Insulin-induced GLUT4 Translocation Research Articles

Molecular analysis of insulin resistance in isolated ventricular cardiomyocytes of obese Zucker rats.

Isolated ventricular cardiomyocytes obtained from lean and genetically (fa/fa) obese Zucker rats were used to correlate alterations of insulin-induced glucose transport activation and GLUT-4 translocation to possible defects of the insulin signaling cascade. Maximal stimulation with insulin was found to produce an unaltered translocation of GLUT-4 to the plasma membrane (4.2- and 3.7-fold increase for lean and obese rats, respectively). However, a largely reduced sensitivity of 3-O-methylglucose transport could be detected in obese rats at physiological doses of insulin (completely unresponsive at 8 x 10(-11) M compared with 3-fold stimulation of glucose transport in lean controls). Tyrosine phosphorylation of the insulin receptor beta-subunit and the insulin receptor substrate 1 (IRS-1) was stimulated identically in cardiomyocytes from both lean and obese rats. Labeling of cells with [33P]orthophosphate revealed a marked increase in the serine and/or threonine phosphorylation of IRS-1 in the obese group (370% of lean controls), with a concomitant reduction in IRS-1 abundance (30-40%). The reduced sensitivity of glucose transport at 8 x 10(-11) M insulin was then found to correlate to a completely blunted response of IRS-1-associated phosphatidylinositol 3-kinase activity in cardiomyocytes from obese rats. Those data show that cardiac insulin resistance of obesity involves defective insulin signaling at low concentrations of the hormone, whereas GLUT-4 translocation is fully operative in the isolated cell. It is suggested that hyperphosphorylation of IRS-1 may significantly contribute to the pathogenesis of insulin resistance in the heart.

Proteolytic cleavage of cellubrevin and vesicle-associated membrane protein (VAMP) by tetanus toxin does not impair insulin-stimulated glucose transport or GLUT4 translocation in rat adipocytes.

Acute insulin stimulation of glucose transport in fat and skeletal muscle occurs principally as a result of the hormonal induced translocation of the GLUT4 glucose transporter from intracellular vesicular stores to the plasma membrane. The precise mechanisms governing the fusion of GLUT4 vesicles with the plasma membrane are very poorly understood at present but may share some similarities with synaptic vesicle fusion, as vesicle-associated membrane protein (VAMP) and cellubrevin, two proteins implicated in the process of membrane fusion, are resident in GLUT4-containing vesicles isolated from rat and murine 3T3-L1 adipocytes respectively. In this study we show that proteolysis of both cellubrevin and VAMP, induced by electroporation of isolated rat adipocytes with tetanus toxin, does not impair insulin-stimulated glucose transport or GLUT4 translocation. The hormone was found to stimulate glucose uptake by approx. 16-fold in freshly isolated rat adipocytes. After a single electroporating pulse, the ability of insulin to activate glucose uptake was lowered, but the observed stimulation was nevertheless nearly 5-fold higher than the basal rate of glucose uptake. Electroporation of adipocytes with 600 nM tetanus toxin resulted in a complete loss of both cellubrevin and VAMP expression within 60 min. However, toxin-mediated proteolysis of both these proteins had no effect on the ability of insulin to stimulate glucose transport which was elevated approx. 5-fold, an activation of comparable magnitude to that observed in cells electroporated without tetanus toxin. The lack of any significant change in insulin-stimulated glucose transport was consistent with the finding that toxin-mediated proteolysis of both cellubrevin and VAMP had no detectable effect on insulin-induced translocation of GLUT4 in adipocytes. Our findings indicate that, although cellubrevin and VAMP are resident proteins in adipocyte GLUT4-containing vesicles, they are not required for the acute insulin-induced delivery of GLUT4 to the plasma membrane.

GTPase activating protein activity for Rab4 is enriched in the plasma membrane of 3T3-L1 adipocytes. Possible involvement in the regulation of Rab4 subcellular localization.

The small guanosine 5'-triphosphate (GTP)ase Rab4 has been suggested to play a role in insulin-induced GLUT4 translocation. Under insulin stimulation, GLUT4 translocates to the plasma membranes, while Rab4 leaves the GLUT4-containing vesicles and becomes cytosolic. Rab proteins cycle between a GTP-bound active form and a guanosine 5'-diphosphate (GDP)-bound inactive form. The intrinsic GTPase activity of Rab proteins is low and the interconversion between the two forms is dependent on accessory factors. In the present work, we searched for a GTPase activating protein (GAP) for Rab4 in 3T3-L1 adipocytes. We used a glutathione-S-transferase (GST)-Rab4 protein which possesses the properties of a small GTPase (ability to bind GDP and GTP and to hydrolyse GTP) and can be isolated in a rapid and efficient way. This GAP activity was observed in 3T3-L1 adipocyte lysates, and was able to accelerate the hydrolysis of the [alpha-32P]GTP bound to GST-Rab4 into [alpha-32P]GDP. This activity, tentatively called Rab4-GAP, was also present in 3T3-L1 fibroblasts. The Rab4-GAP activity was present in total membrane fractions and nearly undetectable in cytosol. Following subcellular fractionation, Rab4-GAP was found to be enriched in plasma membranes when compared to internal microsomes. Insulin treatment of the cells had no effect on the total Rab4-GAP activity or on its subcellular localization. Taking our results together with the accepted model of Rab cycling in intracellular traffic, we propose that Rab4-GAP activity plays a role in the cycling between the GTP- and GDP-bound forms of Rab4, and thus possibly in the traffic of GLUT4-containing vesicles.

Mechanisms Behind Insulin Resistance in Rat Skeletal Muscle After Oophorectomy and Additional Testosterone Treatment

The absence of female sex hormones, as well as testosterone treatment of oophorectomized (OVX) female rats has been demonstrated to result in decreased whole-body insulin-mediated glucose uptake. The cellular mechanism behind this insulin resistance and the role of low levels of female sex hormones as a risk factor for development of peripheral insulin resistance are not yet fully clarified. We assessed the protein expression of GLUT4 and glycogen synthase, as well as insulin-induced translocation of GLUT4 to the plasma membrane, in soleus skeletal muscle from control rats, OVX rats, and OVX rats treated for 8 weeks with testosterone (OVX + T). Whole-body insulin-mediated glucose uptake assessed by the hyperinsulinemic-euglycemic clamp procedure was 25% lower in OVX rats (P < 0.001) and addition of testosterone treatment further decreased insulin-mediated glucose uptake in OVX + T rats by 48% (P < 0.001) compared with controls. GLUT4 protein expression in soleus muscles was unaltered in the OVX and OVX + T rats compared with controls. Insulin induced a 3.7-fold increase (P < 0.05) in the plasma membrane content of GLUT4 in soleus muscle from control rats, whereas plasma membrane content of GLUT4 in soleus muscle from OVX or OVX + T rats was unaltered in response to insulin. Glycogen synthase protein expression in muscle homogenates was decreased by 25% in the OVX group (P < 0.05) and by 37% in the OVX + T group (P < 0.05) when compared with the control group. Insulin receptor and tyrosine kinase activities in the basal and insulin-stimulated states did not differ between the OVX and OVX + T rats. In conclusion, the absence of female sex hormones appears to decrease insulin-mediated whole-body glucose uptake via an impaired insulin-stimulated translocation of GLUT4 to the plasma membrane and by decreased protein expression of glycogen synthase. Testosterone treatment further impairs whole-body insulin-mediated glucose uptake, presumably by additional impairment of glycogen synthase expression.

A Synthetic Peptide Corresponding to the Rab4 Hypervariable Carboxyl-terminal Domain Inhibits Insulin Action on Glucose Transport in Rat Adipocytes

The present study was conducted to examine the involvement of Rab4, a low molecular weight GTP-binding protein, in the action of insulin on glucose transport. A synthetic peptide corresponding to the Rab4 hypervariable carboxyl-terminal domain, Rab4-(191-210), was successfully transferred into rat adipocytes by electroporation and inhibited insulin-stimulated glucose transport by about 50% without affecting the basal transport activity. In contrast, synthetic peptides corresponding to the Rab3C and Rab3D carboxyl-terminal hypervariable domain had little effect on insulin action on glucose transport. The Rab4-(191-210) peptide also reduced insulin-induced GLUT4 translocation from the intracellular pool to the plasma membrane. Furthermore, the Rab4-(191-210) peptide reduced both insulin-induced glucose transport and GLUT4 translocation in the presence of a major histocompatibility complex class I antigen-derived peptide, D(k)-(62-85), which is a potent inhibitor of GLUT4 internalization, suggesting that the peptide inhibited exocytotic recruitment of GLUT4-containing vesicles. The Rab4-(191-210) peptide also inhibited GTP gamma S-stimulated glucose transport. In addition, insulin-stimulated glucose transport was inhibited by the addition of anti-Rab4 antibody. These results suggest that Rab4 protein plays a crucial role in insulin action on GLUT4 translocation, especially in exocytotic recruitment by the hormone of the glucose transporter to the plasma membrane from the intracellular retention pool.

Insulin-induced redistribution of GLUT4 glucose carriers in the muscle fiber. In search of GLUT4 trafficking pathways.

Insulin rapidly stimulates glucose transport in muscle fiber. This process controls the utilization of glucose in skeletal muscle, and it is deficient in various insulin-resistant states, such as non-insulin-dependent diabetes mellitus. The effect of insulin on muscle glucose transport is mainly due to the recruitment of GLUT4 glucose carriers to the cell surface of the muscle fiber. There is increasing evidence that the recruitment of GLUT4 carriers triggered by insulin affects selective domains of sarcolemma and transverse tubules. In contrast, GLUT1 is located mainly in sarcolemma and is absent in transverse tubules, and insulin does not alter its cellular distribution in muscle fiber. The differential distribution of GLUT1 and GLUT4 in the cell surface raises new questions regarding the precise endocytic and exocytic pathways that are functional in the muscle fiber. The current view of insulin-induced GLUT4 translocation is based mainly on studies performed in adipocytes. These studies have proposed the existence of intracellular compartments of GLUT4 that respond to insulin in a highly homogeneous manner. However, studies performed in skeletal muscle have identified insulin-sensitive as well as insulin-insensitive intracellular GLUT4-containing membranes. These data open a new perspective on the dynamics of intracellular GLUT4 compartments in insulin-sensitive cells.

Insulin and insulin-like growth factor I (IGF-I) stimulate GLUT4 glucose transporter translocation in Xenopus oocytes.

1. The heterologous expression of glucose transporters GLUT4 and GLUT1 in Xenopus oocytes has been shown to cause a differential targeting of these glucose-carrier isoforms to cellular membranes and a distinct induction of glucose transport activity. In this study we have evaluated the effect of insulin and insulin-like growth factor I (IGF-I) on glucose uptake and glucose transporter distribution in Xenopus oocytes expressing mammalian GLUT4 and GLUT1 glucose carriers. 2. Insulin and IGF-I stimulated 2-deoxyglucose uptake in GLUT4-expressing oocytes, but not in GLUT1-expressing oocytes or in water-injected oocytes. The stimulatory effect of insulin and IGF-I on 2-deoxyglucose uptake in GLUT4-expressing oocytes occurred via activation of the IGF-I receptor. 3. Subcellular-fractionation studies indicated that insulin and IGF-I stimulated translocation of GLUT4 to the cell surface of the oocyte. 4. Incubation of intact oocytes with insulin stimulated phosphatidylinositol 3-kinase activity, an effect that was blocked by the additional presence of wortmannin. Furthermore, wortmannin totally abolished the insulin-induced stimulation of 2-deoxyglucose uptake in GLUT4-expressing oocytes. 5. In this study, both the insulin-induced GLUT4 carrier translocation and GLUT4-dependent insulin-stimulated glucose transport have been reconstituted in the Xenopus oocyte. These observations, together with the fact that wortmannin, as found in adipocytes, inhibits insulin-stimulated glucose transport in oocytes, suggest that the heterologous expression of GLUT4 in oocytes is a useful experimental model by which to study the cell biology of insulin-induced GLUT4 translocation.

Different Signaling Roles of SHPTP2 in Insulin-induced GLUT1 Expression and GLUT4 Translocation

Insulin activates hexose transport via at least two mechanisms: a p21ras-dependent pathway, leading to an increase in the amount of cell surface GLUT1; and a metabolic, p21ras-independent pathway, leading to translocation of the insulin-responsive transporter GLUT4 to the cell surface. Following insulin stimulation, SHPTP2, a non-transmembrane protein-tyrosine phosphatase, associates with insulin receptor substrate 1 via its Src homology 2 (SH2) domains. Microinjection of a glutathione S-transferase fusion protein encoding the N- and C-terminal SH2 domains of SHPTP2 (GST-NC-SH2) or anti-SHPTP2 antibodies into NIH-3T3 fibroblasts overexpressing the insulin receptor blocks insulin-induced DNA synthesis. Microinjection of either GST-NC-SH2 or anti-SHPTP2 antibodies into 3T3-L1 adipocytes inhibited the insulin-stimulated increase in expression of GLUT1. In contrast, translocation of GLUT4 to the cell surface was unaffected by either GST-NC-SH2 or anti-SHPTP2 antibodies. These data confirm a role for SHPTP2 in insulin-stimulated mitogenesis and indicate that whereas SHPTP2 is necessary for insulin-stimulated expression of GLUT1, it is not required for activation of the metabolic pathway leading to GLUT4 translocation.

Requirement for Phosphoinositide 3-Kinase in Insulin-Stimulated GLUT4 Translocation in 3T3-L1 Adipocytes

Insulin stimulates glucose transport in muscle and fat cells by inducing the redistribution of a specific glucose transporter, GLUT4 from intracellular vesicles to the cell surface. Phosphoinositide (PI) 3-kinase has been implicated as a key intermediate in insulin-stimulated glucose transport by studies that have examined the effects of wortmannin and LY294002, which are thought to be specific inhibitors of this enzyme. However, the specificity of these compounds for PI 3-kinase has recently been questioned. Epidermal growth factor, which activates mitogen-activated protein kinase in mouse 3T3-L1 adipocytes, has now been shown to have no effect on PI 3-kinase activity or GLUT4 translocation in these cells. Furthermore, microinjection of a dominant negative mutant of the 85-kDa subunit of PI 3-kinase, which lacks a binding site for the catalytic 110-kDa subunit, inhibited GLUT4 translocation induced by insulin in 3T3-L1 adipocytes; microinjection of the wild-type protein had no effect. These observations indicate that PI 3-kinase is necessary for insulin-induced GLUT4 translocation and glucose transport in adipocytes.

Insulin-induced translocation of the glucose transporter GLUT4 in cardiac muscle: studies on the role of small-molecular-mass GTP-binding proteins.

Subcellular fractions obtained from rat cardiac ventricular tissue were used to elucidate a possible functional relationship between small-molecular-mass G-proteins and the insulin-responsive glucose transporter GLUT4. Proteins were separated by SDS/PAGE and transferred to nitrocellulose membranes. Incubation with [alpha-32P]GTP revealed the presence of two major distinct GTP-binding protein bands of 24 and 26 kDa in both plasma and microsomal membranes. Immunoadsorption of microsomal membranes to anti-GLUT4 antibodies was used to isolate GLUT4-enriched membrane vesicles. This material was found to contain a much decreased amount of small G-proteins, with the exclusive presence of the 24 kDa species. Insulin treatment in vivo had no effect on the microsomal membrane content of small GTP-binding proteins, but significantly decreased the 24 kDa species in GLUT4-enriched vesicles by 36 +/- 5% (n = 3). This correlated with a decreased (30-40%) recovery of GLUT4-enriched vesicles from insulin-treated animals. Western-blot analysis of microsomal membranes with a panel of antisera against rab GTP-binding proteins indicated the presence of rab4A, with a molecular mass of 24 kDa, whereas rab1A, rab2 and rab6 were not observed. rab4A was barely detectable in GLUT4-enriched vesicles; however, insulin produced an extensive shift of rab4A from the cytosol and the microsomal fraction to the plasma membrane with a parallel increase in GLUT4. These data show that a small GTP-binding protein is co-localized with GLUT4 in an insulin-responsive intracellular compartment, and strongly suggest that this protein is involved in the exocytosis of GLUT4 in cardiac muscle. Furthermore, the observed translocation of rab4A is compatible with insulin-induced endosome recycling processes, possibly including the glucose transporters.

Growth factor-induced stimulation of hexose transport in 3T3-L1 adipocytes: Evidence that insulin-induced translocation of glut4 is independent of activation of MAP kinase

We have examined the effect of growth factors on the rate of hexose transport in 3T3-L1 adipocytes. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) were found to stimulate deoxyglucose transport by about 2-fold. The concentrations of EGF and PDGF which elicited half maximal responses were 100 and 350 pM, respectively. The increases in transport rate were acute effects; the stimulations were evident whithin minutes of exposure to growth factors. By contrast, insulin stimulated deoxyglucose transport ∼16-fold over similar time periods. We have measured the appearance of both the insulin-responsive glucose transporter (GLUT4) and the erytghrocyte-type glucose transporter (GLUT1) at the cell surface in response to insulin, EGF and PDGF. We show that both EGF and PDGF inducea 2-fold increase in GLUT1 at the cell surface, but both these growth factors were without effect on GLUT4 levels at the cell surface. In contrast, insulin induced a 13-fold increase in cell surface GLUT4. We further show that insulin, EGF and PDGF all activate MAP kinase as determine by a shift in electrophoretic mobility of this protein on SDS-PAGE. However, since the large translocation of GLUT4 to the cell surface is specific for insulin, we suggest that activation of MAP kinase is not the sole requisite for this process.

Chloroquine inhibits glucose-transporter recruitment induced by insulin in rat adipocytes independently of its action on endomembrane pH

In adipocytes, stimulation of glucose transport by insulin is mediated largely by translocation of the GLUT4 isoform of glucose transporters from an intracellular store to the plasma membrane. Most endomembrane compartments are endowed with H(+)-pumping ATPases, and the resulting luminal acidification is thought to play a role in vesicular traffic. Chloroquine (Clq), a permeant weak base, was used to test whether endomembrane pH is an important factor in GLUT4 translocation. Under conditions chosen to optimize Clq uptake, the weak base precluded insulin-induced GLUT4 translocation and the associated stimulation of glucose transport. Clq also effectively dissipated the delta pH of acidic endomembrane compartments, assessed fluorimetrically. To define whether the intracellular GLUT4 storage compartment is acidic, immunoadsorption and immunoblotting experiments were performed to determine whether glucose transporters and vacuolar-type H+ pumps coexist in the same membranes. Unexpectedly, H+ pumps were not detectable in vesicles bearing GLUT4. Moreover, dissipation of endomembrane delta pH by monensin failed to inhibit insulin-stimulated GLUT4 translocation and hexose transport. Finally, the inhibitory effect of Clq persisted in the presence of monensin. We conclude that GLUT4 resides in an intracellular compartment devoid of H+ pumps. The insertion of this compartment into the plasmalemma is not regulated by transmembrane pH gradients. Clq impairs the stimulation of glucose transport by blocking translocation of GLUT4 by a pH-independent mechanism. Clq may provide a useful tool to elucidate the signalling or fusion steps involved in insulin-induced GLUT4 translocation.

Direct demonstration of insulin-induced GLUT4 translocation to the surface of intact cells by insertion of a c-myc epitope into an exofacial GLUT4 domain

Stimulation of glucose transport is the main physiological effect of insulin in target tissues. This effect is linked to translocation of the GLUT4 glucose transporter from an intracellular pool to the cell surface. To elucidate the molecular mechanisms involved in this effect, we developed a simple direct sensitive method to detect GLUT4 immunologically on the cell surface. cDNA containing GLUT4 inserted by a c-myc epitope in the first ectodomain (GLUT4myc) was constructed without disrupting the functions of GLUT4 and was expressed in 3T3-L1 and Chinese hamster ovary fibroblast cells. In response to insulin, the GLUT4myc expressed in 3T3-L1 adipocytes was translocated to the cell surface from the intracellular pool, as shown by assays of exofacial antibody binding against the myc epitope and of the uptake of 2-deoxyglucose. Insulin, guanosine 5'-O-(3-thiotriphosphate), guanylyl imidodiphosphate, NaF, and phorbol 12-myristate 13-acetate also induced the translocation of GLUT4myc in Chinese hamster ovary cells coexpressing the human insulin receptor.

Effects of ML-9 on insulin stimulation of glucose transport in 3T3-L1 adipocytes.

Treatment of 3T3-L1 adipocytes with insulin resulted in activation of 2-deoxyglucose transport activity and translocation of glucose transporters (GLUT4 and GLUT1) from the cytoplasmic space to the plasma membrane. ML-9 (a myosin light chain kinase inhibitor) inhibited insulin stimulation of 2-deoxyglucose transport activity by 80% at 100 microM (IC50 = 27 microM) without affecting 2-deoxyglucose transport activity in the basal state. The inhibition was independent of extracellular Ca2+ concentration and almost fully reversible at 40 microM ML-9. ML-9 did not inhibit insulin-stimulated tyrosine phosphorylation of 95-kDa protein in the wheat germ agglutinin-purified preparation and of 95- and 160-kDa proteins in intact cells. However, ML-9 inhibited insulin-induced translocation of both GLUT4 and GLUT1 in a dose-dependent manner. The dose-response curves were similar to those observed for the inhibition of insulin stimulation of 2-deoxyglucose transport activity. Neither insulin nor ML-9 affected the phosphorylation state of both heavy and light chains of myosin. Therefore, it seems likely that ML-9 inhibits the insulin-induced translocation of glucose transporters at a step beyond the insulin receptor kinase activity by a mechanism different from that affecting phosphorylation of the myosin light chain. Phosphorylating activity of microtubule-associated protein 2 and myelin basic protein was stimulated by insulin, and this stimulation was not affected by ML-9. ML-9, however, inhibited the phosphorylating activity in vitro and insulin stimulation of the phosphorylating activity of ribosomal protein S6 in intact cells in a dose-dependent manner similar to that observed for the inhibition of insulin stimulation of glucose transport. These results suggest that mitogen-activated protein kinase may be one of the constituents in intracellular insulin signaling to the glucose transport system.

Phosphorylation state of the GLUT4 isoform of the glucose transporter in subfractions of the rat adipose cell: effects of insulin, adenosine, and isoproterenol.

The acute effects of insulin, adenosine, and isoproterenol on the activity, subcellular distribution, and phosphorylation state of the GLUT4 glucose transporter isoform were investigated in rat adipocytes under conditions carefully controlled to monitor changes in cAMP-dependent protein kinase (A-kinase) activity. In contrast to GLUT1, which has not been shown to be phosphorylated even when cells are exposed to any of the above agents, GLUT4 was partially phosphorylated (0.1-0.2 mol/mol) when the activity of the A-kinase was suppressed, and remained unchanged in response to insulin. Isoproterenol elicited a 64% inhibition of insulin-stimulated glucose transport activity in the absence, but not the presence, of adenosine receptor agonists. However, in either the presence or the absence of agonists, A-kinase was activated as assessed by examining the phosphorylation of the major adipocyte A-kinase substrate, perilipin. Similarly, under either condition, phosphorylation of GLUT4 was enhanced 1.4-fold in the intracellular membranes, but no significant change was observed in the plasma membrane. In the absence of adenosine receptor agonists, isoproterenol exerted a small (14%) but significant inhibition of the insulin-induced translocation of GLUT4 but had no effect on the translocation of GLUT1. Thus, changes in the phosphorylation state and/or subcellular distribution of GLUT4 cannot account for the inhibition of insulin-stimulated glucose activity induced by isoproterenol.

Immunoelectron microscopic demonstration of insulin-stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxyl-terminal epitope of intracellular GLUT4.

Polyclonal antibodies to the amino- or carboxyl-terminated peptide sequences of the GLUT4 transporter protein were used in immunoelectron microscopic studies to demonstrate the location and insulin-induced translocation of GLUT4 in intact isolated rat adipocytes. Labeling of untreated adipocytes with the amino-terminal antibody revealed 95% of GLUT4 was intracellular, associated with plasma membrane invaginations or vesicles contiguous with or within 75 nm of the cell membrane. Insulin treatment increased plasma membrane labeling approximately 13-fold, to 52% of the total transporters, and decreased intracellular labeling proportionately. In contrast, labeling of untreated adipocytes with the carboxyl-terminal antibody or with a monoclonal antibody (1F8) that binds to the carboxyl terminus of GLUT4 detected fewer transporters, only approximately 40% of which were intracellular. In insulin-treated cells, plasma membrane labeling increased approximately 20-fold, but the total number of labeled transporters also increased approximately 13-fold. The number of intracellular transporters was not changed. The insulin-induced increase in plasma membrane labeling was reversible. Thus, the vast majority of GLUT4 transporters in untreated adipocytes are intracellular in invaginations or vesicles attached or close to the plasma membrane. Insulin treatment causes translocation of transporters to the plasma membrane, which involves flow of transporters from invaginations to the cell surface and possible fusion of subplasma membrane vesicles with the plasma membrane. Differences in the labeling of intracellular transporters by peptide antibodies suggested the carboxyl-terminal epitope of intracellular transporters was masked. The unmasking of the carboxyl terminus during translocation to the plasma membrane may be part of the mechanism by which insulin stimulates glucose transport in rat adipocytes.