Glucose is transported by a family of related integral membrane proteins 1 . Unlike other members of this family, the insulin-responsive glucose trans- porter isoform GLUT4 is predomi- nantly, but not exclusively, found in adipose tissue and skeletal and cardiac muscle. In these tissues, insulin in- creases glucose uptake by regulating the intracellular trafficking of the GLUT4 protein in three key steps: (1) segregation of GLUT4 into specialized vesicles or compartments; (2) traffick- ing of these vesicles to the plasma mem- brane; and (3) subsequent fusion to expose GLUT4 at the cell surface (reviewed in Refs 2,3). In the basal state, GLUT4 cycles slowly between the plasma membrane and multiple intracellular compart- ments, with the vast majority of the transporter residing within the cell interior. Activation of the insulin recep- tor stimulates the rate of GLUT4 vesicle exocytosis, concomitant with a smaller decrease in the rate of GLUT4 endo- cytosis, resulting in increased glucose uptake 4,5 . The insulin-mediated increase in exocytosis is predominant in GLUT4 translocation, as a complete inhibition of GLUT4 endocytosis only results in a partial increase in plasma membrane- associated GLUT4 protein, without affecting the extent of insulin-stimu- lated GLUT4 translocation 6 . However, the precise molecular signaling events and protein components responsible for GLUT4 vesicle cycling, and the regulation of docking and fusion with the plasma membrane, are largely undefined. After biogenesis, the GLUT4 vesicles are sequestered at specific sites in the cell. This intracellular holding pattern in resting cells appears to depend crucially on the C-terminal sequences of GLUT4. This finding is based on studies using GLUT1–GLUT4 chimeras 7 , as well as experiments in which the microinjec- tion of C-terminal peptides induced the translocation of GLUT4 to the cell sur- face 8 . Although this C-terminal tail is likely to mediate the tethering of GLUT4 vesicles, the intracellular bind- ing partner and the mechanism of insulin-stimulated vesicle release have not been elucidated. One recent finding indicates that this domain of GLUT4 can interact directly with the glycolytic enzyme aldolase, which can also bind with actin 9 . Thus, aldolase can mediate the interaction of GLUT4 with the cytoskeleton, long known to be crucial for insulin-stimulated glucose transport 10 . The disruption of the aldolase–GLUT4 interaction by aldolase substrates results in the dissociation of the ternary complex, thus inhibiting insulin-stimu- lated glucose transport and providing a potential negative feedback signal for glucose transport. A primary role for phosphatidyl inosi- tol 3′-kinase (PI3′-K) stimulation in GLUT4 translocation has been suggested by experiments in which inhibition of PI3′-K was shown to ablate insulin- stimulated glucose uptake completely (reviewed in Ref. 2). In addition, overex- pression of the genes encoding PI3′-K or its downstream protein kinase target Akt increased glucose uptake independently of insulin 11,12 . Because other growth fac- tors stimulate this pathway but do not increase glucose transport, insulin might differentially activate specific intracellu- lar pools of PI3′-K (Refs 13,14). Studies have also indicated that the PI3′-K- dependent activation of protein kinase C ζ (PKC-ζ) might be a crucial signal trans- duction event necessary for GLUT4 translocation 15 . More recently, a cell-per- meable version of phosphatidyl inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P 3 ], a lipid signaling product of PI3′-K, was shown to stimulate glucose uptake in 3T3-L1 adipocytes treated with insulin and the PI3′-K inhibitor wortmannin. The PtdIns(3,4,5)P 3 derivative had no effect alone, confirming that although PI3′-K activation is required, an addi- tional, undefined signaling pathway is also involved 16 . • GLUT4-containing Vesicles Traffic to the Plasma Membrane in Response to Insulin There is considerable uncertainty about the precise intracellular localiz- ation and trafficking pathways of the GLUT4-containing vesicles. Immuno- electron microscopy has demonstrated the predominant localization of GLUT4 protein in tubulo-vesicular elements beneath the plasma membrane, with the remaining protein localized to the trans-Golgi network (TGN), clathrin- coated vesicles and endosome struc- tures. Several studies have demon- strated a separate subpopulation of GLUT4-containing vesicles away from 408 1043-2760/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(99)00201-5 TEM Vol. 10, No. 10, 1999 Matthew J. Brady, Jeffrey E. Pessin and Alan R. Saltiel Despite intense investigation, major gaps remain in our understanding of the cellular mechanisms that underlie the actions of insulin, as well as the regulation of the enzymes and transport proteins crucial to the orderly control of glucose metabolism. In recent years, the compartmentalization of signaling molecules and metabolic enzymes has been suggested to play an important role in ensuring metabolic balance. We will discuss exam- ples of recent findings, suggesting that spatial compartmentalization and protein translocation might be the keys to understanding the specificity of insulin in the regulation of glucose metabolism. Spatial Compartmentalization in the Regulation of Glucose Metabolism by Insulin M.J. Brady and A.R. Saltiel are at the Depart- ment of Cell Biology, Parke-Davis Pharmaceu- tical Research Division/Warner Lambert Co., 2800 Plymouth Rd, Ann Arbor, MI 48105, USA; and J.E. Pessin is at the Department of Physi- ology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.