IDENTIFICATION AND CHARACTERIZATION OF 2 DISTINCT INTRACELLULAR GLUT4POOLS IN RAT SKELETAL-MUSCLE - EVIDENCE FOR AN ENDOSOMAL AND AN INSULIN-SENSITIVE GLUT4 COMPARTMENT

In skeletal muscle, acute insulin treatment results in the recruitment of the GLUT4 glucose transporter from intracellular vesicular structu res to the plasma membrane. The precise nature of these intracellular GLUT4 stores has, however, remained poorly defined. Using an establish ed skeletal-muscle fractionation procedure we present evidence for the existence of two distinct intracellular GLUT4 compartments. We have s hown that after fractionation of crude muscle membranes on a discontin uous sucrose gradient the majority of the GLUT4 immunoreactivity was l argely present in two sucrose fractions (30 and 35%, w/w, sucrose; den oted F30 and F35 respectively) containing intracellular membranes of d ifferent buoyant densities. Here we show that these fractions containe d 44 +/- 6 and 49 +/- 7 % of the crude membrane GLUT4 reactivity respe ctively, and could be further discriminated on the basis of their immu noreactivity against specific subcellular antigen markers. Membranes f rom the F30 fraction were highly enriched in transferrin receptor (TfR ) and annexin II, two markers of the early endosome compartment, where as they were significantly depleted of both GLUT1 and the alpha 1-subu nit of (Na++K+)-ATPase, two cell-surface markers. Insulin treatment re sulted in a significant reduction in GLUT4 content in membranes from t he F35 fraction, whereas the amount of GLUT4 in the less dense (F30) f raction remained unaffected by insulin. Immunoprecipitation of GLUT4-c ontaining vesicles from both intracellular fractions revealed that TfR was present in GLUT4 vesicles isolated from membranes from the F30 fr action. In contrast, GLUT4 vesicles from the F35 fraction were devoid of TfR. The aminopeptidase, vp165, was present in GLUT4 vesicles from both F30 and F35; however, vesicles isolated from F30 contained over t wice as much vp165per unit of GLUT4 than those isolated from F35. The biochemical co-localization of vp165/GLUT4 was further substantiated b y double-immunogold labelling of ultrathin muscle sections. Overall, o ur data indicate the presence of at least two internal GLUT4 pools: on e possibly derived from an endosomal recycling compartment, and the ot her representing a specialized insulin-sensitive GLUT4 storage pool. B oth pools contain vp165.