Syntaxin 6 regulates Glut4 trafficking in 3T3-L1 adipocytes

Insulin stimulates the movement of glucose transporter-4 (Glut4)-containing vesicles to the plasma membrane of adipose cells. We investigated the role of post-Golgi t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the trafficking of Glut4 in 3T3-L1 adipocytes. Greater than 85% of syntaxin 6 was found in Glut4-containing vesicles, and this t-SNARE exhibited insulin-stimulated movement to the plasma membrane. In contrast, the colocalization of Glut4 with syntaxin 7, 8, or 12/13 was limited and these molecules did not translocate to the plasma membrane. We used adenovirus to overexpress the cytosolic domain of these syntaxin's and studied their effects on Glut4 traffic. Overexpression of the cytosolic domain of syntaxin 6 did not affect insulin-stimulated glucose transport, but increased basal deGlc transport and cell surface Glut4 levels. Moreover, the syntaxin 6 cytosolic domain significantly reduced the rate of Glut4 reinternalization after insulin withdrawal and perturbed subendosomal Glut4 sorting; the corresponding domains of syntaxins 8 and 12 were without effect. Our data suggest that syntaxin 6 is involved in a membrane-trafficking step that sequesters Glut4 away from traffic destined for the plasma membrane. We speculate that this is at the level of traffic of Glut4 into its unique storage compartment and that syntaxin 16 may be involved.     

Effect of phenylarsine oxide on insulin-dependent protein phosphorylation and glucose transport in 3T3-L1 adipocytes

We have reported previously that phenylarsine oxide (PAO) blocks insulin-stimulated glucose transport in 3T3-L1 adipocytes (Frost, S. C., and Lane, M. D. (1985) J. Biol. Chem. 260, 2646-2652). As shown in the present study, the locus of inhibition is post-receptor. Insulin stimulated the extent of receptor autophosphorylation in solution and in the intact cell by approximately 4-fold. PAO had no effect on this activity. Using reduced and carboxamidomethylated lysozyme as a substrate for the tyrosine-specific receptor, insulin stimulated the rate of receptor kinase-catalyzed substrate phosphorylation by 2-fold; PAO had no effect on this stimulation. However, the insulin-stimulated, serine-specific phosphorylation of two endogenous phosphoproteins (pp24 and pp240) in the intact cell was blocked by 25 microM PAO. These complementary in situ and in vitro studies demonstrate that the inhibition by PAO must be distal to the insulin receptor's protein tyrosine kinase activity.     

Nigericin inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes

We used nigericin, a K+/H+ exchanger, to test whether glucose transport in 3T3-L1 adipocytes was modulated by changes in intracellular pH. Our results showed that nigericin increased basal but decreased insulin-stimulated glucose uptake in a time- and dose-dependent manner. Whereas the basal translocation of GLUT1 was enhanced, insulin-stimulated GLUT4 translocation was inhibited by nigericin. On the other hand, the total amount of neither transporter protein was altered. The finding that insulin-stimulated phosphoinositide 3-kinase (PI 3-kinase) activity was not affected by nigericin implies that nigericin exerted its inhibition at a step downstream of PI 3-kinase activation. At maximal dose, nigericin rapidly lowered cytosolic pH to 6.7; however, this effect was transient and cytosolic pH was back to normal in 20 min. Removal of nigericin from the incubation medium after 20 min abolished its enhancing effect on basal but had little influence on its inhibition of insulin-stimulated glucose transport. Moreover, lowering cytosolic pH to 6.7 with an exogenously added HCl solution had no effect on glucose transport. Taken together, it appears that nigericin may inhibit insulin-stimulated glucose transport mainly by interfering with GLUT4 translocation, probably by a mechanism not related to changes in cytosolic pH.     

Effects of cellular ATP depletion on glucose transport and insulin signaling in 3T3-L1 adipocytes

Glucosamine induced insulin resistance in 3T3-L1 adipocytes, which was associated with a 15% decrease in cellular ATP content. To study the role of ATP depletion in insulin resistance, we employed sodium azide (NaN3) and dinitrophenol (DNP), which affect mitochondrial oxidative phosphorylation, to achieve a similar 15% ATP depletion. Unlike glucosamine, NaN3 and DNP markedly increased basal glucose transport, and the increased basal glucose transport was associated with increased GLUT-1 content in the plasma membrane without changes in total GLUT-1 content. These agents, like glucosamine, did not affect the early insulin signaling that is implicated in insulin stimulation of glucose transport. In cells with a severe 40% ATP depletion, basal glucose transport was similarly elevated, and insulin-stimulated glucose transport was similar in cells with 15% ATP depletion. In these cells, however, early insulin signaling was severely diminished. These data suggest that cellular ATP depletion by glucosamine, NaN3, and DNP exerts differential effects on basal and insulin-stimulated glucose transport and that ATP depletion per se does not induce insulin resistance in 3T3-L1 adipocytes.     

Mechanism of inhibition of insulin-stimulated glucose transport by 4-bromocrotonic acid in 3T3-L1 adipocytes

We have demonstrated previously that 4-bromocrotonic acid (Br-C4) inhibited insulin-stimulated glucose transport by interfering with GLUT4 translocation. In the present study, we further examined the underlying mechanism involved. Since insulin-induced insulin receptor substrate-1-associated phosphatidylinositol (PI) 3-kinase activity was not altered by Br-C4, we determined and found insulin activation of protein kinase B (PKB) and protein kinase Clambda (PKClambda) were both inhibited. However, time-course studies showed that only the inhibition of PKB activation correlated with the inhibition of insulin-stimulated glucose transport. In concert, insulin-stimulated Ser(473/474) phosphorylation on PKB(alpha/beta) were similarly decreased by Br-C4. The finding that okadaic acid-stimulated glucose transport and PKClambda activity were both inhibited by Br-C4 suggested that the effect of Br-C4 on Ser(473/474) phosphorylation was not mediated by protein phosphatase 2A. Moreover, whereas Br-C4 nearly abolished insulin-stimulated integrin-linked kinase (ILK) activity, it only inhibited insulin-stimulated PKB activity by 20%, implying that ILK was not the major kinase for Ser(473/474) phosphorylation. Taken together, these results support the notion that PKB is involved in insulin-stimulated glucose transport. In addition, Br-C4 seems to inhibit insulin-stimulated glucose transport via inhibiting insulin activation of PKB, probably by interfering with insulin activation of an upstream kinase responsible for the phosphorylation of Ser(473/474) residue.     

Syntaxin 16 controls the intracellular sequestration of GLUT4 in 3T3-L1 adipocytes

The regulated delivery of Glut4-containing vesicles to the plasma membrane is a specialised example of regulated membrane trafficking. Present models favour the transporter trafficking through two inter-related endosomal cycles. The first is the proto-typical endosomal system. This is a fast trafficking event that, in the absence of insulin, serves to internalise Glut4 from the plasma membrane. Once in this pathway, Glut4 is further sorted into a slowly recycling pathway that operates between recycling endosomes, the trans Golgi network, and a population of vesicles often referred to as Glut4-storage vesicles. Little is known about the molecules that regulate these distinct sorting steps. Here, we have studied the role of Stx16 in Glut4 trafficking. Using two independent strategies, we show that Stx16 plays a crucial role in Glut4 traffic in 3T3-L1 adipocytes. Over-expression of a mutant form of Stx16 devoid of a transmembrane anchor was found to significantly slow the reversal of insulin-stimulated glucose transport. Depletion of Stx16 using antisense approaches profoundly reduced insulin-stimulated glucose transport but was without effect on cell surface transferrin receptor levels, and also reduced the extent of Glut4 translocation to the plasma membrane in response to insulin. These data support a model in which Stx16 is crucial in the sorting of Glut4 from the fast cycling to the slow cycling intracellular trafficking pathways in adipocytes.     

Insulin-dependent phosphorylation of the insulin receptor-protein kinase and activation of glucose transport in 3T3-L1 adipocytes

Insulin stimulates hexose transport and phosphorylation of the insulin receptor in monolayer cultures of intact 3T3-L1 adipocytes. To assess the phosphorylation state of the receptor in situ, cells were equilibrated with [32P]orthophosphate and then disrupted under denaturing conditions which preserved the phosphorylation state of the receptor established in the cell. The insulin receptor, isolated by lectin adsorption and two-dimensional nonreducing/reducing polyacrylamide gel electrophoresis, occurred as a single oligomeric species with an apparent alpha 2 beta 2 subunit composition. This oligomeric structure was not altered by treating cells with insulin. Only the beta-subunit of the receptor was phosphorylated; [32P]phosphoserine and [32P] phosphotyrosine were both identified in the beta-subunit from cells in the unstimulated state, but only [32P] phosphotyrosine increased in cells stimulated with insulin. Neither insulin-like growth factors I nor II stimulated insulin receptor beta-subunit phosphorylation, although both activated hexose transport. Upon the addition of insulin, [32P]orthophosphate incorporated into the beta-subunit increased 4.5-fold (7-fold with respect to [32P]tyrosine) and was complete within 1 min (t1/2 = 8 s). Following the removal of insulin from the monolayers, [32P]beta-subunit fell to the basal level (t1/2 = 2.5 min); there was no lag phase before either transition. The tyrosine protein kinase activity, measured in vitro with a model substrate, was higher with immunoaffinity-purified insulin receptor from insulin-stimulated cells than from cells in the basal state. Hexose transport rate, measured using 3-O-[methyl-14C]glucose, was half-maximally stimulated at 2 nM insulin. A 1-min latency period followed insulin addition, after which a 7-fold increase in the steady-state rate of hexose uptake was achieved within 5 min. Upon the removal of insulin, hexose transport continued at the stimulated steady-state rate for 2.5 min and then declined to the basal rate with a half-time of 8 min. These kinetic experiments in situ and protein kinase activity measurements in vitro support the hypothesis that beta-subunit phosphorylation is an intermediate step linking insulin binding to the increased glucose transport rate.     

SNAP23 promotes insulin-dependent glucose uptake in 3T3-L1 adipocytes: possible interaction with cytoskeleton

The acutestimulation of glucose uptake by insulin in fat and muscle cells isprimarily the result of translocation of facilitative glucosetransporter 4 (GLUT-4) from an internal compartment to the plasmamembrane. Here, we investigate the role of SNAP23 (a 23-kDa moleculeresembling the 25-kDa synaptosome associated protein) in GLUT-4translocation and glucose uptake in 3T3-L1 adipocytes. Microinjectionof a polyclonal antibody directed to the carboxy terminus of SNAP23inhibited GLUT-4 incorporation into the membrane in response toinsulin, whereas microinjection of full-length recombinant SNAP23enhanced the insulin effect. Introduction of recombinant SNAP23 intochemically permeabilized cells also enhanced insulin-stimulated glucosetransport. These results indicate that SNAP23 is required forinsulin-dependent, functional incorporation of GLUT-4 into the plasmamembrane and that the carboxy terminus of the protein is essential forthis process. SNAP23 is therefore likely to be a fusion catalyst alongwith syntaxin-4 and vesicle-associated membrane protein (VAMP)-2.Furthermore, the endogenous content of SNAP23 appears tobe limiting for insulin-dependent GLUT-4 exposure at the cell surface.A measurable fraction of SNAP23 was sedimented with cytoskeletalelements when extracted with Triton X-100, unlike VAMP-2 andsyntaxin-4, which were exclusively soluble in detergent. We hypothesizethat SNAP23 and its interaction with the cytoskeleton may be targetsfor regulation of GLUT-4 traffic.     

Insulin regulation of the two glucose transporters in 3T3-L1 adipocytes

The amounts of the brain type and muscle type glucose transporters (designated Glut 1 and 4, respectively) in 3T3-L1 adipocytes have been determined by quantitative immunoblotting with antibodies against their carboxyl-terminal peptides. There are about 950,000 and 280,000 copies of Glut 1 and 4, respectively, per cell. Insulin caused the translocation of both types of transporters from an intracellular location to the plasma membrane. The insulin-elicited increase in cell surface transporters was assessed by labeling the surface transporters with a newly developed, membrane-impermeant, photoaffinity labeling reagent for glucose transporters. The increases in Glut 1 and 4 averaged 6.5- and 17-fold, respectively, whereas there was a 21-fold in hexose transport. These results indicate that the translocation of Glut 4 could largely account for the insulin effect on transport rate, but only if the intrinsic activity of Glut 4 is much higher than that of Glut 1. The two transporters are colocalized intracellularly: vesicles (average diameter 72 nm) isolated from the intracellular membranes by immunoadsorption with antibodies against Glut 1 contained 95% of the Glut 4 and, conversely, vesicles isolated with antibodies against Glut 4 contained 85% of the Glut 1.     

ArPIKfyve-PIKfyve interaction and role in insulin-regulated GLUT4 translocation and glucose transport in 3T3-L1 adipocytes

Insulin activates glucose transport by promoting translocation of the insulin-sensitive fat/muscle-specific glucose transporter GLUT4 from an intracellular storage compartment to the cell surface. Here we report that an optimal insulin effect on glucose uptake in 3T3-L1 adipocytes is dependent upon expression of both PIKfyve, the sole enzyme for PtdIns 3,5-P(2) biosynthesis, and the PIKfyve activator, ArPIKfyve. Small-interfering RNAs that selectively ablated PIKfyve or ArPIKfyve in this cell type depleted the PtdIns 3,5-P(2) pool and reduced insulin-activated glucose uptake to a comparable degree. Combined loss of PIKfyve and ArPIKfyve caused further PtdIns 3,5-P(2) ablation that correlated with greater attenuation in insulin responsiveness. Loss of PIKfyve-ArPIKfyve reduced insulin-stimulated Akt phosphorylation and the cell surface accumulation of GLUT4 or IRAP, but not GLUT1-containing vesicles without affecting overall expression of these proteins. ArPIKfyve and PIKfyve were found to physically associate in 3T3-L1 adipocytes and this was insulin independent. In vitro labeling of membranes isolated from basal or insulin-stimulated 3T3-L1 adipocytes documented substantial insulin-dependent increases of PtdIns 3,5-P(2) production on intracellular membranes. Together, the data demonstrate for the first time a physical association between functionally related PIKfyve and ArPIKfyve in 3T3-L1 adipocytes and indicate that the novel ArPIKfyve-PIKfyve-PtdIns 3,5-P(2) pathway is physiologically linked to insulin-activated GLUT4 translocation and glucose transport.     

Glucosamine-induced insulin resistance in 3T3-L1 adipocytes

To study molecular mechanisms for glucosamine-induced insulin resistance, we induced complete and reversible insulin resistance in 3T3-L1 adipocytes with glucosamine in a dose- and time-dependent manner (maximal effects at 50 mM glucosamine after 6 h). In these cells, glucosamine impaired insulin-stimulated GLUT-4 translocation. Glucosamine (6 h) did not affect insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 and -2 and weakly, if at all, impaired insulin stimulation of phosphatidylinositol 3-kinase. Glucosamine, however, severely impaired insulin stimulation of Akt. Inhibition of insulin-stimulated glucose transport was correlated with that of Akt activity. In these cells, glucosamine also inhibited insulin stimulation of p70 S6 kinase. Glucosamine did not alter basal glucose transport and insulin stimulation of GLUT-1 translocation and mitogen-activated protein kinase. In summary, glucosamine induced complete and reversible insulin resistance in 3T3-L1 adipocytes. This insulin resistance was accompanied by impaired insulin stimulation of GLUT-4 translocation and Akt activity, without significant impairment of upstream molecules in insulin-signaling pathway.     

Molecular regulation of GLUT-4 targeting in 3T3-L1 adipocytes

Insulin stimulates glucose transport in muscle and adipose tissue by triggering the movement of the glucose transporter GLUT-4 from an intracellular compartment to the cell surface. Fundamental to this process is the intracellular sequestration of GLUT-4 in nonstimulated cells. Two distinct targeting motifs in the amino and carboxy termini of GLUT-4 have been previously identified by expressing chimeras comprised of portions of GLUT-4 and GLUT-1, a transporter isoform that is constitutively targeted to the cell surface, in heterologous cells. These motifs-FQQI in the NH2 terminus and LL in the COOH terminus- resemble endocytic signals that have been described in other proteins. In the present study we have investigated the roles of these motifs in GLUT-4 targeting in insulin-sensitive cells. Epitope-tagged GLUT-4 constructs engineered to differentiate between endogenous and transfected GLUT-4 were stably expressed in 3T3-L1 adipocytes. Targeting was assessed in cells incubated in the presence or absence of insulin by subcellular fractionation. The targeting of epitope-tagged GLUT-4 was indistinguishable from endogenous GLUT-4. Mutation of the FQQI motif (F5 to A5) caused GLUT-4 to constitutively accumulate at the cell surface regardless of expression level. Mutation of the dileucine motif (L489L490 to A489A490) caused an increase in cell surface distribution only at higher levels of expression, but the overall cells surface distribution of this mutant was less than that of the amino- terminal mutants. Both NH2- and COOH-terminal mutants retained insulin- dependent movement from an intracellular to a cell surface locale, suggesting that neither of these motifs is involved in the insulin- dependent redistribution of GLUT-4. We conclude that the phenylalanine- based NH2-terminal and the dileucine-based COOH-terminal motifs play important and distinct roles in GLUT-4 targeting in 3T3-L1 adipocytes.     

Chronic treatment with insulin selectively down-regulates cell-surface GLUT4 glucose transporters in 3T3-L1 adipocytes

A new method for photoaffinity labeling of glucose transporters has been used to compare the effects of glucose-starvation, acute-insulin, and chronic-insulin treatments on the cell-surface glucose transporters in 3T3-L1 adipocytes. Starvation alone increased the cell-surface levels of GLUT1 and GLUT4 by approximately 4- and approximately 2-fold, respectively. As shown by Calderhead, D, M., Kitagawa, K., Tanner, L.T., Holman, G.D., and Lienhard, G.E. (1990) J. Biol. Chem. 265, 13800-13808) acute-insulin treatment increased cell-surface GLUT1 and GLUT4 by approximately 5- and approximately 15-fold respectively. In contrast to this, chronic-insulin treatment gave a further 3-4-fold increase in both cell-surface and total cellular GLUT1, but availability of GLUT4 at the cell-surface was down-regulated to half the level found in the acute treatment but with no change in the total cellular level. This effect occurred in starved and non-starved cells and suggests that starvation, acute-insulin, and chronic-insulin treatments regulate glucose transporter availability through independent mechanisms. The down-regulation of GLUT4 reached a maximally reduced cell-surface level in 6 h while the rise in GLUT1 reached a maximum after 24-48 h. The rise in GLUT1 appeared to compensate for the decline in cell-surface GLUT4 as glucose transport activity was further increased during the long term treatment with insulin. The down-regulation of GLUT4 due to the chronic-insulin treatment is associated with a marked resistance of the cells to restimulate glucose transport and particularly to recruit further GLUT4 to the cell-surface following an additional insulin treatment. The defect appears to be in the signaling mechanism that is responsible for translocation.     

Chemerin enhances insulin signaling and potentiates insulin-stimulated glucose uptake in 3T3-L1 adipocytes

To explore a novel adipokine, we screened adipocyte differentiation-related gene and found that TIG2/chemerin was strongly induced during the adipocyte differentiation. Chemerin was secreted by the mature 3T3-L1 adipocytes and expressed abundantly in adipose tissue in vivo as recently described. Intriguingly, the expression of chemerin was differently regulated in the liver and adipose tissue in db/db mice. In addition, serum chemerin concentration was decreased in db/db mice. Chemerin and its receptor/ChemR23 were expressed in mature adipocytes, suggesting its function in autocrine/paracrine fashion. Finally, chemerin potentiated insulin-stimulated glucose uptake concomitant with enhanced insulin signaling in the 3T3-L1 adipocytes. These data establish that chemerin is a novel adipokine that regulates adipocyte function.     

Differential regulation of secretory compartments containing the insulin-responsive glucose transporter 4 in 3T3-L1 adipocytes

Insulin and guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) both stimulate glucose transport and translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes. Previous studies suggest that these effects may be mediated by different mechanisms. In this study we have tested the hypothesis that these agonists recruit GLUT4 by distinct trafficking mechanisms, possibly involving mobilization of distinct intracellular compartments. We show that ablation of the endosomal system using transferrin-HRP causes a modest inhibition ( approximately 30%) of insulin-stimulated GLUT4 translocation. In contrast, the GTPgammaS response was significantly attenuated ( approximately 85%) under the same conditions. Introduction of a GST fusion protein encompassing the cytosolic tail of the v-SNARE cellubrevin inhibited GTPgammaS-stimulated GLUT4 translocation by approximately 40% but had no effect on the insulin response. Conversely, a fusion protein encompassing the cytosolic tail of vesicle-associated membrane protein-2 had no significant effect on GTPgammaS-stimulated GLUT4 translocation but inhibited the insulin response by approximately 40%. GTPgammaS- and insulin-stimulated GLUT1 translocation were both partially inhibited by GST-cellubrevin ( approximately 50%) but not by GST-vesicle-associated membrane protein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes with GTPgammaS caused a marked accumulation of Rab4 and Rab5 at the cell surface, whereas other Rab proteins (Rab7 and Rab11) were unaffected. These data are consistent with the localization of GLUT4 to two distinct intracellular compartments from which it can move to the cell surface independently using distinct sets of trafficking molecules.     

Cytoplasmic Ca2+ during differentiation of 3T3-L1 adipocytes. Effect of insulin and relation to glucose transport

The cytoplasmic concentration of ionized Ca2+ [( Ca2+]i) was determined in 3T3-L1 cells during their differentiation from fibroblasts to adipocytes, suspended and loaded with the fluorescent Ca2+ indicators quin2 or indo-1. In undifferentiated fibroblasts, as well as in differentiated adipocytes up to day 9, [Ca2+]i was steady around 170 nM, and it increased significantly only in old adipocytes (day 12). During differentiation, stimulation of glucose uptake by insulin increased from a few percent to severalfold. Stimulation of uptake was already apparent after 10 min of addition of the hormone, and 10 nM insulin produced maximal stimulation in 30 min. Insulin (10(-6) M) added to quin2- or indo-1-loaded, suspended adipocytes had no detectable effect on [Ca2+]i for at least 10 min. In contrast, addition of the general anesthetic halothane increased [Ca2+]i from 172 to 251 nM in 3 min. In EGTA solution, the Ca2+ ionophore ionomycin elicited release of Ca2+ from intracellular stores that resulted in a transient increase in [Ca2+]i. A smaller but measurable Ca2+ release from intracellular stores (increasing [Ca2+]i by 20 nM) resulted upon addition of 20 micrograms/ml phosphatidic acid. In contrast, insulin did not produce any detectable release of Ca2+ from intracellular stores. Incubation of 3T3-L1 adipocytes with insulin in the presence of EGTA (the latter in excess over the Ca2+ concentration of the medium) did not prevent the stimulation of hexose uptake by the hormone, indicating that extracellular Ca2+ does not play a role in the insulin response. Furthermore, incubation of cells with quin2/AM in EGTA medium during exposure to insulin did not prevent stimulation of hexose uptake. Under these conditions it is demonstrated that intracellular quin2 suffices to chelate cytoplasmic Ca2+ even if releasable Ca2+ from intracellular stores were to pour into the cytoplasm. Thus, quin2 effectively lowers [Ca2+]i without impairing insulin action. It is concluded that insulin does not produce changes in [Ca2+]i and that chelating intracellular Ca2+ does not prevent stimulation of hexose uptake by insulin. These results suggest that it is unlikely that changes in [Ca2+]i may play a role in the transduction of information in insulin stimulation of glucose uptake in 3T3-L1 adipocytes.     

Inhibition by insulin of resistin gene expression in 3T3-L1 adipocytes.

Expression of the gene encoding resistin, a low molecular weight protein secreted from adipose tissue postulated to link obesity and type II diabetes, was examined in 3T3-L1 adipocytes. Resistin mRNA was detected in 3T3-L1 cells by day 3 following induction of differentiation into adipocytes; by day 4 the level of resistin mRNA peaked and remained high. The PPARgamma activators, rosiglitazone or darglitazone, reduced the level of resistin mRNA. Dexamethasone upregulated resistin mRNA level, but no effect was observed with the beta(3)-adrenoceptor agonist, BRL 37344. A substantial reduction in resistin mRNA level was observed with insulin, which induced decreases at physiological concentrations. Insulin may be a major inhibitor of resistin production, and this does not support a role for resistin in insulin resistance.     

Tunicamycin-mediated depletion of insulin receptors in 3T3-L1 adipocytes.

Tunicamycin, an antibiotic that inhibits protein glycosylation, elicited a rapid depletion of insulin binding activity at the surface of 3T3-L1 adipocytes. Disappearance of insulin receptors occurred more rapidly in the presence of tunicamycin than when protein synthesis was inhibited by cycloheximide and was accompanied by a diminution in sensitivity of the adipocytes to the acute effects of insulin and anti-insulin receptor antibody on hexose uptake and metabolism.