Twenty cytochalasins were tested for binding to and for inhibition of glucose transport in human erythrocyte membrane. In this membrane three cytochalasin B (CB) binding sites have been identified. All but three of the cytochalasins bind at site II. On the other hand, only nine of them, which are structurally closely related, bind at site I and inhibit glucose transport. For site I (and site III) binding and glucose transport inhibitory activities (a) the macrocyclic ring in the cytochalasin molecule must be at least 13-membered, (b) the nature of the aromatic ring at C-10 is not important, (c) the C-20-C-23 region makes a major contribution, and (d) the C-5-C-7 segment has a relatively minor influence. These findings do not support a proposed mechanism which involves 24, C-23, C-20, and C-1 oxygen atoms for interaction of CB with glucose carrier. The structural requirements for site II activity are less stringent. The size and the structure of the macrocyclic ring and the nature of the aromatic residue at C-10 modulate this activity only slightly, if at all. Modifications in the C-5-C-7 region of the molecule, however, result in substantial changes in this activity.

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Insulin recruits GLUT4 from an intracellular location to the plasma membrane in rat adipocytes. The process involves multiple intracellular compartments and multiple protein functions, details of which are largely unknown partly due to our inability to separate individual GLUT4 compartments. Here, by hypotonic lysis, differential centrifugation, and glycerol density gradient sedimentation, we separated intracellular GLUT4 compartments in rat adipocytes into three fractions: plasma membrane-containing fraction T and plasma membrane-free fractions H and L. The GLUT4 contents in fractions T, H, and L were ϳ25, 56, and 18% of total GLUT4, respectively, in basal adipocytes and 55, 42, and 3-4% in insulin-stimulated adipocytes. The plasma membrane GLUT4 contents estimated separately further revealed that intracellular GLUT4 in fraction T amounts to ϳ20% in both basal and insulin-stimulated adipocytes. Organelle-specific marker and membrane traffic-related protein distribution data suggested that intracellular GLUT4 in fraction T represents sorting endosomes, whereas GLUT4 in fractions H and L represents storage endosomes and exocytic vesicles, respectively. The subcellular fractionation without homogenization described here should be useful in identifying the role of the individual GLUT4 compartments and the associated proteins in insulin-induced GLUT4 recruitment in rat adipocytes.The uptake of glucose by muscle and adipose cells is a tightly insulin-regulated process, mediated primarily by the GLUT4 facilitative glucose transporter isoform (recently reviewed in Refs. 1-3). GLUT4 in these cells is mostly (Ͼ90%) sequestered in an intracellular pool, and insulin stimulates glucose uptake by recruiting GLUT4 from this intracellular pool to the plasma membrane (2, 4, 5). It is also known that GLUT4 constantly recycles between the plasma membrane and the intracellular pool by endocytosis and exocytosis and that insulin causes GLUT4 recruitment through modulation of these processes (6 -8). Immunoelectron microscopic studies have revealed that GLUT4 in adipocytes is associated with several morphologically distinct membrane structures, including tubulovesicular elements, small vesicles, clathrin-coated vesicles, and plasma membrane invaginations (9, 10). These data support the notion that the intracellular GLUT4 pool of adipocytes is composed of several distinct compartments or organelles. We do not yet know, however, exactly what roles these various GLUT4 compartments play in GLUT4 recycling and sequestration and how they are regulated by insulin.The intracellular GLUT4 pool in rat adipocytes has been isolated after cell homogenization by immunoadsorption of microsomal fractions with anti-GLUT4 antibodies. The GLUT4 pool thus isolated was shown to be a homogeneous population of small vesicles of uniform size, 50 -70 nm in diameter (11,12). The GLUT4-containing vesicles show significant biochemical differences from other intracellular membrane compartments such as synaptic vesicles and secretory vesicles (13). Biochemical an...

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