Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes

Following the differentiation of 3T3-L1 preadipocytes insulin acutely activates the rate of 2-deoxy-[1-14C]glucose uptake in the mature 3T3-L1 adipocyte by 15- to 20-fold. Phenylarsine oxide, a trivalent arsenical that forms stable ring complexes with vicinal dithiols, prevents insulin-activated hexose uptake in a concentration-dependent manner (Ki = 7 microM) but has no inhibitory effect on basal hexose uptake. 2,3-Dimercaptopropanol at a level nearly stoichiometric to that of phenylarsine oxide prevents or rapidly reverses the inhibition of hexose uptake; 2-mercaptoethanol, even in high stoichiometric excess over the arsenical, does not reverse inhibition of hexose uptake. When phenylarsine oxide is added after adipocytes have been fully activated by insulin, 2-deoxy-[1-14C]glucose uptake rate decays slowly at a rate corresponding to that caused by the withdrawal of insulin (t1/2 = 10 min). Using the same conditions under which phenylarsine oxide blocked activation, the Km for deoxyglucose uptake, the rate at which 125I-insulin became cell-associated, and the 125I-insulin binding isotherm for solubilized insulin receptor were not affected by phenylarsine oxide. These results support the transporter translocation model for insulin-activated hexose transport and implicate vicinal sulfhydryl groups in a post-insulin binding event essential for the translocation of glucose transporters to the plasma membrane.     

Effect of phenylarsine oxide on fluid phase endocytosis: further evidence for activation of the glucose transporter

We have shown previously that insulin stimulates fluid phase endocytosis in 3T3-L1 adipocytes (Gibbs et al., 1986). Using [14C]sucrose as an endocytotic marker, we show here that phenylarsine oxide, a trivalent arsenical which binds neighboring dithiols, blocked not only insulin-stimulated fluid phase endocytosis, but basal endocytosis as well. The Ki for this process was 6 microM in the presence or absence of insulin and the time required for inhibition was less than 2.5 min, the limit of detection in our assay system. These results can be compared with the inhibitory effect of phenylarsine oxide on insulin-stimulated glucose transport. Although the Ki for insulin-stimulated transport (7 microM) was similar to that for inhibition of endocytosis, basal glucose transport was not affected by the inhibitor. Further, when cells were prestimulated with insulin causing maximal stimulation of the glucose transport rate, phenylarsine oxide induced a time-dependent reduction to the basal rate (t 1/2 of 10 min), despite the fact that endocytosis was blocked immediately. This observation suggests that if the transporter is recycled by an exocytotic/endocytotic mechanism, it is distinct from fluid-phase endocytosis/exocytosis, which is a vesicle-mediated process, and provides further evidence that the transporter may undergo intrinsic activation/inactivation which does not require vesicle movement.     

Effect of phenylarsine oxide on protein synthesis in 3T3-L1 adipocytes

In this report, we show that insulin stimulated the incorporation of tracer [3H]leucine into protein of 3T3-L1 adipocytes within 2 min of insulin addition. The concentration of insulin required to elicit 50% activation was 4nM. Phenylarsine oxide, an inhibitor of insulin-stimulated glucose transport, blocked not only insulin-stimulated protein synthesis but constitutive protein synthesis as well (Ki, 3 microM). Importantly, protein synthesis was not required for insulin-activated glucose transport since cycloheximide added either before or after insulin had no effect on the stimulated rates of glucose transport.     

Nocodazole inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes via a microtubule-independent mechanism

Insulin stimulates glucose transport in adipocytes and muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Recent reports have shown that the microtubule-depolymerizing agent nocodazole inhibits insulin-stimulated glucose transport, implicating an important role for microtubules in this process. In the present study we show that 2 microm nocodazole completely depolymerized microtubules in 3T3-L1 adipocytes, as determined morphologically and biochemically, resulting in dispersal of the perinuclear GLUT4 compartment and the Golgi apparatus. However, 2 microm nocodazole did not significantly effect either the kinetics or magnitude of insulin-stimulated glucose transport. Consistent with previous studies, higher concentrations of nocodazole (10-33 microm) significantly inhibited basal and insulin-stimulated glucose uptake in adipocytes. This effect was not likely the result of microtubule depolymerization because in the presence of taxol, which blocked nocodazole-induced depolymerization of microtubules as well as the dispersal of the perinuclear GLUT4 compartment, the inhibitory effect of 10-33 microm nocodazole on insulin-stimulated glucose uptake prevailed. Despite the decrease in insulin-stimulated glucose transport with 33 microm nocodazole we did not observe inhibition of insulin-stimulated GLUT4 translocation to the cell surface under these conditions. Consistent with a direct effect of nocodazole on glucose transporter function we observed a rapid inhibitory effect of nocodazole on glucose transport activity when added to either 3T3-L1 adipocytes or to Chinese hamster ovary cells at 4 degrees C. These studies reveal a new and unexpected effect of nocodazole in mammalian cells which appears to occur independently of its microtubule-depolymerizing effects.     

Differential regulation of NHE1 phosphorylation and glucose uptake by inhibitors of the ERK pathway and p90RSK in 3T3-L1 adipocytes

Insulin stimulates trafficking of GLUT4 to the cell surface for glucose uptake into target cells, and phosphorylation of Ser703 of the Na⁺/H⁺ exchanger NHE1, which activates proton efflux. The latter has been proposed to facilitate optimal glucose uptake into cardiomyocytes. We found that the insulin-stimulated phosphorylation of Ser703 of NHE1 is mediated by p90RSK but not directly coupled to glucose uptake in 3T3-L1 adipocytes in the short-term. Inhibiting Erk1/2 activation prevented NHE1 phosphorylation but not glucose uptake in 3T3-L1 adipocytes. In contrast, both NHE1 phosphorylation and insulin-stimulated uptake of glucose into 3T3-L1 adipocytes were blocked by inhibitors of the N-terminal kinase domain of p90RSK, namely BI-D1870 and SL0101, but not the FMK inhibitor of the C-terminal kinase domain of p90RSK, though in our hands FMK did not inhibit p90RSK in 3T3-L1 adipocytes. Further experiments were consistent with phosphorylation of AS160 by PKB/Akt mediating insulin-stimulated trafficking of GLUT4 to the plasma membrane. BI-D1870 and SL0101 however, inhibited glucose uptake without blocking GLUT4 translocation. While BI-D1870 partially inhibited insulin-stimulated PKB activation in these cells, this only partially inhibited AS160 phosphorylation and did not block GLUT4 trafficking, suggesting that p90RSK might regulate glucose transport after GLUT4 translocation. Moreover, BI-D1870 also prevented PMA-induced glucose transport in 3T3-L1 adipocytes further suggesting a role for p90RSK in regulating uptake of glucose into the cells. Kinetic experiments are consistent with SL0101 being a direct competitor of 2-deoxyglucose entry into cells, and this compound might also inhibit uptake of glucose into cells via inhibiting p90RSK, as revealed by comparison with the inactive form of the inhibitor. Taken together, we propose that BI-D1870 and SL0101 might exert their inhibitory effects on glucose uptake in 3T3-L1 adipocytes at least partially through a p90RSK dependent step after GLUT4 becomes associated with the plasma membrane.     

On the mechanism for neomycin reversal of wortmannin inhibition of insulin stimulation of glucose uptake

Although a number of studies and approaches have indicated that activation of the Ser/Thr kinase called Akt/protein kinase B is critical for the insulin-stimulated increase of glucose uptake in adipocytes, other studies have indicated that this enzyme may play an ancillary role. For example, a recent study indicated that neomycin would allow insulin-stimulated Glut4 translocation and glucose transport in the presence of the phosphatidylinositol (PI) 3-kinase inhibitor, wortmannin, a known inhibitor of Akt activation (James, D. J., Salaun, C., Brandie, F. M., Connell, J. M. C., and Chamberlain, L. H. (2004) J. Biol. Chem. 279, 20567-20570). To better understand this observation, we examined a number of downstream targets of Akt. As previously reported, treatment of 3T3-L1 adipocytes with neomycin prevented the wortmannin inhibition of insulin-stimulated glucose transport. However, in the presence of neomycin, wortmannin did not inhibit the insulin-stimulated phosphorylation of several downstream targets of Akt including a proline-rich Akt substrate of 40 kDa, ribosomal protein S6, and glycogen synthase kinase-3. In addition, neomycin did not prevent the ability of a structurally unrelated PI 3-kinase inhibitor, LY294002, to inhibit the insulin-stimulated activation of glucose uptake. Moreover, neomycin reversed the inhibitory effect of wortmannin but not LY294002 on insulin stimulation of Akt kinase activity. Finally, neomycin was found to inactivate in vitro the PI 3-kinase inhibitory actions of wortmannin but not LY294002. These results indicate that the effects of neomycin in adipocytes are not mediated via its ability to sequester phosphatidylinositol 4,5-bisphosphate but are instead caused by the ability of neomycin to inactivate wortmannin.     

Insulin and Chromium Picolinate Induce Translocation of CD36 to the Plasma Membrane Through Different Signaling Pathways in 3T3-L1 Adipocytes,and with a Differential Functionality of the CD36

Chromium picolinate (CrPic) has been indicated to activate glucose transporter 4 (GLUT4) trafficking to the plasma membrane (PM) to enhance glucose uptake in 3T3-L1 adipocytes. In skeletal and heart muscle cells, insulin directs the intracellular trafficking of the fatty acid translocase/CD36 to induce the uptake of cellular long-chain fatty acid (LCFA). The current study describes the effects of CrPic and insulin on the translocation of CD36 from intracellular storage pools to the PM in 3T3-L1 adipocytes in comparison with that of GLUT4. Immunofluorescence microscopy and immunoblotting revealed that both CD36 and GLUT4 were expressed and primarily located intracellularly in 3T3-L1 adipocytes. Upon insulin or CrPic stimulation, PM expression of CD36 increased in a similar manner as that for GLUT4; the CrPic-stimulated PM expression was less strong than that of insulin. The increase in PM localization for these two proteins by insulin paralleled LCFA ([1-¹⁴C]palmitate) or [³H]deoxyglucose uptake in 3T3-L1 adipocytes. The induction of the PM expression of GLUT4, but not CD36, or substrate uptake by insulin and CrPic appears to be additive in adipocytes. Furthermore, wortmannin completely inhibited the insulin-stimulated translocation of GLUT4 or CD36 and prevented the increased uptake of glucose or LCFA in these cells. Taken together, for the first time, these findings suggest that both insulin and CrPic induce CD36 translocation to the PM in 3T3-L1 adipocytes and that their translocation-inducing effects are not additive. The signaling pathway inducing the translocations is different, apparently resulting in a differential activity of CD36.     

Iodophenylarsine oxide and arsenical affinity chromatography: new probes for dithiol proteins. Application to tubulins and to components of the insulin receptor-glucose transporter signal transduction pathway.

In our studies of the effects of the trivalent arsenical phenylarsine oxide on insulin-dependent hexose uptake in 3T3-L1 adipocytes, we needed direct methods to study arsenical-protein interactions. In this report, we describe two such new tools. The first is the radiolabeled covalent affinity reagent 4-[125I]iodophenylarsine oxide. This compound has effects on 3T3-L1 adipocytes similar to those of phenylarsine oxide both with respect to effects of hexose uptake and the accumulation of pp15, a phosphotyrosine-containing putative mediator of insulin action. Iodophenylarsine oxide labels numerous proteins in intact cells in a concentration-dependent, but apparently insulin-independent fashion. The second tool is trivalent arsenical affinity chromatography, which we use to show novel direct interactions between trivalent arsenicals and several proteins from 3T3-L1 adipocytes including the insulin-responsive glucose transporter GLUT4, the insulin proreceptor, and both the alpha and beta subunits of tubulin. The non-insulin-dependent glucose transporter GLUT1, the mature insulin receptor, and the fatty acid-binding protein 422(aP2) do not show strong interactions with arsenical resin. These results provide a new chemical approach to the study of both insulin-dependent hexose transport and tubulin function.     

Differential effects of palmitate on glucose uptake in rat-1 fibroblasts and 3T3-L1 adipocytes.

Non-esterified fatty acids are thought to be one of the causes for insulin resistance. However, the molecular mechanism of fatty acid-induced insulin resistance is not clearly known. In this study, we first examined the effect of palmitate on insulin signaling in 3T3-L1 adipocytes. We found that 1h treatment with 1 mmol/l palmitate had no effect on insulin binding, tyrosine phosphorylation of insulin receptors, 185 kDa proteins and Shc, and PI3 kinase activity in 3T3-L1 adipocytes. Then, the effects of palmitate on MAP kinase activity and glucose uptake in fully differentiated 3T3-L1 adipocytes were compared with those in poorly differentiated 3T3-L1 cells and in HIRc-B cells. Palmitate treatment had no effect on MAP kinase activity in fully differentiated 3T3-L1 adipocytes, while it inhibited MAP kinase in poorly differentiated 3T3-L1 cells and HIRc-B cells. Glucose transport in 3T3-L1 adipocytes treated with palmitate for 1 h, 4 h and 16 h was higher than that in control cells, but palmitate treatment caused a rightward shift of the insulin-dose responsive curve for glucose uptake in HIRc-B cells. Palmitate treatment did not significantly affect basal and insulin-stimulated GLUT4 translocation. When the cells were treated with PD98059, a specific MEK inhibitor, insulin-stimulated glucose uptake was not affected in 3T3-L1 adipocytes, while it was almost completely inhibited in HIRc-B cells. These results suggest the primary effect of palmitate on adipocytes may not involve insulin resistance of adipocytes themselves.     

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.     

Activation of cell surface glucose transporters measured by photoaffinity labeling of insulin-sensitive 3T3-L1 adipocytes.

Several studies have demonstrated that the intrinsic catalytic activity of cell surface glucose transporters is highly regulated in 3T3-L1 adipocytes expressing GLUT1 (erythrocyte/brain) and GLUT4 (adipocyte/skeletal muscle) glucose transporter isoforms. For example, inhibition of protein synthesis in these cells by anisomycin or cycloheximide leads to marked increases in hexose transport without a change in the levels of cell surface glucose transporter proteins (Clancy, B. M., Harrison, S. A., Buxton, J. M., and Czech, M. P. (1991) J. Biol. Chem. 266, 10122-10130). In the present work the exofacial hexose binding sites on GLUT1 and GLUT4 in anisomycin-treated 3T3-L1 adipocytes were labeled with the cell-impermeant photoaffinity reagent [2-3H]2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine [( 2-3H] ATB-BMPA) to determine which isoform is activated by protein synthetic blockade. As expected, a 15-fold increase in 2-deoxyglucose uptake in response to insulin was associated with 1.7- and 2.6-fold elevations in plasma membrane GLUT1 and GLUT4 protein levels, respectively. Anisomycin treatment of cultured adipocytes for 5 h produced an 8-fold stimulation of hexose transport but no increase in the content of glucose transporters in the plasma membrane fraction as measured by protein immunoblot analysis. Cell surface GLUT1 levels were also shown to be unaffected on 3T3-L1 adipocytes in response to anisomycin using an independent method, the binding of an antiexofacial GLUT1 antibody to intact cells. In contrast, anisomycin fully mimicked the action of insulin to stimulate (about 4-fold) the radiolabeling of GLUT1 transporters specifically immunoprecipitated from intact 3T3-L1 adipocytes irradiated after incubation with [2-3H] ATB-BMPA. Photolabeling of GLUT4 under these conditions was also significantly enhanced (1.8-fold) by anisomycin treatment, but this effect was only 15% of that caused by insulin. These results suggest that: 1) the photoaffinity reagent [2-3H]ATB-BMPA labels those cell surface glucose transporters present in a catalytically active state rather than total cell surface transporters as assumed previously and 2) inhibition of protein synthesis in 3T3-L1 adipocytes stimulates sugar transport primarily by enhancing the intrinsic catalytic activity of cell surface GLUT1, and to a lesser extent, GLUT4 proteins.     

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.     

Effect of vanadate on the cellular accumulation of pp15, an apparent product of insulin receptor tyrosine kinase action

The possible involvement of a 15-kDa phosphotyrosyl protein, pp15, in insulin action was investigated by using the insulin-mimetic agent, vanadate. Vanadate, a phosphotyrosine phosphatase inhibitor, was found to mimic insulin in 3T3-L1 adipocytes by three criteria. First, kinetic and concentration-dependence studies verified the insulin-like effect of vanadate in activating 2-deoxyglucose uptake. Insulin had an additive activating effect at a submaximal vanadate concentration, but showed no further activation at a saturating vanadate concentration. The trivalent arsenical, phenylarsine oxide (PAO) which forms complexes with vicinal dithiols, markedly inhibited vanadate-activated hexose transport in agreement with our previous studies in which PAO abolished the insulin-activated component of sugar uptake. Second, in situ phosphorylation experiments showed that vanadate activated tyrosine phosphorylation of the insulin receptor's beta-subunit. Exposure of vanadate-treated cells to PAO further increased the level of beta-subunit phosphorylation. The increased level of phosphorylation in the presence of PAO occurred only on tyrosyl residues. Third, vanadate caused the accumulation of a phosphorylated 15-kDa protein in the presence of PAO, but not in its absence. The characteristics of this protein were identical to those of pp15: 1) both proteins behaved identically by two-dimensional gel electrophoresis, 2) digestion of both proteins with trypsin gave rise to apparently identical phosphopeptides, and 3) both proteins contained phosphotyrosine as the only phosphoamino acid. The results indicate that both vanadate and insulin stimulate the accumulation of pp15 in the presence of PAO. The dithiol,2,3-dimercaptopropanol, but not a monothiol, reversed the effects of PAO on the inhibition of vanadate-induced hexose transport and the accumulation of pp15, thus implicating a vicinal dithiol in these actions of vanadate and insulin. Our results support the hypothesis that turnover of the phosphoryl group of pp15, a product of insulin receptor tyrosine kinase action, is coupled to signal transmission to the glucose transport system.     

Suitability of 2-deoxyglucose for measuring initial rates of glucose uptake in isolated adipocytes

The suitability of [3H]-2-deoxyglucose from measuring initial rates of glucose uptake in isolated rat adipocytes was assessed using three approaches. Basal and insulin-stimulated rates of glucose uptake were directly compared in 2 sec and 5 min assays using [14C]-3-O-methylglucose, [3H]-2-deoxyglucose, and [3H]-D-glucose. Equilibrium kinetics of 2-deoxyglucose uptake were compared with those of 3-O-methylglucose through impairment of hexokinase activity by depleting cellular energy with 2,4-dinitrophenol. The equivalence of these glucose analogues in a dynamic system was assessed by measuring the lag time preceding insulin stimulation of glucose uptake, insulin activation rates, and the T 1/2 of insulin activation. Our results demonstrate that no fundamental difference exists in the initial transport of 3-O-methylglucose, 2-deoxyglucose, and D-glucose.     

α-Lipoic acid ameliorates impaired glucose uptake in LYRM1 overexpressing 3T3-L1 adipocytes through the IRS-1/Akt signaling pathway

Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.     

The nitric oxide-donating derivative of acetylsalicylic acid, NCX 4016, stimulates glucose transport and glucose transporters translocation in 3T3-L1 adipocytes

NCX 4016 is a nitric oxide (NO)-donating derivative of acetylsalicylic acid. NO and salicylate, in vivo metabolites of NCX 4016, were shown to be potential actors in controlling glucose homeostasis. In this study, we evaluated the action of NCX 4016 on the capacity of 3T3-L1 adipocytes to transport glucose in basal and insulin-stimulated conditions. NCX 4016 induced a twofold increase in glucose uptake in parallel with the translocation of the glucose transporters GLUT1 and GLUT4 to the plasma membrane, leaving unaffected their total expression levels. Importantly, NCX 4016 further increased glucose transport induced by a physiological concentration of insulin. The stimulatory effect of NCX 4016 on glucose uptake appears to be mediated by its NO moiety. Indeed, it is inhibited by a NO scavenger and treatment with acetylsalicylic or salicylic acid had no effect. Although NO is involved in the action of NCX 4016, it did not mainly depend on the soluble cGMP cyclase/protein kinase G pathway. Furthermore, NCX 4016-stimulated glucose transport did not involve the insulin-signaling cascade required to stimulate glucose transport. NCX 4016 induces a small activation of the mitogen-activated protein kinases p38 and c-Jun NH(2)-terminal kinase and no activation of other stress-activated signaling molecules, including extracellular signal-regulated kinase, inhibitory factor kappaB, or AMP-activated kinases. Interestingly, NCX 4016 modified the content of S-nitrosylated proteins in adipocytes. Taken together, our results indicate that NCX 4016 induced glucose transport in adipocytes through a novel mechanism possibly involving S-nitrosylation. NCX 4016 thus possesses interesting characteristics to be considered as a candidate molecule for the treatment of patients suffering from metabolic syndrome and type 2 diabetes.     

Protein targeting to glycogen overexpression results in the specific enhancement of glycogen storage in 3T3-L1 adipocytes

Protein phosphatase-1 (PP1) plays an important role in the regulation of glycogen synthesis by insulin. Protein targeting to glycogen (PTG) enhances glycogen accumulation by increasing PP1 activity against glycogen-metabolizing enzymes. However, the specificity of PTG's effects on cellular dephosphorylation and glucose metabolism is unclear. Overexpression of PTG in 3T3-L1 adipocytes using a doxycycline-controllable adenoviral construct resulted in a 10-20-fold increase in PTG levels and an 8-fold increase in glycogen levels. Inclusion of 1 microg/ml doxycycline in the media suppressed PTG expression, and fully reversed all PTG-dependent effects. Infection of 3T3-L1 adipocytes with the PTG adenovirus caused a marked dephosphorylation and activation of glycogen synthase. The effects of PTG seemed specific, because basal and insulin-stimulated phosphorylation of a variety of signaling proteins was unaffected. Indeed, glycogen synthase was the predominant protein whose phosphorylation state was decreased in 32P-labeled cells. PTG overexpression did not alter PP1 protein levels but increased PP1 activity 6-fold against phosphorylase in vitro. In contrast, there was no change in PP1 activity measured using myelin basic protein, suggesting that PTG overexpression specifically directed PP1 activity against glycogen-metabolizing enzymes. To investigate the metabolic consequences of altering PTG levels, glucose uptake and storage in 3T3-L1 adipocytes was measured. PTG overexpression did not affect 2-deoxy-glucose transport rates in basal and insulin-stimulated cells but dramatically enhanced glycogen synthesis rates under both conditions. Despite the large increases in cellular glucose flux upon PTG overexpression, basal and insulin-stimulated glucose incorporation into lipid were unchanged. Cumulatively, these data indicate that PTG overexpression in 3T3-L1 adipocytes discretely stimulates PP1 activity against glycogen synthase and phosphorylase, resulting in a marked and specific increase in glucose uptake and storage as glycogen.     

Genistein directly inhibits GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

The isoflavone-derivative genistein is commonly applied as an inhibitor of tyrosine kinases. In this report we analyze the effect of genistein on insulin-stimulated glucose uptake in 3T3-L1 adipocytes. In these cells insulin-induced glucose uptake is primarily mediated by the GLUT4 glucose transporter. We observed that pre-treatment with genistein did not affect insulin-induced tyrosine kinase activity of the insulin receptor or activation of protein kinase B. On the other hand, genistein acted as a direct inhibitor of insulin-induced glucose uptake in 3T3-L1 adipocytes with an IC(50) of 20 microM. We conclude that apart from acting as a general tyrosine kinase inhibitor, genistein also affects the function of other proteins such as the GLUT4 transporter. These data suggest that caution must be applied when interpreting data on the involvement of tyrosine kinase activity in glucose uptake in 3T3-L1 adipocytes.     

FATP1 mediates fatty acid-induced activation of AMPK in 3T3-L1 adipocytes

Fatty acid transport proteins are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. FATP-dependent production of AMP was evaluated using FATP4 proteoliposomes, and fatty acid-dependent activation of AMP-activated protein kinase (AMPK) was assessed in 3T3-L1 adipocytes. Insulin-stimulated fatty acid influx (palmitate or arachidonate) into cultured adipocytes resulted in an increase in the phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase. Consistent with the activation of AMPK, palmitate uptake into 3T3-L1 adipocytes resulted in an increase in intracellular [AMP]/[ATP]. The fatty acid-induced increase in AMPK activation was attenuated in a cell line expressing shRNA targeting FATP1. Taken together, these results demonstrate that, in adipocytes, insulin-stimulated fatty acid influx mediated by FATP1 regulates AMPK and provides a potential regulatory mechanism for balancing de novo production of fatty acids from glucose metabolism with influx of preformed fatty acids via phosphorylation of acetyl-CoA carboxylase.     

Apelin stimulates glucose uptake through the PI3K/Akt pathway and improves insulin resistance in 3T3-L1 adipocytes

Apelin, a cytokine mainly secreted by adipocytes, is closely related with insulin resistance. The underlying molecular mechanisms of how apelin affects insulin resistance, however, are poorly understood. This study aimed to investigate the effect of apelin on glucose metabolism and insulin resistance in 3T3-L1 adipocytes. After 10 ng/ml TNF-α treatment for 24 h, insulin-stimulated glucose uptake was reduced by 47% in 3T3-L1 adipocytes. Apelin treatment improved glucose uptake in a time- and dose-dependent manner. Treatment of 1,000 nM apelin for 60 min maximally augmented glucose uptake in insulin-resistant 3T3-L1 adipocytes. Furthermore, apelin pre-incubation also increased adipocytes' insulin-stimulated glucose uptake, and PI3K/Akt pathway were involved in these effects. In addition, immunocytochemistry staining and western blotting analysis indicated that apelin could increase glucose transporter 4 translocation from the cytoplasm to the plasma membrane. Apelin also increased the anti-inflammatory adipokine adiponectin mRNA expression while reducing that of pro-inflammatory adipokine interleukin-6 in insulin-resistant 3T3-L1 adipocytes. These results suggest that apelin stimulates glucose uptake through the PI3K/Akt pathway, promotes GLUT4 translocation from the cytoplasm to the plasma membrane, and modulates inflammatory responses in insulin-resistant 3T3-L1 adipocytes.