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Endocrinology Vol. 145, No. 11 4912-4916
Copyright © 2004 by The Endocrine Society

Regulation of Insulin-Stimulated Muscle Glucose Uptake in the Conscious Mouse: Role of Glucose Transport Is Dependent on Glucose Phosphorylation Capacity

Patrick T. Fueger, Holli S. Hess, Deanna P. Bracy, R. Richard Pencek, Kelly A. Posey, Maureen J. Charron and David H. Wasserman

Department of Molecular Physiology and Biophysics (P.T.F., H.S.H., D.P.B., R.R.P., K.A.P., D.H.W.) and Mouse Metabolic Phenotyping Center (D.P.B., D.H.W.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and Department of Biochemistry (M.J.C.), Albert Einstein College of Medicine, Bronx, New York 10461

Address all correspondence and requests for reprints to: Patrick T. Fueger, Duke University Medical Center, Department of Pharmacology and Cancer Biology, 4321 Medical Park Drive, Suite 200, Durham, North Carolina 27704. E-mail: patrick.fueger{at}duke.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous work suggests that normal GLUT4 content is sufficient for increases in muscle glucose uptake (MGU) during hyperinsulinemia, because glucose phosphorylation is the more formidable barrier to insulin-stimulated MGU. It was hypothesized that a partial ablation of GLUT4 would not impair insulin-stimulated MGU when glucose phosphorylation capacity is normal but would do so when glucose phosphorylation capacity is increased. Thus, chow-fed C57BL/6J mice with a GLUT4 partial knockout (GLUT4+/–), hexokinase II overexpression (HKTg), or both (HKTg + GLUT4+/–) and wild-type littermates were studied. Carotid artery and jugular vein catheters were implanted for sampling and infusions at 4 months of age. After a 5-d recovery, 5-h fasted mice (n = 8–11/group) underwent a 120-min saline infusion or insulin clamp (4 mU/kg·min insulin with glucose maintained at 165 mg/dl) and received a 2-deoxy[3H]glucose bolus to provide an index of MGU (Rg) for the soleus, gastrocnemius, and superficial vastus lateralis. Basal Rg from all muscles studied from saline-infused mice were not changed by any of the genetic modifications. HKTg mice had augmented insulin-stimulated Rg in all muscles studied compared with remaining genotypes. Insulin-stimulated Rg was not impaired in any of the muscles studied from GLUT4+/– mice. However, the enhanced insulin-stimulated Rg created by HK overexpression was ablated in HKTg + GLUT4+/– mice. Thus, a 50% reduction of normal GLUT4 content in the presence of normal HK activity does not impair insulin-stimulated MGU. However, when the glucose phosphorylation barrier is lowered by HK overexpression, GLUT4 availability becomes a limitation to insulin-stimulated MGU.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SKELETAL MUSCLE IS the primary site for insulin-stimulated glucose disposal. Discerning how the muscle regulates glucose influx and defining the specific impairments to this process are critical to understanding the pathophysiology of insulin resistance and type 2 diabetes. Park and colleagues (1, 2, 3) demonstrated in the 1950s that glucose transport across the sarcolemma is the primary limitation to muscle glucose uptake (MGU) in the basal state and that intracellular glucose phosphorylation to glucose-6-phosphate by a hexokinase isozyme (HK) becomes a limitation during hyperinsulinemia. Implicit in these observations is that deficits to muscle glucose transport and/or phosphorylation may result in impairments of glucose influx depending on the condition. More recent work, however, using genetically manipulated mice that either overexpress (4, 5, 6) or underexpress (7, 8, 9) GLUT4 (the GLUT family member that translocates to the sarcolemma upon stimulation by insulin or exercise) has led to the contention that glucose transport is the rate-limiting step for MGU during insulin-stimulated as well as basal conditions. In fact, a partial deletion of GLUT4 has been reported to create a monogenic form of insulin resistance (7). There is controversy as to whether muscle membrane transport represents a single rate-limiting step for glucose uptake as glucose phosphorylation can become a significant barrier to MGU in states characterized by high glucose fluxes such as hyperinsulinemia and exercise (10, 11, 12, 13, 14). In line with the concept that glucose phosphorylation becomes increasingly important when muscle is stimulated is the observation that mice with a partial ablation of HK II have impaired exercise-stimulated MGU in oxidative muscles (15). The role that glucose transport and phosphorylation play may depend on conditions such as nutritional state and physiological state and may be influenced by technical and experimental considerations.

Transgenic manipulations to the proteins that catalyze steps of metabolic pathways have provided new insight into the regulation of these pathways. Transgenes that alter both glucose transport and phosphorylation can be employed individually or simultaneously to characterize which sites are barriers to MGU during various physiological conditions. To this end, chow-fed C57BL/6J mice with or without a partial ablation of GLUT4 can be used to determine the extent to which glucose transport is a barrier to MGU during basal and insulin-stimulatory conditions. In addition, the presence or absence of muscle-specific HK II overexpression can be combined with manipulations to GLUT4 to determine the impact of lowering the glucose phosphorylation barrier on glucose influx in vivo. To maintain normal physiology, lean mice were studied in the conscious state with a surgical catheterization technique that permits access to the arterial and venous circulation without restraining or stressing the animal by handling. It was hypothesized that, in otherwise healthy animals, a partial ablation of GLUT4 would not impair insulin-stimulated MGU when glucose phosphorylation capacity is normal but would do so when glucose phosphorylation capacity is increased.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse maintenance and genotyping
All procedures performed were approved by the Vanderbilt University Animal Care and Use Committee. Male FVB/NJ mice that selectively overexpress HK II (HKTg) in skeletal muscle using a transgene containing the human HK II cDNA driven by the rat muscle creatine kinase promoter that yields a 3-, 5-, and 7-fold increase in HK activity in the soleus, gastrocnemius, and superficial vastus lateralis (SVL) muscles, respectively, were obtained from Dr. David E. Moller (11) and backcrossed onto the C57BL/6J background for at least seven generations. Male mice with a complete ablation of the GLUT4 gene (16) were bred with HKTg females to obtain heterozygous GLUT4 knockout mice with (HKTg + GLUT4+/–) or without (GLUT4+/–) the HK II transgene. Mice from this mating were bred to create offspring with one of four genotypes: wild-type (WT), GLUT4+/–, HKTg, and HKTg + GLUT4+/–. Littermates were separated by gender after a 3-wk weaning period and were maintained in microisolator cages. Genotyping was performed with the PCR on genomic DNA obtained from a tail biopsy and isolated by a DNeasy Tissue Kit (QIAGEN, Valencia, CA). All mice were fed standard chow containing 5.5% fat (5001 laboratory rodent diet; Purina, Richmond, IN) ad libitum, were handled at least twice per week, and were studied at approximately 4 months of age.

Surgical procedures
The surgical procedures used for implanting chronic catheters were similar to those described previously (13, 17, 18). Briefly, mice of either sex were anesthetized with pentobarbital (70 mg/kg body weight). The right jugular vein and the left common carotid artery were catheterized for infusions and arterial blood sampling, respectively. The catheters were exteriorized and sealed with stainless steel plugs. Animals were housed individually after surgery, and body weight was recorded daily. Line patency was maintained by flushing daily with 10–50 µl saline containing 200 U/ml heparin and 5 mg/ml ampicillin.

In vivo metabolic experiments
Mice were allowed to recover for approximately 5 d. Mice were studied only when body weight was restored to within 10% of presurgery body weight and exhibited normal feeding and grooming behaviors. Conscious, unrestrained mice were fasted for 5 h. Approximately 1 h before an experiment, Micro-Renathane (0.033-in. outer diameter) tubing was connected to the catheter leads and infusion syringes. From this point until the end of the experiment, mice were not handled and were allowed to move freely. After a 1-h acclimation period, a 150-µl baseline arterial blood sample was drawn at t = –90 min for the measurement of arterial blood glucose (HemoCue, Mission Viejo, CA), hematocrit, and plasma insulin and nonesterified fatty acids (NEFAs). The remaining red blood cells were washed with 0.9% saline containing 10 U/ml heparin and reinfused. An infusion of saline (n = 11 WT, 8 GLUT4+/–, 11 HKTg, and 10 GLUT4+/– + HKTg) or 4 mU/kg·min of insulin (n = 11 WT, 8 GLUT4+/–, 11 HKTg, and 10 GLUT4+/– + HKTg) at a rate of 1.375 µl/min was begun at t = –90 min. Euglycemia was maintained during insulin experiments by frequently 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 prevent a marked fall in hematocrit (>5%). The average change in hematocrit for mice in this study was 3.2%. A 150-µl arterial blood sample was obtained at t = 0 min and processed as the baseline blood sample. At t = 5 min, a 12-µCi bolus of 2-deoxy[3H]glucose ([2-3H]DG) was administered to determine Rg, an index of tissue-specific glucose uptake. Arterial blood (40–50 µl) was sampled to determine arterial blood glucose and plasma [2-3H]DG at t = 7, 10, 15, 20, and 30 min. A final 150-µl arterial blood sample was withdrawn at t = 30 min and processed in a manner similar to the baseline blood sample. Mice were then anesthetized with an infusion of sodium pentobarbital, and the soleus, gastrocnemius, and SVL muscles were excised, immediately frozen in liquid nitrogen, and stored at –70 C until future tissue analysis.

Assays for plasma and muscle samples
Immunoreactive insulin was assayed with a double-antibody method (19). NEFAs were measured spectrophotometrically by an enzymatic colorimetric assay (Wako NEFA C kit, Wako Chemicals Inc., Richmond, VA). Plasma samples were deproteinized with Ba(OH)2 (0.3 N) and ZnSO4 (0.3 N), and then [2-3H]DG radioactivity was determined by liquid scintillation counting (Packard TRI-CARB 2900TR, Packard, Meriden, CT) with Ultima Gold (Packard) as scintillant. Frozen muscle samples were homogenized in 0.5% perchloric acid, centrifuged, and neutralized. One supernatant aliquot was counted directly to determine radioactivity from both [2-3H]DG and [2-3H]DGP. A second aliquot was treated with Ba(OH)2 and ZnSO4 to remove [2-3H]DGP (2-deoxy[3H]glucose-6-phosphate) and any tracer incorporated into glycogen and then counted to determine radioactivity from free [2-3H]DG (20). [2-3H]DGP was calculated as the difference between the two aliquots. The accumulation of [2-3H]DGP was normalized to tissue weight and tracer bolus in all experiments. Rg, an index of tissue-specific glucose uptake, was calculated as previously described (21). Muscle glycogen was determined by the method of Chan and Exton (22) on the contralateral gastrocnemius and SVL muscles. Soleus glycogen content was not determined because both muscles were used for the assay to determine Rg.

Immunoblotting
Total HKII protein content was determined on representative gastrocnemius and SVL muscles. Muscles were homogenized in a solution containing 10% glycerol, 20 mM Na-pyrophosphate, 150 mM NaCl, 50 mM HEPES (pH 7.5), 1% Nonidet P-40, 20 mM ß-glycerophosphate, 10 mM NaF, 2 mM EDTA (pH 8.0), 2 mM phenylmethylsulfonyl fluoride, 1 mM CaCl2, 1 mM MgCl2, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM Na2VO3, and 3 mM benzamide. After centrifugation (1 h at 4500 x g), pellets were discarded and supernatants were retained for protein determination using a Pierce BCA protein assay kit (Rockford, IL). Proteins (30 µg) were separated on a SDS-PAGE gel and then transferred to a polyvinylidene difluoride membrane. Membranes were blocked, blotted with rabbit {alpha}-HKII (1:1000; Chemicon International, Temecula, CA), and incubated with {alpha}-rabbit-horseradish peroxidase (1:20,000; Pierce, Rockford, IL). Densitometry was performed using ImageJ software (National Institutes of Health, Bethesda, MD).

Statistical analysis
Data are presented as means ± SEM. Differences between groups were determined by ANOVA followed by Tukey’s post hoc tests. The significance level was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Basal metabolism
Fasting arterial blood glucose was lower in GLUT4+/– and HKTg + GLUT4+/– mice compared with WT and HKTg mice (156 ± 5 and 152 ± 6 vs. 174 ± 6 and 174 ± 4 mg/dl). NEFAs were also lower in GLUT4+/– and HKTg + GLUT4+/– mice compared with WT and HKTg mice (1.0 ± 0.1 and 1.0 ± 0.1 vs. 1.5 ± 0.1 and 1.5 ± 0.1 mmol/liter). Plasma insulin concentrations, however, did not differ between WT, GLUT4+/–, HKTg, and HKTg + GLUT4+/– mice (24 ± 2, 26 ± 3, 20 ± 2, and 22 ± 3 µU/ml, respectively).

Arterial blood glucose concentrations did not change significantly throughout the course of the 120-min saline infusion (data not shown). Basal Rg (Table 1Go) was not altered in soleus, gastrocnemius, or SVL muscles by any of the genetic manipulations. Muscle glycogen was lower in GLUT4+/– and HKTg + GLUT4+/– mice compared with WT and HKTg mice after the 120-min saline infusion.


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  		TABLE 1. Basal Rg and glycogen from 5-h fasted C57BL/6J mice

 
HK II protein content
The HK II transgene more than doubled HK II protein content in the gastrocnemius and increased it approximately 4-fold in the SVL (Table 2Go). HK II protein content was not altered by a partial GLUT4 knockout.


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  		TABLE 2. Muscle HK II content (arbitrary units) from C57BL/6J mice

 
Insulin-stimulated metabolism
Arterial blood glucose concentrations did not differ between groups throughout the insulin clamp period (Fig. 1AGo). The glucose infusion rates (GIRs) required to maintain euglycemia are shown in Fig. 1BGo. A partial deletion of GLUT4 did not decrease GIR compared with WT control mice. Although HK II overexpression significantly increased GIR compared with GLUT4+/– and HKTg + GLUT4+/– mice, it did not do so compared with WT mice.



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  		FIG. 1. Arterial blood glucose concentration (A) and average GIR (B) during the last 30 min of a 120-min insulin clamp experiment in chronically catheterized, conscious mice. WT, GLUT4+/–, HKTg, and HKTg + GLUT4+/– were catheterized and allowed to recover from surgery for approximately 5 d. Mice were fasted for 5 h and subjected to a 120-min hyperinsulinemic-euglycemic clamp (4 mU/kg·min insulin with arterial blood glucose clamped at ~165 mg/dl). GIR data are the average glucose infused required to maintain euglycemia during the final 30 min of the 120-min experiment. Data are means ± SEM for eight to 11 mice per group. *, P < 0.05 vs. GLUT4+/– and HKTg + GLUT4+/–.

 
A partial GLUT4 ablation did not impair insulin-stimulated Rg in soleus, gastrocnemius, or SVL muscles (Fig. 2Go). HK II overexpression enhanced insulin-stimulated Rg in all three muscles studied. This enhancement of insulin-stimulated Rg created by HK II overexpression, however, was abrogated by the addition of a partial GLUT4 ablation in each muscle studied.



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  		FIG. 2. Tissue-specific glucose uptake in intact skeletal muscles from chronically catheterized, conscious mice during an insulin clamp. WT, GLUT4+/–, HKTg, and HKTg + GLUT4+/– were catheterized and allowed to recover from surgery for approximately 5 d. After a 5-h fast and a bolus injection of 12 µCi of [2-3H]DG during an insulin clamp, Rg was calculated as described in Materials and Methods for the soleus (A), gastrocnemius (B), and SVL (C) muscles. Data are means ± SEM for eight to 11 mice per group. *, P < 0.05 vs. WT, GLUT4+/–, and HKTg + GLUT4+/–; {dagger}, P < 0.05 vs. WT.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the basal state (i.e. during saline infusion), neither a partial GLUT4 ablation nor HK II overexpression altered Rg in any of the muscles studied. Because HK II overexpression had no effect on basal MGU here and in previous reports (10, 11, 13), it is reasonable to conclude that glucose phosphorylation is not a site of resistance to glucose influx in the basal state. GLUT1 (23, 24) or GLUT4 (4, 5, 6) overexpression has been previously shown to augment basal MGU, suggesting that glucose transport is the primary barrier to MGU in the basal state. Because Rg during the saline infusion was similar between WT and GLUT4+/– mice, it can be stated that any GLUT1 and half of the normal GLUT4 content are sufficient for basal muscle glucose transport and uptake. Similar results were noted in isolated MGU studies under basal conditions (9).

The hyperinsulinemia significantly increased MGU in all mice and required exogenous glucose at rates greater than 50 mg/kg·min during the insulin clamp. WT mice exhibited a more than 2-fold increase in Rg during the insulin clamp. A partial GLUT4 knockout did not limit the muscle’s ability to take up glucose during a 4 mU/kg·min insulin clamp. In this study, GLUT4+/– mice did not have a marked reduction in GIR during the insulin clamp. In contrast, Rossetti et al. (7) reported reductions in whole-body and muscle glucose disposal in GLUT4+/– mice. The differences in the phenotypes between the previous report and the present study are potentially due to the differences in adiposity between the mice, the selected insulin infusion rate, and the mouse strains. The 4- to 5-month-old mice in the previous study weighed approximately 37 g compared with approximately 25 g in this study. A difference in the fat composition of the diet (~5.5% here and not reported in the previous study) could account for the alterations in body weight. One could, in fact, argue that the previous report studied mice in the transition from healthy animals to those with frank insulin resistance because plasma insulin concentrations were higher than normal values. The implications of these conflicting results are that under normal physiological conditions, a 50% reduction in GLUT4 does not create a barrier to MGU. However, in a condition characterized by insulin resistance such as occurs with high fat feeding and increased weight gain, a decrease in glucose transport may create a significant barrier to insulin-stimulated MGU.

The data presented here in C57BL/6J mice are consistent with the previous reports in that HK II overexpression had no effect on Rg in saline-infused mice (11) but is augmented by insulin-stimulated Rg (13). Importantly, these results in the mouse that demonstrate that insulin shifts the primary barrier of MGU from glucose transport to a distal step are consistent with data from rats (12, 14, 25) and humans (26). Here we show that only when the barrier to glucose phosphorylation is minimized by HK II overexpression does a 50% reduction in GLUT4 create a glucose transport barrier to insulin-stimulated MGU. This was evident from the observation that the augmented insulin-stimulated Rg in HKTg mice is absent when GLUT4 content is reduced by 50%.

Although the partial ablation of GLUT4 had no effect on MGU as indicated by normal basal and insulin-stimulated Rg, it did create a unique metabolic phenotype. GLUT4+/– mice had decreased plasma NEFAs and muscle glycogen content. Both of these observations are potential compensatory mechanisms to restore normal glucose influx to the muscle. Lowering NEFAs with Acipimox has been shown to be an effective means of reversing muscle insulin resistance (27), and decreasing muscle glycogen by exercising enhances glucose disposal during insulin stimulation (28, 29). Rossetti et al. (7) also report a slight reduction in glycogen content in GLUT4+/– mice and a reduction in glycogen synthesis in vivo, yet glycogen synthase activity is not altered. One can speculate that there have been adaptive neuroendocrine changes or adaptation to metabolic pathways responsible for long-chain fatty acid flux and glycogen turnover. If NEFAs and muscle glycogen were elevated to levels found in WT mice, it is possible that this would result in impairments to MGU under basal and stimulated conditions in GLUT4+/– mice. Despite this metabolic milieu that lends itself toward increased insulin sensitivity (i.e. lower NEFAs and glycogen content), HK II overexpression had no effect on MGU when combined with a reduction in GLUT4 content. This further emphasizes that glucose transport can become a barrier to insulin-stimulated glucose influx in muscles where glucose phosphorylation capacity is increased.

Interestingly, the effects observed in muscle do not always correlate with whole-body responses. For example, although HK II overexpression markedly increased insulin-stimulated Rg, it did not significantly alter disappearance of [2-3H]DG from the plasma (data not shown) or GIR. GIR and arterial [2-3H]DG disappearance reflect contributions from all tissues and are not sensitive reflections of tissue-specific MGU.

In summary, glucose transport is not a significant barrier to insulin-stimulated MGU even with a 50% reduction of normal GLUT4 content. When the glucose phosphorylation barrier is decreased by HK II overexpresion, insulin-stimulated MGU increases. However, when the glucose phosphorylation barrier is lowered by HK II overexpression, GLUT4 availability becomes a limitation to insulin-stimulated MGU. The implication from these results is that the most effective means for increasing muscle glucose disposal will need to target multiple steps of the MGU pathway.


   Acknowledgments
 
We thank Wanda Snead and Greg Poffenberger of the Vanderbilt Hormone Assay Core for performing the insulin assays and the Vanderbilt Mouse Metabolic Phenotyping Center for technical assistance.


   Footnotes
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-54902, R01 DK-47425, and U24 DK-59637.

Abbreviations: GIR, Glucose infusion rate; GLUT4+/–, GLUT4 partial knockout mice; [2-3H]DG, 2-deoxy[3H]glucose; [2-3]DGP, 2-deoxy[3H]glucose-6-phosphate; HK, hexokinase isozyme; HKTg, mice overexpressing HK II; MGU, muscle glucose uptake; NEFA, nonesterified fatty acid; SVL, superficial vastus lateralis.

Received April 12, 2004.

Accepted for publication July 20, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Glucose kinetics and exercise tolerance in mice lacking the GLUT4 glucose transporter
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D. H. Wasserman and P. T. Fueger
Point-Counterpoint: Glucose phosphorylation is/is not a significant barrier to muscle glucose uptake by the working muscle
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A. Bertoldo, R. R. Pencek, K. Azuma, J. C. Price, C. Kelley, C. Cobelli, and D. E. Kelley
Interactions Between Delivery, Transport, and Phosphorylation of Glucose in Governing Uptake Into Human Skeletal Muscle
Diabetes, November 1, 2006; 55(11): 3028 - 3037.
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D. B. Olsen, M. Sacchetti, F. Dela, T. Ploug, and B. Saltin
Glucose clearance is higher in arm than leg muscle in type 2 diabetes
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