©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of an Exercise-sensitive Pool of Glucose Transporters in Skeletal Muscle (*)

(Received for publication, June 19, 1995; and in revised form, September 11, 1995)

Lise Coderre (§) Konstantin V. Kandror (§) Gino Vallega Paul F. Pilch (¶)

From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Augmentation of glucose transport into skeletal muscle by GLUT4 translocation to the plasma and T-tubule membranes can be mediated independently by insulin and by contraction/exercise. Available data suggest that separable pools of intracellular GLUT4 respond to these two stimuli. To identify and characterize these pools, we fractionated skeletal muscle membranes in a discontinuous sucrose density gradient. Fractions of 32 and 36% sucrose exhibited the highest enrichment of GLUT4 and were independently responsive to insulin and exercise, respectively. The combination of the two stimuli depleted both GLUT4 fractions simultaneously. Both vesicle populations contained the gp160 aminopeptidase, whose expression had previously been shown to be specific to muscle and fat and restricted to GLUT4 vesicles in the latter tissue. In muscle, gp160 translocates exactly as does GLUT4 in response to insulin and exercise. The contraction- and insulin-sensitive GLUT4 pools also contained secretory component-associated membrane protein/glucose transporter vesicle triplet but not GLUT1 and caveolin. Immunoadsorption of the two pools followed by silver staining did not reveal any obvious difference in their major protein components. On the other hand, sedimentational analysis in sucrose velocity gradients revealed that the insulin-sensitive GLUT4 vesicles had a larger sedimentation coefficient than the exercise-sensitive vesicles. Thus, the separation of the two intracellular GLUT4 pools should be useful in dissecting what are likely to be different signal transduction pathways that mediate their translocation to the cell surface.


INTRODUCTION

In skeletal muscle, stimulation of glucose uptake by insulin is achieved by recruiting GLUT4 vesicles from their intracellular site to the plasma membrane and to the T-tubules (Hirshman et al., 1990; Douen et al., 1990; Goodyear et al., 1991; Marette et al., 1992). A similar recruitment mechanism is thought to be responsible for the increased glucose uptake seen in response to exercise (Douen et al., 1990; Goodyear et al., 1991) and hypoxia (Cartee and Holloszy, 1990; Cartee et al., 1991). However, there are likely to be mechanistic differences between the stimulation of glucose transport by insulin and exercise. Thus, it has been recently demonstrated that the fungal metabolite wortmannin blocks insulin-stimulated glucose transport in muscle but has no effect on contraction or hypoxia-stimulated glucose uptake (Yeh et al., 1995). Furthermore, it has been known for some time that the effects of insulin and exercise are additive with regard to glucose transport in muscle (Zorzano et al., 1986; Constable et al., 1988; Wallberg-Henriksson et al., 1988; Ploug et al., 1990, 1992) as are their effects on GLUT4 appearance at the plasma membrane (Gao et al., 1994). Finally, it has been demonstrated in denervated muscle (Turinsky, 1987) and in muscles of obese Zucker rats (Brozinick et al., 1992; Dolan et al., 1993; King et al., 1993; Brozinick et al., 1994) that muscle contraction can stimulate glucose transport even when the ability of insulin to do so is impaired. Taken together, these data suggest that muscle may possess two different pools of GLUT4 vesicles, one insulin-sensitive and one exercise-sensitive. Although some evidence has been presented supporting the existence of two such pools (Douen et al., 1990), others have not obtained data consistent with this postulate (Goodyear et al., 1991), and the pools have heretofore not been isolated or characterized.

The likely reason for this is the difficulty in isolating relatively pure membrane fractions from muscle, a tissue consisting of multiple fiber types and a complex subcellular membrane structure. The cell surface includes T-tubules as well as sarcolemma, and the abundant myofibrils hinder facile membrane purification. Nevertheless, many investigators have fractionated skeletal muscle membranes to study GLUT4 translocation, and a comparison of some of the different methods for membrane isolation from this source has been performed by Fushiki et al.(1989). They concluded that the method of Grimditch et al.(1985) gives a good preparation for studying the insulin-dependent GLUT4 enrichment in the plasma membrane, whereas the procedure of Klip et al.(1987) is preferable for the analysis of GLUT4-containing microsomal membranes. However, the latter procedure, while providing results supportive of two transporter pools translocating to the plasma membrane (Douen et al., 1990), employs only 25, 30, and 35% sucrose fractions, and it did not result in identification of an intracellular GLUT4 fraction that responded to exercise/contraction. Given this background, we determined that adding 32 and 38% sucrose layers to this published protocol (Klip et al., 1987) allowed us to clearly separate intracellular GLUT4 vesicles that responded independently to exercise and insulin. Here we present a biochemical characterization of these pools.


EXPERIMENTAL PROCEDURES

Animals

Male Sprague-Dawley rats (175-200 g) were purchased from the Charles River Breeding Laboratory (Wilmington, MA). The animals were fasted overnight and divided into three groups: untreated controls, those injected with insulin (1.5 units/rat) via the portal vein 8 min prior to being sacrificed, and those exercised. The latter consisted of a 30-min swimming period in a water bath maintained at 37 °C. For all three groups, the rats were anesthetized with sodium pentobarbital (60 mg/kg of body weight by intraperitoneal injection), insulin was administered or not, hind limb muscles were removed, and membranes were immediately isolated. These surgical procedures were approved by the Institutional Animal Care and Use Committee of Boston University School of Medicine.

Preparation of Membranes from Skeletal Muscle

Membranes from mixed skeletal muscle were prepared essentially according to Klip et al.(1987). Red and white gastrocnemius and soleus muscles from rat hind limb were rapidly removed and trimmed of connective tissue, fat, and nerves. The muscles (2.5-3.0 gm/rat) were then minced and homogenized for 5 s by Polytron in a buffer containing 20 mM HEPES, 250 mM sucrose, 5 mM EDTA, 1 µM leupeptin, 1 µM pepstatin, 1 µM aprotinin A, pH 7.2, at 4 °C. The homogenate was centrifuged at 1,200 times g for 10 min at 4 °C. The supernatant was saved, and the pellet was resuspended, homogenized, and centrifuged again at 1,200 times g for 10 min. The two supernatants were then combined and centrifuged at 9,000 times g for 10 min at 4 °C. The resulting supernatant was then centrifuged at 200,000 times g for 140 min at 4 °C. The pellet was then resuspended in 7 ml of 38% sucrose (w/v) and 5 ml each of 25, 30, 32, and 36% sucrose solutions were layered on the top. The 30% sucrose layer was adjusted to 7 ml in order to minimize plasma membrane contamination with intracellular membranes. The gradient was centrifuged at 68,000 times g for 14 h. Membranes from the gradient were then collected, diluted with homogenization buffer, and centrifuged again at 200,000 times g for 140 min. The resulting pellets were resuspended in the homogenization buffer, and total membrane protein content was determined by the BCA protein assay (Pierce).

Fractionation of Microsomes in Sucrose Velocity Gradient

Microsomes from the 32 and 36% sucrose fractions were resuspended in phosphate-buffered saline, and an equal amount of protein from each fraction was loaded on a 4.6-ml 10-30% continuous sucrose gradient containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 2 mM dithiothreitol. Membranes were centrifuged at 48,000 rpm for 55 min at 4 °C. Membranes from the gradient were then collected into 200-µl fractions(25, 26, 27, 28, 29, 30) starting from the bottom of the tube. Protein content and refractive index were measured. The position of GLUT4 was determined by Western blotting.

Immunoadsorption of GLUT4 Vesicles

Purified anti-GLUT4 antibody 1F8 (James et al., 1988) as well as nonspecific IgG were coupled to acrylamide beads (Reacti-gel GF2000, Pierce) at a concentration of 1 mg of antibody/ml of resin according to the manufacturer's instructions. Microsomes from the 32 and 36% sucrose fractions were incubated with each antibody for 16 h at 4 °C. The beads were collected by centrifugation, washed three times with phosphate-buffered saline, and eluted with sample buffer (Laemmli, 1970). Portions of the eluate were used for silver stain and for Western blotting.

Gel Electrophoresis and Immunoblotting

Membranes were subjected to electrophoresis in 10% polyacrylamide gels containing 0.1% sodium dodecyl sulfate (Laemmli, 1970), transferred to an Immobilon-P membrane (Millipore, Bedford, MA), which was treated with 5% nonfat dry milk to block nonspecific interactions of antibodies. The membranes were then incubated with anti-GLUT4 antibody (James et al., 1988), a monoclonal antibody directed against SCAMPs/GTV3 (^1)(Thoidis et al., 1993), and a monoclonal antibody for the dihydropyridine receptor (a gift of Dr. Kevin Campbell, University of Iowa). Rabbit polyclonal antibody recognizing gp160 aminopeptidase (Kandror and Pilch, 1994) and GLUT-1 (a gift of Dr. C. Carter-Su, University of Michigan) were also used. The antigen-antibody complexes were detected with either I-goat anti-mouse antibody, I-protein A, or horseradish peroxidase-conjugated antibodies and an enhanced chemiluminescence detection system (DuPont NEN). When horseradish peroxidase-conjugated secondary antibodies were used, the autoradiograph was scanned in a computing densitometer (Molecular Dynamics) and expressed in arbitrary units. For some experiments both approaches were utilized with virtually identical results.

Materials

Electrophoresis chemicals were obtained from National Diagnostics (Atlanta, GA), and collagenase, aprotinin, leupeptin, and pepstatin were purchased from Boehringer Mannheim. I-goat anti-mouse antibody and I-protein A were obtained from DuPont NEN. Other reagents were from Sigma.

Statistical Analysis

The statistical analysis of the immunoblots was performed using an unpaired Student's t test for comparing the experimental group with the control.


RESULTS

Muscle Membrane Fractionation

Fig. 1A shows the distribution of GLUT4, GLUT1, and gp160 aminopeptidase in the discontinuous sucrose density gradient where detection is by Western blotting in all cases. Fig. 1B is the quantitative analysis of the data together with the protein values. The 32 and 36% sucrose fractions are specifically enriched in GLUT4 to the greatest extent of any fractions, and this is also true for gp160 aminopeptidase (Kandror et al., 1994), the latter being co-localized exactly as is GLUT4 in fat cells (Kandror and Pilch, 1994) (see also Fig. 3and Fig. 5). A readily detectable amount of GLUT4 is present in the 25 and 30% sucrose fractions, but this represents only 18% of the total in fractions 25 through 36. Previous studies have shown that the 25% sucrose fraction is enriched in plasma membrane markers (Douen et al., 1990) and that the 30% sucrose fraction may also be slightly enriched in plasma membrane (Douen et al., 1989). We observe that GLUT1 is most abundant in the 25% sucrose fraction consistent with this fraction being enriched in plasma membrane, although much of muscle GLUT1 may derive from the perineural sheath (Handberg et al., 1992). In any case, our interests in this study are focused on the intracellular GLUT4 pools mobilized by insulin and exercise, and therefore, we did not characterize the 25 and 30% sucrose fractions further. We also used antibodies for the T-tubule marker, the dihydropyridine receptor, which overlaps somewhat in distribution with GLUT4 but is most enriched in the 30% sucrose fraction (data not shown). Finally, most (70%) of the protein applied to the gradient appears in the 38% sucrose fraction including 60% of the GLUT4. As shown in Fig. 1B, there is no enrichment of GLUT4 in 38% sucrose fraction as compared with the homogenate, and thus, this fraction very likely represents heterogeneous and impure membrane structures from all parts of the cell. The remaining 30% of GLUT4 distributed in the 32 and 36% sucrose fractions represents the intracellular GLUT4 pools. This value compares with previous studies of intracellular GLUT4 vesicles obtained in 50% yield from muscles frozen in liquid nitrogen, pulverized, homogenized, and fractionated by differential centrifugation (Rodnick et al., 1992).


Figure 1: Distribution of GLUT1, GLUT4, and gp160 aminopeptidase in muscle membrane fractions subjected to discontinuous sucrose density gradient centrifugation. Subfractionation of skeletal muscle membranes was performed as described under ``Experimental Procedures.'' Membrane protein (15 µg) was then subjected to SDS-polyacrylamide gel electrophoresis. Immunoblotting (A) was performed with a polyclonal antibody against GLUT1, a monoclonal anti-GLUT4 antibody (1F8), and an anti-gp160 anti-peptide antibody as described under ``Experimental Procedures,'' and detection of antigen was by appropriate secondary antibody and enhanced chemiluminescence (GLUT4 and gp160) or iodinated protein A (GLUT1). Quantitation (B) was performed as described under ``Experimental Procedures.'' The total membranes applied to the gradient are called the homogenate (H), and the pellet (P) is the bottom of the gradient. The positions of GLUT4 and gp160 are indicated by the closed symbols, GLUT1 by is indicated by open circles, and protein is indicated by open triangles.




Figure 3: Insulin- and exercise-dependent translocation of gp160 aminopeptidase. An experiment was performed that was similar to that of Fig. 2except that anti-gp160 aminopeptidase antibody was used to show translocation of this protein in response to insulin and exercise. Ins refers to membranes from insulin treated animals, and Exe refers to membranes from exercised animals. C, control.




Figure 5: gp160 aminopeptidase and SCAMPs/GTV3 proteins are present in GLUT4-containing vesicles from exercise- and insulin-recruitable pools. Western blotting of a portion of the same preparation used in the previous figure was performed with the appropriate antibodies as described in the previous figure legends and under ``Experimental Procedures.''




Figure 2: Skeletal muscle contains two separate GLUT4 pools: insulin-sensitive and exercise-sensitive. Rats were left untreated(-) or were injected in vivo with 1.5 units of insulin (top, +), subjected to a 30-min swimming period (middle, +), or a combination of both swimming and insulin (see ``Experimental Procedures'') (bottom, +). Membrane protein (50 µg) from the 32 and 36% sucrose fractions was resolved by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting. Detection of GLUT4 was accomplished with monoclonal antibody (1F8) and iodinated secondary antibody. A representative blot is shown in A. B shows the quantitative analysis of autoradiographs from several experiments as determined by excising the appropriate region of the blot and counting it in a counter. The data are expressed in arbitrary units where each fraction was compared with the 36% sucrose fraction, which was set at a value of 1. Each experiment was performed three times on separate occasions, and the results are expressed as the means ± S.E. The asterisks indicate p < 0.05 compared with control.



Selective Response of Intracellular Fractions to Insulin and Exercise

We tested the 32 and 36% sucrose fractions for the ability to respond to insulin and exercise (swimming) as described under ``Experimental Procedures.'' Fig. 2A depicts a representative experiment showing the Western blot analysis from these fractions. It can be seen that GLUT4 is depleted from the 32% sucrose fraction in response to insulin, whereas the 36% sucrose fraction is depleted by exercise, and these responses are specific to each fraction. The combination of both insulin and exercise depletes both fractions simultaneously. The quantitative analysis of three such experiments is shown in Fig. 2B, only for the treatments separately. However, the combination of insulin and exercise resulted in 50% GLUT4 depletion from both fractions (data not shown). Because gp160 aminopeptidase is co-localized with GLUT4 in fat (Kandror and Pilch, 1994) and muscle (Kandror et al., 1995a), we also determined its response to the two stimuli, and as expected, it behaves like GLUT4 (Fig. 3). That is, gp160 aminopeptidase is depleted from the 32% sucrose fraction in response to insulin and from the 36% sucrose fraction in response to exercise. We did not perform the complete analysis of this translocation as we show in Fig. 2for GLUT4.

Immunoadsorption and Silver Staining of GLUT4 Vesicles

In attempts to determine possible biochemical differences that might underlie the differential response of the two vesicle populations, we used immobilized anti-GLUT4 antibody to immunoabsorb transporter-containing vesicles from the 32 and 36% sucrose fractions, and we analyzed their protein composition by silver staining (Fig. 4) and by Western blot (Fig. 5). As shown in Fig. 4, we observed no obvious differences in the composition of the major proteins in vesicles derived from the two fractions, and GLUT4 is clearly visible as a diffuse band. The background (IgG) staining for the 36% sucrose fraction is always higher than that for the 32% fraction, presumably due to its higher overall protein content (Fig. 1B). It is not entirely obvious why the protein composition of the immunoadsorbed vesicles is so similar in the exercise- and insulin-mobilizable pools (see ``Discussion''). In order to obtain clean silver staining, we must thoroughly wash vesicles bound to antibody immobilized on acrylamide beads, and it is possible that this procedure removes a peripheral membrane protein or proteins that regulate insulin versus exercise responsiveness. Alternatively, because it is difficult to obtain GLUT4 vesicles in large quantities and silver staining is not uniform for every protein, we may simply have a detection problem. In fact, the vesicles from the 32% pool differ in their sedimentation properties from those in the 36% fraction (Fig. 6).


Figure 4: Silver staining of vesicles from 32 and 36% sucrose fractions. Membrane protein (0.5 mg) from the fractions of interest were adsorbed with 100 µl of 1F8 or nonspecific IgG beads as described under ``Experimental Procedures.'' The figure is a silver stained 10% polyacrylamide gel with the proteins identified by Western blot indicated by the arrows.




Figure 6: Fractionation of muscle membranes by sucrose velocity gradient centrifugation. Fractionation of GLUT4-containing vesicles and detection of antigen was performed as described under ``Experimental Procedures.'' A shows the autoradiogram, and B shows the densitometric analysis of the blot (closed symbols) expressed as arbitrary units. The open circles are the protein values from the 32% sucrose fraction, and the open triangles represent protein from the 36% fraction. Ins and Exe are as defined in the legend to Fig. 3. Shown is a representative experiment that was perfomed on three independent occasions.



As noted above, GLUT4-enriched vesicles contain a muscle- and fat-specific aminopeptidase, gp160 (Kandror and Pilch, 1994; Kandror et al., 1994), and they also possess three antigenically related integral membrane proteins of unknown function, called SCAMPs/GTV3 for secretory component-associated membrane protein/glucose transporter vesicle triplet (Brand et al., 1991; Thoidis et al., 1993; Laurie et al., 1993). Fig. 5shows a Western blot of immunoisolated vesicles from the 32 and 36% sucrose fraction using three antibodies to these vesicle constituents. Again, there are no striking differences between the two fractions, although the slowest migrating form of SCAMPs/GTV3 appears somewhat less abundant in the insulin-sensitive vesicles. As is the case in fat cells, GLUT1 (Zorzano et al., 1989; Kandror et al., 1995a) and caveolin (Kandror et al., 1995b) are completely excluded from the GLUT4-containing vesicles in both sucrose fractions (data not shown).

Sucrose Velocity Gradient

To further characterize GLUT4 vesicles from the 32 and 36% sucrose fraction, membranes from the two fractions were analyzed by sucrose velocity gradient centrifugation, and their sedimentation coefficients were compared. Fig. 6A shows the raw Western blotting data, and Fig. 6B shows the quantitative analysis of these data along with the protein distribution (closed symbols). GLUT4 vesicles from these fractions have a narrow distribution, and a comparison of the insulin-responsive and exercise-responsive pools reveal close but nonetheless distinct sedimentation coefficients (Fig. 6B). The overall sedimentation properties of each fraction (open symbols) are distinct with shoulders that correspond to the GLUT4 vesicles. Thus, these two GLUT4 vesicle populations can be distinguished on the basis of their densities and their sedimentation coefficients.


DISCUSSION

Previous work by Douen et al.(1990) in skeletal muscle had demonstrated that both insulin and exercise increased GLUT4 in a fraction enriched in plasma membrane markers, whereas only insulin provoked a statistically significant, concomitant decrease in the internal pool that they measured, a 35% sucrose fraction. We modified their fractionation protocol by eliminating the 35% fraction and replacing it with 32 and 36% sucrose fractions, and we also added a 38% sucrose fraction. These relatively minor additions allowed a sufficient increase in the resolution of membrane separation as compared with the prior study (Douen et al., 1990), and this allowed us to isolate two fractions of 32 and 36% sucrose that were highly enriched in GLUT4 (Fig. 1). The former responded to insulin and the latter to exercise with regard to translocation to the cell surface (Fig. 2). We compared the composition of the insulin-sensitive vesicles to the exercise-sensitive pool by silver staining and by Western blotting with antibodies to known vesicular components ( Fig. 4and Fig. 5), but we saw no obvious differences in overall protein composition by either method. We did observe differential sedimentational behavior, however, in the vesicles from the two pools (Fig. 6).

What then is the molecular basis for the different behavior of the insulin- and exercise-sensitive pools? Previous studies from our lab have compared the composition and sedimentational properties of GLUT4-containing vesicles obtained from unstimulated and insulin-stimulated adipocytes with those from muscle exposed to insulin or not (Kandror et al., 1995a). Somewhat surprisingly and despite different methods of tissue homogenization and vesicle isolation, we found no obvious differences in protein composition determined by silver staining, nor did the vesicles from the two tissues behave differently in sucrose velocity and density gradients (Kandror et al., 1995a). We take this as evidence that GLUT4 vesicles are a unique type of endosomal compartment that is the same in muscle and fat. With regard to changes in sedimentation behavior observed here (Fig. 6) and in the previous paper (Kandror et al., 1995a), there are two possible explanations. First, the protein composition may be the same in vesicles isolated from different fractions under different conditions, but the protein to lipid ratio may vary. The presence or absence of specific phospholipids may be an important parameter in GLUT4 vesicle trafficking, and we are currently examining this possibility. With regard to the apparent constant protein composition in GLUT4 vesicles as determined by silver staining, in order to obtain a clean result with this highly sensitive technique, vesicles adsorbed to beads are thoroughly washed, and this washing is very likely to remove peripheral membrane proteins that may or may not be different in insulin versus exercise responsive GLUT4 vesicles. Moreover, GLUT4 vesicles are not abundant and possibly important differences in minor protein components from differentially responsive vesicles will not be detected until specific antibodies to these are available. Thus, we are also working to identify additional protein components in GLUT4 vesicles by the immunological approach previously used to identify GLUT4 (James et al., 1988) and SCAMPs/GTV3 (Thoidis et al., 1993).

Regardless of the reason, GLUT4 vesicles are able to respond differentially to exercise and insulin, most likely by different mechanisms (Yeh et al., 1995). We suggest that this may be, in part, as a result of the association of GLUT4 vesicles with glycogen particles in a saturable manner. That is, this association is governed by the amount of glycogen in the muscle, and a variety of studies provide indirect evidence for this hypothesis. For example, exercise depletes glycogen concomitant with GLUT4 translocation (Fig. 2). Shortly after exercise, rats are more sensitive with respect to insulin-stimulated glucose transport (Zorzano et al., 1986), and we suggest that this is due to the larger available pool of free GLUT4 vesicles resulting from glycogen depletion. Additionally, just after denervation of muscle and before changes in GLUT4 gene expression, glycogen is depleted, and glucose transport is enhanced (Coderre et al., 1992). Finally, transgenic mice overexpressing GLUT1 in muscle have dramatically increased glycogen levels and are insulin-resistant (Gulve et al., 1994). It should be noted that in our current protocol, the homogenization and fractionation conditions (Fig. 1) remove all measurable glycogen prior to this analysis. Therefore, we are in the process of trying other methodology to experimentally verify this possible association of GLUT4 vesicles with glycogen particles.


FOOTNOTES

*
This work was supported by a grant from the United States Public Health Service (DK-44269) and by the Canadian Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors contributed equally.

To whom correspondence should be addressed: Dept. of Biochemistry, 80 East Concord St., Boston University School of Medicine, Boston, MA 02118.

(^1)
The abbreviation used is: SCAMPs/GTV3, secretory component-associated membrane protein/glucose transporter vesicle triplet.


ACKNOWLEDGEMENTS

We thank Neil Ruderman for many helpful discussions and for careful reading of this manuscript and Dr. Kevin Campbell for the gift of anti-dihydropyridine receptor antibody.


REFERENCES

  1. Brand, S. H., Laurie, S. M., Mixon, M. B., and Castle, J. D. (1991) J. Biol. Chem. 266, 18949-18957 [Abstract/Free Full Text]
  2. Brozinick, J. T., Jr., Etgen, G. J., Jr., Yaspelkis, B. B., III, and Ivy, J. L. (1992) J. Appl. Physiol. 73, 382-387 [Abstract/Free Full Text]
  3. Brozinick, J. T., Jr., Etgen, G. J., Jr., Yaspelkis, B. B., III and Ivy, J. L. (1994) Am. J. Physiol. 267, R236-R243
  4. Cartee, G. D., and Holloszy, J. O. (1990) Am. J. Physiol. 258, E390-E-393
  5. Cartee, G. D., Douen, A. G., Ramlal, T., Klip, A., and Holloszy, J. O. (1991) J. Appl. Physiol. 70, 1593-1600 [Abstract/Free Full Text]
  6. Coderre, L., Monfar, M. M., Chen, K. S., Heydrick, S. J., Kurowski, T. G., Ruderman, N. B., and Pilch, P. F. (1992) Endocrinology 131, 1821-1825 [Abstract]
  7. Constable, S. H., Favier, R. J., Cartee, G. D., Young, D. A., and Holloszy, J. O. (1988) J. Appl. Physiol. 64, 2329-2332 [Abstract/Free Full Text]
  8. Dolan, P. L., Tapscott, E. B., Dorton, P. J., and Dohm, G. L. (1993) Biochem. J. 289, 423-426 [Medline] [Order article via Infotrieve]
  9. Douen, A. G., Ramlal, T., Klip, A., Young, D. A., Cartee, G. D., and Holloszy, J. O. (1989) Endocrinology 124, 449-454 [Abstract]
  10. Douen, A. G., Ramlal, T., Rastogi, S., Bilan, P. J., Cartee, G. D., Vranic, M., Holloszy, J. O., and Klip, A. (1990) J. Biol. Chem. 265, 13427-13430 [Abstract/Free Full Text]
  11. Fushiki, T., Wells, J. A., Tapscott, E. B., and Dohm, G. L. (1989) Am. J. Physiol. 256, E580-E587
  12. Gao, J., Ren, J., Gulve, E. A., and Holloszy, J. O. (1994) J. Appl. Physiol. 77, 1597-1601 [Abstract/Free Full Text]
  13. Goodyear, L. J., Hirshman, M. F., and Horton, E. S. (1991) Am. J. Physiol. 261, E795-E799
  14. Grimditch, G. K., Barnard, R. J., Kaplan, S. A., and Sternlicht, E. (1985) Am. J. Physiol. 249, E398-E408
  15. Gulve, E. A., Ren, J.-M., Marshall, B. A., Gao, J., Hansen, P. A., Holloszy, J. O., and Mueckler, M. (1994) J. Biol. Chem. 269, 18366-18370 [Abstract/Free Full Text]
  16. Handberg, A., Kayser, L., Hoyer, P. E., and Vinten, J. (1992) Am. J. Physiol. 262, E721-E727
  17. Hirshman, M. F., Goodyear, L. J., Wardzala, L. J., Horton, E. D., and Horton, E. S. (1990) J. Biol. Chem. 265, 987-991 [Abstract/Free Full Text]
  18. James, D. E., Brown, R., Navarro, J., and Pilch, P. F. (1988) Nature 333, 183-185 [CrossRef][Medline] [Order article via Infotrieve]
  19. Kandror, K. V., and Pilch, P. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8017-8021 [Abstract]
  20. Kandror, K. V., Yu, L., and Pilch, P. F. (1994) J. Biol. Chem. 269, 30777-30780 [Abstract/Free Full Text]
  21. Kandror, K. V., Coderre, L., Pushkin, A. V., and Pilch, P. F. (1995a) Biochem. J. 307, 383-390 [Medline] [Order article via Infotrieve]
  22. Kandror, K. V., Stephens, J. M., and Pilch, P. F. (1995b) J. Cell Biol. 129, 999-1006 [Abstract]
  23. King, P. A., Betts, J. J., Horton, E. D., and Horton, E. S. (1993) Am. J. Physiol. 265, R447-R452
  24. Klip, A., Ramlal, T., Young, D. A., and Holloszy, J. O. (1987) FEBS Lett. 224, 224-230 [CrossRef][Medline] [Order article via Infotrieve]
  25. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  26. Laurie, S. M., Cain, C. C., Lienhard, G. E., and Castle, J. D. (1993) J. Biol. Chem. 268, 19110-19117 [Abstract/Free Full Text]
  27. Marette, A., Burdett, E., Douen, A., Vranic, M., and Klip, A. (1992) Diabetes 41, 1562-1569 [Abstract]
  28. Ploug, T., Stallknecht, B. M., Pedersen, O., Kahn, B. B., Ohkuwa, T., Vinten, J., and Galbo, H. (1990) Am. J. Physiol. 259, E778-E786
  29. Ploug, T., Galbo, H., Ohkuwa, T., Tranum-Jensen, J., and Vinten, J. (1992) Am. J. Physiol. 262, E700-E711
  30. Rodnick, K. J., Slot, J. W., Studelska, D. R., Hanpeter, D. E., Robinson, L. J., Geuze, H. J., and James, D. E. (1992) J. Biol. Chem. 267, 6278-6285 [Abstract/Free Full Text]
  31. Thoidis, G., Kotliar, N., and Pilch, P. F. (1993) J. Biol. Chem. 268, 11691-11696 [Abstract/Free Full Text]
  32. Turinsky, J. (1987) Endocrinology 121, 528-535 [Abstract]
  33. Wallberg-Henriksson, H., Constable, S. H., Young, D. A., and Holloszy, J. O. (1988) J. Appl. Physiol. 65, 909-913 [Abstract/Free Full Text]
  34. Yeh, J.-I., Gulve, E. A., Rameh, L., and Birnbaum, M. J. (1995) J. Biol. Chem. 270, 2107-2111 [Abstract/Free Full Text]
  35. Zorzano, A., Balon, T. W., Goodman, M. N., and Ruderman, N. B. (1986) Am. J. Physiol. 251, E21-E26
  36. Zorzano, A., Wilkinson, W., Kotliar, N., Thoidis, G., Wadzinkski, B. E., Ruoho, A. E., and Pilch, P. F. (1989) J. Biol. Chem. 264, 12358-12363 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.