < 0.05 versus WT, GLUT4 Tg,HK Tg mice. in marked reduction in glycogen content and weight, or suprapharmacological insulin, GLUT4 overexpression increased glucose utilization during an insulin clamp. This contrasted with their own results using a short-term fast (3 4 h) and physiological insulin levels (Ren et al. 1995). Chang et al. (1996) reported an enhancement of insulin-stimulated MGU from isolated muscles overexpressing HKII. We, here and in a previous study (Halseth et al. 1999), demonstrated an increase in insulin-stimulated MGU in vivo when glucose phosphorylation capacity is increased by HKII overexpression. This response was observed in all muscles studied and at a physiological insulin concentration ( 80 µuml 1 ). The effectiveness of the HKII transgene, like the GLUT4 transgene, may also be sensitive to experimental differences. Hansen et al. (2000) observed no effect of the HKII transgene in the extensor digitorum longus during suprapharmacological insulin stimulation in vitro.chang et al. (1996) reported that this muscle was Figure 4. Arterial insulin concentrations during a insulin an clamp experiment at a physiological dose in chronically catheterized, conscious mice Wild-type (WT, ) and mice overexpressing GLUT4 (GLUT4 Tg, ), HKII (HK Tg, ) orboth (GLUT4 Tg + HK Tg, ) were fasted for 5 h and subjected to a 120-min hyperinsulinaemic euglycaemic clamp (4 mu kg 1 min 1 insulin with blood glucose concentration clamped at 165 mg dl 1 ). P < 0.05 versus baseline. the least sensitive to HKII overexpression. This further emphasizes the need to consider the type of muscle when evaluating control of MGU. The comparisons of K g are important because they serve as an index of MGU that is concentration-independent. The glucose concentration-dependent R g data are of value in order to assign a mass to glucose influx. Interpretation can be difficult when glucose concentrations are not equal (e.g. during the saline-infusion experiments). For example, while GLUT4 overexpression significantly augments R g in saline-infused mice, the magnitude of this difference would undoubtedly be greater if the arterial glucose concentrations were identical. Of course the concentration dependence is not a factor during the hyperinsulinaemic euglycaemic clamp experiments. Therefore, if the glucose concentrations were matched for all genotypes in saline-infused conditions, basal R g would be increased and insulin-stimulated component of R g (i.e. R g during insulin clamp minus R g during saline infusion) for GLUT4 overexpressing mice would actually be even lower. This further underscores the transition from glucose transport to glucose phosphorylation as a barrier to MGU during insulin-stimulated conditions. We hypothesized that GLUT4 overexpression would be more effective when the barrier to glucose phosphorylation was lowered by HKII overexpression. This was not the case. The result of combined GLUT4 and HKII overexpression could be a shift in control so that the ability to deliver glucose to the muscle could be limited. The contribution of muscle glucose delivery cannot be directly assessed by the present study. However, recent work from our laboratory demonstrated that GLUT4 overexpression in combination with HKII overexpression enhances exercise-stimulated K g compared to HKII overexpression alone (Fueger et al. 2004b). We hypothesize that the massive hyperaemia associated with exercise markedly reduces the resistance to glucose delivery. Even though insulin too can improve glucose delivery by its own hyperaemic effect (Baron, 1994; Baron et al. 1995; Rattigan et al. 1997, 2001), it cannot do so to the extent seen during exercise (Halseth et al. 1998; Clark et al. 2003). Based on

9 J Physiol Control of insulin-stimulated muscle glucose uptake in vivo 933 Figure 5. Incremental insulin-stimulated tissue-specific glucose uptake in intact skeletal muscles from chronically catheterized, conscious mice Wild-type (WT) or mice overexpressing GLUT4 (GLUT4 Tg ), HKII (HK Tg )orboth (GLUT4 Tg + HK Tg ) were chronically catheterized and allowed to recover from surgery for 5 days. Following a 5-h fast, mice were subjected to a 120-min hyperinsulinaemic euglycaemic clamp. A bolus of 12 µci [2-3 H]DG was injected in order to calculate gastrocnemius (A and C) and superficial vastus lateralis (SVL, B and D) K g and R g, indices of tissue-specific glucose uptake. Data are calculated as the incremental increase in K g or R g following insulin stimulation from basal K g or R g,respectively, and are reported as means ± S.E.M. for 8 14 mice per group. P < 0.05 versus WT and GLUT4 Tg ; P < 0.05 versus WT. this, we speculate that glucose delivery exerts significant control of MGU during insulin stimulation when GLUT4 and HKII are overexpressed together. This is supported by the reduction in muscle glucose seen in GLUT4 Tg + HK Tg gastrocnemius and soleus muscles. A striking correlation exists between indices of MGU and total content of the proteins overexpressed in the current study. In the basal state, total GLUT4 content appeared to determine the K g and R g measured in saline-infused mice. In contrast, it is the total HKII content that dictated the insulin-stimulated K g and R g. One might even argue that the reduced HKII content in GLUT4 Tg mice limited insulin-stimulated MGU. The results of the current study provide support for the concept that metabolic pathways are regulated by multiple steps rather than limited to a single rate-limiting step and that the relative contributions of these steps are influenced by the physiological conditions (Fell & Thomas, 1995). Skeletal muscle glucose transport is the primary barrier to glucose influx during the basal state. However, this study shows that, during the high flux state created by stimulation with physiological hyperinsulinaemia, Figure 6. Incremental insulin-stimulated tissue-specific glucose uptake in intact skeletal muscles from chronically catheterized, conscious mice Wild-type (WT) or mice overexpressing GLUT4 (GLUT4 Tg ), HKII (HK Tg ), or both (GLUT4 Tg + HK Tg ) were chronically catheterized and allowed to recover from surgery for 5 days. Following a 5-h fast, mice were subjected to a 120-min hyperinsulinaemic euglycaemic clamp. A bolus of 12 µci [2-3 H]DG was injected in order to calculate soleus (B) K g and R g, indices of tissue-specific glucose uptake. Data are calculated as the incremental increase in K g or R g following insulin stimulation from basal K g or R g,respectively, and are reported as means ± S.E.M. for 8 14 mice per group. P < 0.05 versus WT and GLUT4 Tg ; P < 0.05 versus GLUT4 Tg.

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