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