states such as non-insulin-dependent (type II) diabetes (8) and certain forms of muscular dystrophy (9). Thus, it is imperative to establish the cellular basis by which glucose transport is stimulated in muscle. The recent elucidation of multiple glucose transporter isoforms has proKENNETH J. RODNICK, PHD ROBERT C. PIPER, BS vided a major advance in our underJAN W. SLOT, PHD standing of the regulation of glucose DAVID E. JAMES, PHD transport. A family of facilitative glucose transporters has been characterized by molecular cloning and sequencing (10). There is considerable amino acid seGlucose transport is the rate-limiting step for glucose utilization in muscle. In muscle quence homology between these proand adipose tissue, glucose transport is acutely regulated by such factors as insulin teins and, structurally, they are very simand exercise. Translocation of glucose transporters (GLUT4) from an intracellular ilar. The five members of this family that domain to the cell surface is the major mechanism for this regulation. Using immu- have been described include: GLUT1 nocytochemistry, the intracellular distribution of GLUT4 under resting conditions is (11), which is found in many tissues, similar in adipocytes and myocytes. GLUT4 is concentrated in tubulovesicular including brain, placenta, human erythstructures either in the trans-Golgi region or in the cytosol, often close to the cell rocytes, and transformed cells; GLUT2 surface but not on the cell surface. After stimulation, cell surface GLUT4 labeling is (12), which is found in liver, kidney, intestine, and pancreas; GLUT3 (13), increased by as much as 40-fold. GLUT4 is chronically regulated by altered gene expression. Neural and/or contractile which is expressed in human brain and activity regulates GLUT4 expression in muscle: J) GLUT4 levels differ among muscles at low levels in many cells; GLUT4 (14of different fiber type; 2) GLUT4 levels in muscle are increased with exercise training 18), which is expressed primarily in and decreased with denervation; and 3) cultured muscle cells, which lack an intact muscle and fat; and, GLUT5 (19), which nerve supply, express very low levels of GLUT4. GLUT4 expression appears to be is expressed at high levels in small intesregulated in parallel with many oxidative enzymes in muscle, suggesting that there tine and kidney. may be a unified developmental program that determines the overall metabolic Distinct tissue distribution of properties of a particular muscle. Preliminary evidence suggests that impaired GLUT4 these transporters provides the basis for expression in muscle is not the primary defect associated with insulin resistance. the major functional differences between Nevertheless, it is conceivable that the adaptive increase in muscle GLUT4 that is them. GLUT1 is expressed at high levels found with exercise training may have beneficial effects in insulin-resistant states such in the endothelial cells that make up the as non-insulin-dependent diabetes. blood-brain barrier (20) and in placenta

Interaction of Insulin and Exercise on Glucose Transport in Muscle

I

n view of its mass (40% of body weight), its metabolic demands, and its responsiveness to insulin, skeletal muscle constitutes a major site for in vivo glucose disposal in the postabsorptive state (1,2). Glucose transport is of major importance in regulating muscle glucose

disposal because it is the initial step in this process, it is rate-limiting (3,4), and it undergoes rapid modulation by such factors as exercise and insulin (2,5-7). Furthermore, impaired insulin-stimulated glucose transport in skeletal muscle is characteristic of a number of disease

FROM THE DEPARTMENTS OF MEDICINE AND CELL BIOLOGY AND PHYSIOLOGY, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS, MISSOURI; AND THE DEPARTMENT OF CELL BIOLOGY, MEDICAL SCHOOL, UNIVERSITY OF UTRECHT, UTRECHT, NETHERLANDS. ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO DAVID E. JAMES, PHD, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, DEPARTMENT OF CELL BIOLOGY AND PHYSIOLOGY, BOX 8228, 660 SOUTH EUCLID AVENUE, ST. LOUIS, MO 63110.

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(10). This would ensure that these critical organs are supplied with glucose even when the blood glucose level is low. GLUT2 is enriched in cells of the kidney, intestine, and liver that have a high gluconeogenic capacity, and in the (3-cells of the pancreas (12). Glucose transport in liver has a much higher Km for glucose (15-30 mM) than that observed in other cell types (21). Thus, whereas most transporters will be close to saturation at physiological glucose concentrations, GLUT2 will be well below its maximal transport velocity within this range. Thus, GLUT2 may function as part of a "glucose sensor" regulating insulin secre-

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tion in (3-cells and glucose output in hepatocytes. GLUT4 is expressed only in cells that exhibit insulin-regulatable glucose transport, and in these cells it is translocated from an intracellular pool to the cell surface in response to insulin (14,22). This type of regulation appears to be unique to muscle and fat because in most other cell types the native transporter is constitutively expressed on the cell surface (23-25). Thus, an essential feature of GLUT4 function is its specific targeting to the intracellular compartment. ACUTE REGULATION OF GLUCOSE TRANSPORT— Considerable evidence suggests that glucose transport in muscle and fat is regulated on a minute-by-minute basis by shuttling glucose transporters to and from the surface of the cell. Glucose transport in adipose tissue is regulated primarily by growth factors, such as insulin. In muscle, a number of variables regulate glucose transport aside from insulin. In particular, exercise (6,26-31) and hypoxia (29) have a profound stimulatory effect on muscle glucose transport. Unfortunately, very little is known about the signal transduction pathway(s) that regulates glucose transport in these tissues. Nevertheless, the recent identification of different transporter isoforms constitutes a major advance that may well lead to further progress in this area in the near future. We now know where the transporter resides in muscle and fat cells, that the increase in transporters at the cell surface following stimulation is much greater than previously suspected, and that multiple transporter isoforms are expressed in muscle and fat cells. As described later, these advances are caused by the availability of specific antibodies that distinguish between the different transporter isoforms.

Translocation Most of what is currently known about translocation of transporters is based on studies in primary rat adipocytes. The

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translocation hypothesis originally arose from subcellular fractionation studies in fat cells showing that plasma membranes isolated from insulin-treated cells had a fivefold higher concentration of transporters compared with basal membranes (32,33). Using specific antisera, it has been shown that insulin increases the cell surface concentration of GLUT4 by at least 10-fold (34). An effect of this magnitude may account for the insulinstimulated transport response (20- to 30-fold) in adipocytes. It has been argued that translocation is the major mechanism by which insulin stimulates glucose transport in fat cells. It has been more difficult to obtain similar data in skeletal muscle. Translocation is implicated in the regulation of transport in muscle because both exercise and insulin stimulate the Vmax for glucose transport with little if any change in the Km (6,7,28,30,31). Consistent with this, a number of studies have reported that insulin or contractile activity increase the number of glucose transporters in the plasma membrane fraction of either skeletal or cardiac muscle by two- to threefold (35-42). However, this effect is much less than the increase in glucose transport measured under the same conditions (5- to 10-fold), and so it has been concluded that there must be an additional effect of stimulation to increase the "intrinsic activity" of a given number of transporters to facilitate glucose entry into the cell (31,35,38). Alternatively, this discrepancy could be caused by the difficulty in isolating sarcolemma and transverse tubules (43) from the intracellular vesicles containing GLUT4. This difficulty can be circumvented using immunoelectron microscopy. We have recently performed quantitative immunocytochemistry using several different GLUT4-specific antibodies in brown adipose tissue obtained from rats that were injected with insulin or not (44). In the absence of insulin, only 1% of total cell GLUT4 labeling was observed at the cell surface. Intracellular labeling of GLUT4 occurred primarily in

tubulovesicular structures both in the trans-Golgi reticulum and scattered throughout the cytosol. After insulin stimulation, there was a decrease in intracellular labeling and a 40-fold increase in labeling at the cell surface. An effect of this magnitude is adequate to account for the typical response to insulin that has been observed in these cells. Based on these data, we propose that the much lower increase in cell surface transporters that is observed in muscle using subcellular fractionation is a function of the techniques that have been used to make these observations. As described later, we are currently in the process of characterizing the distribution of GLUT4 using immunocytochemistry in cardiac (45) and skeletal muscle and, hopefully, these studies will clarify this issue. Intracellular location of GLUT4 in cardiac muscle Figure 1 shows immunolabeling of GLUT4 in nonstimulated cardiac muscle from rat. GLUT4 is visualized using gold-conjugated protein A in combination with an anti-GLUT4 antibody raised against a carboxy terminal peptide. Intracellular GLUT4 labeling was concentrated in an area of the cardiac cell near the nucleus (nucleus not shown), where the myofibrils break free of the plasma membrane. This region of the cell typically contains many mitochondria (m) and the Golgi (g) apparatus. GLUT4 was most abundant in tubulovesicular elements on the trans side of the Golgi stacks (referred to as the trans-Golgi reticulum) and in similar tubulovesicular elements clustered in the cytosol often just beneath the cell surface (marked by arrowhead). Several small vesicles, which were quite densely labeled for GLUT4 (5-10 gold particles/vesicle), are shown at the top of the figure very close to the plasma membrane of the myocyte. Similar densely labeled vesicles were also observed at the Z-line in cardiac muscle sometimes close to the transverse tubules (Fig. 1). However, very little GLUT4 label was observed on the plasma mem-

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g

tt Figure 1—Electron micrograph of cardiac muscle from a basal rat, immunolabeled with anti-GLUT4 antisera followed by protein A-labeled gold. GLUT! is located in small tubulovesicular structures beneath the plasma membrane (PM), as marked by the arrowheads and near the Golgi complex (g). Inset indicates labeling of structures between the myofibrils, at the Z-line close to the surface connected transverse tubules (tt). m, Mitochondria.

brane or in the transverse tubule of the myocyte in the nonstimulated condition. We have also observed a similar distribution of GLUT4 in soleus muscle sections from nonstimulated rats (J.W.S.,

Geuze H, D.E.J., unpublished observations). Thus, these morphological data (44-46; Fig. 1) indicate that the intracellular location of GLUT4 is well conserved between different insulin-sensitive

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tissues and support the hypothesis that glucose transport is stimulated in both muscle and fat by translocation of these vesicles to the cell surface. It remains to be seen if other constituents in these ves-

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icles are similar in fat and muscle, and also if the signaling mechanism that triggers their movement to the cell surface is the same between tissues. Targeting of GLUT4 to the specialized transverse tubules is one potential difference between muscle and fat. The transverse tubules are a series of membrane folds extending inward from the plasma membrane of the cell, wrapping around the myofibrils. It is through these tubules that the action potential spreads from the nerve terminal, eventually leading to increased calcium release from the sarcoplasmic reticulum and muscle contraction. The presence of GLUT4 in and around the transverse tubules suggests that this organelle may have an important role in mediating the uptake of important nutrients, such as glucose. It will be important to examine the targeting of GLUT4 to the transverse tubules vs. the sarcolemmal membrane in stimulated muscle because it has been shown that these membranes have a distinct protein composition (47).

Multiple transporter isoforms are expressed in muscle Although GLUT4 is nominally referred to as the major glucose transporter isoform expressed in muscle (14), the expression of other isoforms in muscle tissue has also been reported (10). However, the latter refer to mRNA or protein measurements using intact muscle tissue as starting material. The potential for contamination with other cell types, such as fibroblasts and endothelial cells, is considerable using this approach, and verification using immunocytochemistry is critical. Nevertheless, as shown in Fig. 2, GLUT1 levels are reasonably high in muscle homogenates consistent with specific expression in myocytes. Thus, it cannot be assumed that GLUT4 is the only transporter in muscle, and therefore, the role of other transporters needs to be addressed. Two questions, concerning the functional role of multiple transporter

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isoforms in muscle, need to be considered. What is the contribution of each isoform to total glucose transport? How are these isoforms regulated by insulin and exercise? Figure 2 shows an immunoblot of GLUT1 and GLUT4 in muscle homogenates using saturating concentrations of specific polyclonal antibodies. This method, which has been described in detail elsewhere (48), enables a quantitative comparison between two different proteins that are expressed in the same tissue. Using this method, we find that among muscles of different oxidative capacity, GLUT4 protein levels are 1.5to 4-fold higher than GLUT1. It is noteworthy that Calderhead et al. (49), using an independent method of quantitation, reported a GLUT4:GLUT1 ratio in rat muscle of 3-4:1. These measurements are consistent with estimates of GLUT1 and GLUT4 mRNA by quantitative northern blot analysis of skeletal muscle (L. Koranyi and A. Permutt, unpublished observations). Thus, although GLUT4 may be the major transporter isoform in muscle, the level of other transporter isoforms such as GLUT1 may make a significant contribution to total transport. A major functional difference between GLUT1 and GLUT4 in insulinsensitive cells is the preferential targeting of GLUT1 to the plasma membrane, even under basal conditions. This is clearly shown in Fig. 3, which illustrates the distribution of GLUT1 and GLUT4 in 3T3-L1 adipocytes using immunofluorescence microscopy. Preferential targeting of GLUT1 to the plasma membrane is also observed in insulin-sensitive rat adipocytes (50,51), skeletal muscle (22), and non-insulin-sensitive cells, such as fibroblasts (25,52,53), HepG2 cells (25), and brain microvessels (24). Despite this difference, insulin stimulates translocation of both transporters to the plasma membrane in 3T3-L1 adipocytes, as shown by the depletion of the intracellular GLUT1 pool and the appearance of GLUT4 in the plasma membrane (Fig. 3). Both the kinetics and dose response

GLUT 4 CONTENT

GLUT 1 CONTENT

30

20

10-

Figure 2—GLUT1 and GLUT4 protein content in various rat skeletal muscles. Tissues were homogenized in 40 volumes of an ice-cold buffer containing 20 mM HEPES, 1 mM EDTA, and 250 mM sucrose (pH 7.4). Total protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting using rabbit polyclonal serum against specific peptid.es, which correspond to the 12 terminal amino acids within the C terminus of GLUT1 and GLUT4. Immunoreactive glucose transporters were visualized using 125l-labeled anti-rabbit igG, and the amount of labeled transporter on each autoradiogram was determined by densitometry. A representative autoradiogram is shown above each bar graph. EDL, extensor digitorum longus.

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-INS

+ INS

GLUT 4

GLUT 1

Figure 3—Confocal immunofiuorescence localization of GLUTl and GLUT4 in 3T3-L1 adipocytes. Basal deft) or insulin-treated (right) 3T3-L1 adipocytes were fixed with paraformaldehyde, permeabilized with Triton X-100, and immunolabeled with anti-GLUT4 (top) or anti-GLUTl (bottom) C-terminal tail rabbit antisera. Transporters were visualized withfiuoresceinisothiocyanate-conjugated antirabbit secondary and imaged by scanning laser confocal microscopy and digital enhancement. INS, insulin.

curves for insulin-stimulated movement of the two different transporters are indistinguishable in 3T3-L1 adipocytes (48), suggesting that the mechanism for these effects is very similar. The higher levels of GLUTl in the membrane of insulin-sensitive cells under basal conditions (22,48,49,51; Fig. 3) raises the possibility that GLUTl may function as the basal glucose transporter in muscle and GLUT4 may function only under stimulated conditions (i.e., exercise or insulin). In fact, in a number of cases, the relative tissue expression of GLUTl appears to correlate with basal glucose transport, and GLUT4 levels reflect transport under stimulated conditions. Both GLUT4 lev-

els and insulin-stimulated glucose transport are decreased in adipocytes of fasted and streptozotocin-diabetic animals, whereas GLUTl and basal transport are relatively unaffected (54,55). Among different skeletal muscles, there is little variation in GLUTl levels or in basal glucose utilization (56,57; Fig. 2). In contrast, there is considerable variation in GLUT4 levels and insulin-stimulated glucose utilization among different muscles. Cardiac muscle, which expresses much higher levels of GLUTl compared with skeletal muscles (Fig. 2), also exhibits very high rates of basal glucose utilization (57). In support of this correlation, we have shown that fasting resulted in a marked reduction in basal glucose up-

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take (90%) and in GLUTl levels (60%) in rat heart, whereas GLUT4 levels and insulin-stimulated glucose uptake were unaltered (E.W. Kraegen and D.E.J., unpublished observations). Chronic exercise training is accompanied by increased expression of GLUT4 (58) in muscle and increased insulin-stimulated glucose uptake (59-61), whereas both GLUTl and basal uptake do not change (Fig. 4). Collectively, these data support a model where GLUTl is constitutively expressed at the plasma membrane of all cells, including muscle and fat cells and is well suited to function as a basal transporter. However, it is important to note that the specific expression of GLUTl in either myocytes or adipocytes has not been

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Glucose transport in muscle

GLUT 4 CONTENT 400 n

Interaction between insulin and exercise on glucose transport

Exercise and insulin both increase the rate of glucose entry into muscle, despite 2 major differences in the eventual metaQ. 200bolic fate of the sugar. However, it is not 2 known if these two different stimuli augm CM ment glucose transport by similar or disa. 100o tinct mechanisms. Several lines of evidence suggest that the mechanisms are distinct: 1) in contrast to insulin, the Control ET Control ET exercise-induced increase in glucose transport persists for several hours after cessation of exercise (62,63); 2) the time GLUT 1 CONTENT course for stimulation of transport ap** pears more rapid for contractile activity than insulin (64); 3) insulin is not re3quired for the exercise-induced increase in glucose transport (28,63,65,66), and 2exercise does not stimulate the tyrosine kinase activity of the insulin receptor (67); and 4) the glucose transport response to insulin plus exercise in muscle may be additive with respect to the response with each stimulus alone Control ET Control ET (63,66,68). Although there is some conPLANTARIS SOLEUS flict concerning the latter observation (28,69), these data raise the interesting Figure 4—GLUT4 and GLUT1 protein conpossibility that two separate pools of incentration in hindlimb muscles from control and tracellular transporters exist in muscle; exercise-trained (ET) rats. Exercise-trained rats one sensitive to insulin and the other to ran voluntarily in exercise wheel cages for 6 wk. exercise (22). Clearly, additional studies Immunoblotting techniques and protein quantification are described in Fig. 2 and ref 58. Resultsare needed to identify and characterize are shown as means ± SEfor 8 rats. *P < 0.01 the intracellular pools of glucose transvs. control plantaris; **P < 0.05 compared with porters in skeletal muscle in order to plantaris. understand the interaction between insulin and exercise. 300-

shown. Most studies have involved the measurement of GLUT1 mRNA or protein in tissue homogenates that contain many different cell types (e.g., endothelial cells). Thus, additional studies involving immunolocalization are required to verify the cell-specific expression of GLUT1. It will also be of interest to determine if other members of the facilitated transporter family are expressed in muscle when specific antibodies become available.

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Although there is considerable evidence to suggest that exercise and insulin stimulate transport by separate pathways in muscle, little progress has been made concerning the nature of these mechanisms. It appears that translocation of GLUT4 to the cell surface is the major mechanism (45). However, it is not certain if this is a function of accelerated exocytosis or decreased internalization of GLUT4. Making this distinction will be important in the search for potential mechanisms.

CHRONIC REGULATION OF GLUCOSE TRANSPORT— Alterations in the function or expression of any one of a number of gene products that regulate the proper targeting of GLUT4 in muscle either under basal or insulinstimulated conditions may disrupt glucose homeostasis. However, with the recent cloning of GLUT4, a number of studies have focused on this molecule. Whereas altered expression of GLUT4 in muscle and/or fat appears to occur in various conditions, such as fasting (70) and streptozotocin diabetes (54,55,71), the specificity of such changes with respect to the overall defect is not at all clear. Furthermore, the expression of GLUT4 in muscle does not appear to be altered in type II diabetes (72). Thus, other molecules related to GLUT4 function must be involved. Nevertheless, there are interesting aspects to the control of GLUT4 expression in muscle. Chronic stimulation of muscle at low frequency for a period of weeks increases the expression of many genes that regulate oxidative metabolism, whereas many of the genes that regulate glycolytic metabolism are switched off (73). It is likely that GLUT4 is a part of the gene package that determines the overall oxidative phenotype of muscle.

Skeletal muscle is a heterogenous tissue Skeletal muscle is a heterogenous tissue composed of at least three distinct muscle fiber types. In both rats and humans, there are no skeletal muscles containing just one fiber type. The three major classes of muscle fibers include: fasttwitch glycolytic (FG), fast-twitch oxidative glycolytic (FOG), and slow-twitch oxidative (SO). These different fiber types are classified primarily on the basis of differences in contractile properties and metabolic potential (74-77). The fiber types can also be classified according to their capacity for insulin-responsive glucose utilization and the relative proportions of glucose transporter isoforms.

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FG fibers have a low respiratory capacity, low capillarity and blood flow, a high glycogenolytic capacity, and a high myosin ATPase activity. FOG fibers have a high respiratory capacity, a high capillarity, a high glycogenolytic capacity, and high myosin ATPase activity. SO fibers have a high respiratory capacity, high capillarity and blood flow, low glycogenolytic capacity, and a low myosin ATPase activity. The capacity for respiration and glycogenolysis is reflected in the levels of the major enzymes that regulate these pathways. These differences have been described in detail by others (75,76,78). This heterogeneity in enzyme composition depicts the unique functions of the different muscle fibers. FG fibers are suited for rapid and extreme bursts of activity because of their high capacity for glycogenolysis and anaerobic glycolysis. These fibers do not express high levels of the enzymes that are essential for oxidizing glucose. FOG and SO fibers, on the other hand, express high levels of most oxidative enzymes but not of the glycolytic enzymes. Thus, these fibers are suited for prolonged exercise involving primarily the utilization of extramuscular glucose and free fatty acids. Another major difference between these muscle fibers is in their capacity for insulin-stimulated glucose utilization (56,79,80). Using the hyperinsulinemic glucose clamp technique in rats combined with tracer administration, the following ranking with respect to insulin sensitivity and responsiveness of 2-deoxyglucose uptake has been established: heart > soleus > red quadriceps, red gastrocnemius > extensor digitorum longus, plantaris > white quadriceps, white gastrocnemius. As indicated in Fig. 2, we have observed an identical ranking with respect to the expression of GLUT4 in these different muscles. In contrast, there was much less variation in GLUT1 levels among these different muscles with the notable exception of the heart, which exhibited the highest GLUT1 and GLUT4 levels of any muscle examined. The heterogeneity in the expression of

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GLUT4 among different muscle fibers is particularly interesting because it follows the heterogeneity in hexokinase levels (75,78). As previously noted, a distinct characteristic of different fibers is based on glycolytic vs. oxidative capacity. However, the expression of hexokinase, like GLUT4, follows the pattern of oxidative enzymes rather than the glycolytic enzymes. Thus, both hexokinase and GLUT4 can probably be viewed as oxidative-type enzymes in muscle, which have an important role in regulating the utilization of circulating glucose. The heterogeneous composition of skeletal muscle implies that fiber type must be considered in studies of muscle metabolism. This is particularly relevant to studies using human muscle biopsies because of variation in fiber type from one sample to another. In fact, it is conceivable that variations in fiber composition between individuals may contribute to variations in whole-body insulin action. Exercise training A chronic increase in muscle contractile activity results in altered expression of many genes involved in regulating metabolism (73,75,78). Studies of crossreinervation of muscle or chronic stimulation of muscle at a controlled frequency have indicated that the twitch characteristics and oxidative capacity of a particular muscle fiber are very dependent on the intrinsic contractile pattern (73). In accordance with this concept, endurance-type exercise training increases the expression of many oxidative enzymes in muscle, whereas the expression of glycolytic enzymes is decreased (75,78). However, once again hexokinase is an exception because exercise training results in increased levels of this protein in muscle by 30-170%. As a consequence of these adaptations, the overall oxidative capacity of the muscle is improved. In addition, the ability of insulin to stimulate muscle glucose utilization also improves following prolonged training (59-61). We have shown that GLUT4

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expression in rat muscle is increased by about 60% after 6 wk of voluntary running in exercise wheel cages (Fig. 4, ref. 58). Similar to GLUT4, hexokinase was increased in rat muscle after voluntary running (81). These effects are localized to those muscles selectively recruited during exercise and are probably caused by the contractile activity per se as opposed to a systemic effect. Furthermore, there is a certain degree of specificity with respect to this effect because there was no change in muscle GLUT1 levels following exercise training. Thus, the expression of GLUT4 and hexokinase, but not GLUT1, appears to be regulated in parallel with many of the oxidative enzymes in exercise-trained muscle. Presumably, all of these adaptations are fundamental to the improved insulin action that is observed following exercise training. Denervation Just as increased contractile activity in the form of exercise training increases the oxidative capacity and insulin sensitivity of muscle, decreased contractile activity has the opposite effect (82-86). Impaired insulin-stimulated glucose transport in muscle is a characteristic feature of both limb immobilization (83) and denervation (84-86). These effects occur very rapidly (1-2 days) and are localized only to muscles with reduced contractile activity. In addition, many of the oxidative enzymes in muscle are also decreased by 50-75% between 1 and 15 days postdenervation (82). In support of the notion that GLUT4 expression is regulated in parallel with a family of contractile-regulated muscle genes, its expression was significantly decreased in denervated muscle (86). Three days after lesion of the sciatic nerve, insulinstimulated 2-deoxyglucose uptake in soleus was decreased by 70%, and GLUT4 levels were decreased by 50% (Fig. 5). Muscle cell culture lines One of the major problems in studying regulation of glucose transport in skeletal

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Glucose transport in muscle

2-Deoxyglucose Uptake

• •

Control

1 d DNV

Basal Insulin

3d DNV

GLUT 4 CONTENT

400

300-

200-

100-

Control

1 d DNV

3 d DNV

muscle has been the inability to reproduce insulin-dependent glucose transport to a similar extent to that observed in vivo in a cell culture model. For example, L6 muscle cells (87) or BC3H1 muscle cells (88) typically exhibit only a 50-200% increase in glucose transport after a maximal insulin stimulation. One possible explanation for this relatively poor response to insulin is that L6 cells Q.C. Lawrence, unpublished observations) and BC3H1 cells, C2 cells, and G8 cells (49) express very low levels of GLUT4 compared with adult skeletal muscle. It would be of interest to examine GLUT4 expression in myocytes cocultured with neurons to ascertain if neural input is a critical factor in regulating the expression of this molecule. Such experiments may also be useful in distinguishing between the role of the nerve per se vs. the contractile output of the muscle. It is noteworthy that, in most of the myocyte lines described, only a small percentage of cells in the dish contract. We have also found that primary cultures of rat myotubes, the entire population of which contract throughout the period of culture, also do not express GLUT4 (D.EJ. and J.C. Lawrence, unpublished observations). Thus, these data suggest that the pattern of contractile activity and/or neurotrophic factors must be involved in regulating GLUT4 expression. Because of these limitations, it is likely that such myocyte cell lines are not suitable model systems for studying glucose metabolism in muscle.

Group

Figure 5—Effect of denervation on 2-deoxyglucose uptake and GLUT4 content in soleus muscle. Muscles were denervated (DNV) for 1 or 3 days. 2-Deoxyglucose uptake was measured in the absence or presence of 2000 [iU/ml insulin. GLUT4 content was determined as described in Fig. 4. Values are presented as mean ± SEfor 6-15 muscles. *P < 0.05 vs. basal control; tP < 0.01 vs. insulin control; **P < 0.001 vs. control.

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CONCLUSIONS— Translocation of GLUT4 from intracellular organelles to the cell surface appears to be the major mechanism for stimulation of glucose transport in muscle and adipocytes. Muscle also expresses significant levels of GLUT1, the functional role of which remains to be determined. Based on differential subcellular distribution of GLUT1 compared with GLUT4 and differences in the expression of these two transporters in muscle, we propose that GLUT1 functions as the basal transporter and

GLUT4 as the acutely regulated transporter. Several important questions concerning the acute regulation of glucose transport remain to be answered. What is the relative subcellular distribution of GLUT1 and GLUT4 in muscle? Are these transporters targeted to the transverse tubules or to the sarcolemma following stimulation? The electron microscopic data indicate that GLUT4 is localized very close to the Z-line in cardiac muscle. Thus, transverse tubules are likely to be involved in this regulation. This may provide considerable advantages in accessing the entire myocyte to incoming glucose, particularly because many of the mitochondria are located in deep areas well removed from the sarcolemma. What are the functional characteristics of the tubulovesicular structures that contain GLUT4 within the cell? Whether this is a unique organelle, the main function of which is to shuttle transporters to the membrane on demand, remains to be determined. Also, what are the signaling pathways by which exercise and insulin stimulate translocation? It is apparent that these two stimuli augment transport by distinct mechanisms. It will be important to establish these mechanisms at the biochemical and cellular levels, and to quantify the level of different transporter isoforms in different regions of the cell following each stimulus. With the recent elucidation of different glucose transporter isoforms, a number of investigators have examined the possibility that altered expression of GLUT4, apparently the major transporter isoform in muscle and fat, may contribute to insulin resistance. However, this does not appear to be the case. In muscle from human type II diabetic patients (72) and genetically obese mice (89), GLUT4 levels were not significantly different from controls, despite marked insulin resistance. In many respects, this is not surprising because, as previously noted, there does not seem to be anything particularly unique about the regulation of GLUT4 expression in muscle. In fact, there is good evidence that the

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expression of GLUT4 is linked to that of a number of important genes in muscle, including oxidative enzymes and hexokinase. Thus, GLUT4 appears to belong to a package of genes that determine the respiratory capacity of a particular muscle fiber. We have been unable to observe conditions under which the expression of GLUT4 in muscle is dissociated from the expression of these other genes. Further studies are needed to ascertain how this group of genes is regulated in muscle. Although impaired expression of GLUT4 does not appear to be a major contributor to insulin resistance, it is possible that an inherited GLUT4 mutation that somehow impairs the function of the molecule could be involved. However, in view of the paucity of information concerning the acute regulation of glucose transport in muscle, it is somewhat premature to speculate about possible defects. A crucial question that remains to be addressed is whether a selective increase in GLUT4 expression in insulin-resistant muscle would lead to improved insulin-stimulated glucose utilization, even if this were not the primary defect. Such an experiment may provide important information about the regulation of intermediary metabolism.

Acknowledgments—This work was supported by the Juvenile Diabetes Foundation, the National Institutes of Health (DK-42503 and Institutional National Research Service Award AG-00078), and the Washington University Diabetes Research and Training Center. We thank David Hanpeter for excellent technical assistance.

3.

4.

5.

6.

7.

8.

9.

10.

References 1. Curtis-Prior PB, Trethewey J, Stewart GA, Hanley T: The contribution of different organs and tissues of the rat to assimilation of glucose. Diabetologia 5:384-91, 1969 2. DeFronzo RA, Jacot E, Jequier E, Maeder E, Wahren J, Felber JP: The effect of insulin on the disposal of intravenous

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