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.

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