Regulation of Myosin Light Chain Kinase during Insulin-Stimulated Glucose Uptake in 3T3-L1 Adipocytes

Myosin II (MyoII) is required for insulin-responsive glucose transporter 4 (GLUT4)-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC) of MyoIIA via myosin light chain kinase (MLCK). The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy) ethane-N,N,N'',N''-tetra acetic acid, (BAPTA) (in the presence of insulin) impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKIIδ did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.     

A novel role for myosin II in insulin-stimulated glucose uptake in 3T3-L1 adipocytes

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles from an intracellular pool to the plasma membrane. The studies presented here show that inhibition of myosin II activity impairs GLUT4-mediated glucose uptake but not GLUT4 translocation to the plasma membrane. We also show that adipocytes express both myosin IIA and IIB isoforms, and that myosin IIA is recruited to the plasma membrane upon insulin stimulation. Taken together, the data presented here represent the first demonstration that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. Based on our findings, we hypothesize that myosin II is activated upon insulin stimulation and recruited to the cell cortex to facilitate GLUT4 fusion with the plasma membrane. The identification of myosin II as a key component of GLUT4-mediated glucose uptake represents an important advance in our understanding of the mechanisms regulating glucose homeostasis.     

The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxy- methyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA-AM. While the ionophores or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degrees C, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/calmodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.     

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.     

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.     

Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles

We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS(97)xS(99)). Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3. Although wild-type Synip (WT-Synip) undergoes an insulin-stimulated dissociation from Syntaxin4, the Synip serine 99 to phenylalanine mutant (S99F-Synip) is resistant to Akt2 phosphorylation and fails to display insulin-stimulated Syntaxin4 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, whereas overexpression of S99F-Synip functioned in a dominant-interfering manner by preventing insulin-stimulated GLUT4 recruitment and plasma membrane fusion. These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip-Syntaxin4 interaction.     

Thyroid hormone promotes insulin-induced glucose uptake by enhancing Akt phosphorylation and VAMP2 translocation in 3T3-L1 adipocytes

The purpose of this study was to test a hypothesis that T3 promotes glucose uptake via enhancing insulin-induced Akt phosphorylation and VAMP2 translocation in 3T3-L1 adipocytes. T3 significantly enhanced insulin-induced phosphorylation of Akt, cytoplasma to cell membrane translocations of vesicle-associated membrane protein 2 (VAMP2) and glucose transporter 4 (GLUT4), and glucose uptake in adipocytes. Akt inhibitor X abolished the promoting effects of T3, suggesting that Akt activation is essential for T3 to enhance these insulin-induced events in adipocytes. Knockdown of VAMP2 using siRNA abrogated the effects of T3 on insulin-induced GLUT4 translocation and glucose uptake, suggesting that VAMP2 is an important mediator of these processes. These data suggest that T3 may promote glucose uptake via enhancing insulin-induced phosphorylation of Akt and subsequent translocations of VAMP2 and GLUT4 in 3T3-L1 adipocytes. Akt phosphorylation is necessary for the promoting effects of T3 on insulin-stimulated VAMP2 translocation. Further, VAMP2 is essential for T3 to increase insulin-stimulated translocation of GLUT4 and subsequent uptake of glucose in adipocytes.     

Unconventional myosin Myo1c promotes membrane fusion in a regulated exocytic pathway

Glucose homeostasis is controlled in part by regulation of glucose uptake into muscle and adipose tissue. Intracellular membrane vesicles containing the GLUT4 glucose transporter move towards the cell cortex in response to insulin and then fuse with the plasma membrane. Here we show that the fusion step is retarded by the inhibition of phosphatidylinositol (PI) 3-kinase. Treatment of insulin-stimulated 3T3-L1 adipocytes with the PI 3-kinase inhibitor LY294002 causes the accumulation of GLUT4-containing vesicles just beneath the cell surface. This accumulation of GLUT4-containing vesicles near the plasma membrane prior to fusion requires an intact cytoskeletal network and the unconventional myosin motor Myo1c. Remarkably, enhanced Myo1c expression under these conditions causes extensive membrane ruffling and overrides the block in membrane fusion caused by LY294002, restoring the display of GLUT4 on the cell exterior. Ultrafast microscopic analysis revealed that insulin treatment leads to the mobilization of GLUT4-containing vesicles to these regions of Myo1c-induced membrane ruffles. Thus, localized membrane remodeling driven by the Myo1c motor appears to facilitate the fusion of exocytic GLUT4-containing vesicles with the adipocyte plasma membrane.     

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.     

Activation of protein kinase C zeta induces serine phosphorylation of VAMP2 in the GLUT4 compartment and increases glucose transport in skeletal muscle

Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKC zeta in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKC zeta to associate specifically with the GLUT4 compartments and that PKC zeta together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKC zeta and GLUT4 recycled independently of one another. To further establish the importance of PKC zeta in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKC zeta (DNPKC zeta) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKC zeta was associated with a marked increase in the activity of this isoform. The overexpressed, active PKC zeta coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKC zeta caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKC zeta induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKC zeta disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKC zeta regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.     

MEK inhibitors impair insulin-stimulated glucose uptake in 3T3-L1 adipocytes

In 3T3-L1 adipocytes, insulin activates three major signaling cascades, the phosphoinositide 3-kinase (PI3K) pathway, the Cbl pathway, and the mitogen-activated protein kinase (MAPK) pathway. Although PI3K and Cbl mediate insulin-stimulated glucose uptake by promoting the translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane, the MAPK pathway does not have an established role in insulin-stimulated glucose uptake. We demonstrate in this report that PI3K inhibitors also inhibit the MAPK pathway. To investigate the role of the MAPK pathway separately from that of the PI3K pathway in insulin-stimulated glucose uptake, we used two specific inhibitors of MAPK kinase (MEK) activity, PD-98059 and U-0126, which reduced insulin-stimulated glucose uptake by approximately 33 and 50%, respectively. Neither MEK inhibitor affected the activation of Akt or PKCzeta/lambda, downstream signaling molecules in the PI3K pathway. Inhibition of MEK with U-0126 did not prevent GLUT4 from translocating to the plasma membrane, nor did it inhibit the subsequent docking and fusion of GLUT4-myc with the plasma membrane. MEK inhibitors affected glucose transport mediated by GLUT4 but not GLUT1. Importantly, the presence of MEK inhibitors only at the time of the transport assay markedly impaired both insulin-stimulated glucose uptake and MAPK signaling. Conversely, removal of MEK inhibitors before the transport assay restored glucose uptake and MAPK signaling. Collectively, our studies suggest a possible role for MEK in the activation of GLUT4.     

Adrenergic receptor stimulation attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes by inhibiting GLUT4 translocation

Activation of the sympathetic nervous system inhibits insulin-stimulated glucose uptake. However, the underlying mechanisms are incompletely understood. Therefore, we studied the effects of catecholamines on insulin-stimulated glucose uptake and insulin-stimulated translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. We found that epinephrine (1 microM) nearly halved insulin-stimulated 2-deoxyglucose uptake. The beta-adrenoceptor antagonist propranolol (0.3 microM) completely antagonized the inhibitory effect of epinephrine on insulin-stimulated glucose uptake, whereas the alpha-adrenoceptor antagonist phentolamine (10 microM) had no effect. When norepinephrine was used instead of epinephrine, the results were identical. None of the individual selective beta-adrenoceptor antagonists (1 microM, beta(1): metoprolol, beta(2): ICI-118551, beta(3): SR-59230A) could counteract the inhibitory effect of epinephrine. Combination of ICI-118551 and SR-59230A, as well as combination of all three selective beta-adrenoceptor antagonists, abolished the effect of epinephrine on insulin-stimulated glucose uptake. After differential centrifugation, we measured the amount of GLUT1 and GLUT4 in the plasma membrane and in intracellular vesicles by means of Western blotting. Both epinephrine and norepinephrine reduced insulin-stimulated GLUT4 translocation to the plasma membrane. These results show that beta-adrenergic (but not alpha-adrenergic) stimulation inhibits insulin-induced glucose uptake in 3T3-L1 adipocytes, most likely via the beta(2)- and beta(3)-adrenoceptor by interfering with GLUT4 translocation from intracellular vesicles to the plasma membrane.     

Regulation of insulin signaling and glucose transporter 4 (GLUT4) exocytosis by phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase, skeletal muscle, and kidney enriched inositol polyphosphate phosphatase (SKIP)

The glucose transporter 4 (GLUT4) is responsible for glucose uptake in the skeletal muscle. Insulin-induced translocation of GLUT4 to the plasma membrane requires phosphatidylinositol 3-kinase activation-mediated generation of phosphatidylinositol 3,4,5-trisphosphate PIP(3) and subsequent activation of Akt. Previous studies suggested that skeletal muscle and kidney enriched inositol polyphosphate phosphatase (SKIP) has negative effects on the regulation of insulin signaling in the skeletal muscle cells. Here, we compared its effects on insulin signaling by selective inhibition of SKIP, SHIP2, and phosphatase and tensin homologue on chromosome 10 (PTEN) by short interfering RNA in the C2C12 myoblast cells. Suppression of SKIP significantly increased the insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate levels and Akt phosphorylation. Furthermore, silencing of SKIP, but not of PTEN, increased the insulin-dependent recruitment of GLUT4 vesicles to the plasma membrane. Taken together, these results imply that SKIP negatively regulates insulin signaling and glucose uptake by inhibiting GLUT4 docking and/or fusion to the plasma membrane.     

Inhibition of IRS-1 phosphorylation and the alterations of GLUT4 in isolated adipocytes from cachectic tumor-bearing rats

Cellular and molecular mechanisms of insulin resistance in isolated adipocytes from methylcholanthrene-induced sarcoma-bearing rats were investigated by measuring 3-O-[14C]methyl glucose transport activity, glucose transporter-4 (GLUT4) protein in both plasma membrane and low-density microsomes, and insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate-1 (IRS-1). Compared to both pair-fed and freely fed controls, tumor-bearing rats (TBR) had a decreased insulin-stimulated glucose transport activity with a lower Vmax and a higher EC50. GLUT4 protein in low-density microsomes from adipocytes maintained at the basal state was less in TBR than in controls. In insulin-stimulated adipocytes, GLUT4 protein in plasma membranes was also less in tumor-bearing rats than in controls. Insulin-induced tyrosine phosphorylation of IRS-1 was less in TBR than controls, but that of the IR was similar among the three groups. These data suggest that the insulin resistance seen in adipose cells of these tumor-bearing rats was caused in part by a decreased amount of GLUT4 protein in both basal and insulin-stimulated states resulting from the selective inhibition of insulin-stimulated phosphorylation of IRS-1.     

Akt mediates insulin induction of glucose uptake and up-regulation of GLUT4 gene expression in brown adipocytes

Insulin acutely stimulated glucose uptake in rat primary brown adipocytes in a PI3-kinase-dependent but p70S6-kinase-independent manner. Since Akt represents an intermediate step between these kinases, this study investigated the contribution of Akt to insulin-induced glucose uptake by the use of a chemical compound, ML-9, as well as by transfection with a dominant-negative form of Akt (DeltaAkt). Pretreatment with ML-9 for 10 min completely inhibited insulin stimulation of (1) Akt kinase activity, (2) Akt phosphorylation on the regulatory residue Ser473 but not on Thr308, and (3) mobility shift in Akt1 and Akt2. However, ML-9 did not affect insulin-stimulated PI3-kinase nor PKCzeta activities. In consequence, ML-9 precluded insulin stimulation of glucose uptake and GLUT4 translocation to plasma membrane (determined by Western blot), without any effect on the basal glucose uptake. Moreover, DeltaAkt impaired insulin stimulation of glucose uptake and GFP-tagged GLUT4 translocation to plasma membrane in transiently transfected immortalised brown adipocytes and HeLa cells, respectively. Furthermore, ML-9 treatment for 6 h down-regulated insulin-induced GLUT4 mRNA accumulation, without affecting GLUT1 expression, in a similar fashion as LY294002. Indeed, co-transfection of brown adipocytes with DeltaAkt precluded the transactivation of GLUT4-CAT promoter by insulin in a similar fashion as a dominant-negative form of PI3-kinase. Our results indicate that activation of Akt may be an essential requirement for insulin regulation of glucose uptake and GLUT4 gene expression in brown adipocytes.     

PACSIN3 overexpression increases adipocyte glucose transport through GLUT1

PACSIN family members regulate intracellular vesicle trafficking via their ability to regulate cytoskeletal rearrangement. These processes are known to be involved in trafficking of GLUT1 and GLUT4 in adipocytes. In this study, PACSIN3 was observed to be the only PACSIN isoform that increases in expression during 3T3-L1 adipocyte differentiation. Overexpression of PACSIN3 in 3T3-L1 adipocytes caused an elevation of glucose uptake. Subcellular fractionation revealed that PACSIN3 overexpression elevated GLUT1 plasma membrane localization without effecting GLUT4 distribution. In agreement with this result, examination of GLUT exofacial presentation at the cell surface by photoaffinity labeling revealed significantly increased GLUT1, but not GLUT4, after overexpression of PACSIN3. These results establish a role for PACSIN3 in regulating glucose uptake in adipocytes via its preferential participation in GLUT1 trafficking. They are consistent with the proposal, which is supported by a recent study, that GLUT1, but not GLUT4, is predominantly endocytosed via the coated pit pathway in unstimulated 3T3-L1 adipocytes.     

NCS-1 inhibits insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes through a phosphatidylinositol 4-kinase-dependent pathway

Expression of NCS-1 (neuronal calcium sensor-1, also termed frequenin) in 3T3L1 adipocytes strongly inhibited insulin-stimulated translocation of GLUT4 and insulin-responsive aminopeptidase. The effect of NCS-1 was specific for GLUT4 and the insulin-responsive aminopeptidase translocation as there was no effect on the trafficking of the cation-independent mannose 6-phosphate receptor or the GLUT1 glucose transporter isoform. Moreover, NCS-1 showed partial colocalization with GLUT4-EGFP in the perinuclear region. The inhibitory action of NCS-1 was independent of calcium sequestration since neither treatment with ionomycin nor endothelin-1, both of which elevated the intracellular calcium concentration, restored insulin-stimulated GLUT4 translocation. Furthermore, NCS-1 did not alter the insulin-stimulated protein kinase B (PKB/Akt) phosphorylation or the recruitment of Cbl to the plasma membrane. In contrast, expression of the NCS-1 effector phosphatidylinositol 4-kinase (PI 4-kinase) inhibited insulin-stimulated GLUT4 translocation, whereas co-transfection with an inactive PI 4-kinase mutant prevented the NCS-1-induced inhibition. These data demonstrate that PI 4-kinase functions to negatively regulate GLUT4 translocation through its interaction with NCS-1.     

A pre-docking role for microtubules in insulin-stimulated glucose transporter 4 translocation

Insulin stimulates glucose uptake by inducing translocation of glucose transporter 4 (GLUT4) from intracellular resides to the plasma membrane. How GLUT4 storage vesicles are translocated from the cellular interior to the plasma membrane remains to be elucidated. In the present study, intracellular transport of GLUT4 storage vesicles and the kinetics of their docking at the plasma membrane were comprehensively investigated at single vesicle level in control and microtubule-disrupted 3T3-L1 adipocytes by time-lapse total internal reflection fluorescence microscopy. It is demonstrated that microtubule disruption substantially inhibited insulin-stimulated GLUT4 translocation. Detailed analysis reveals that microtubule disruption blocked the recruitment of GLUT4 storage vesicles to underneath the plasma membrane and abolished the docking of them at the plasma membrane. These data suggest that transport of GLUT4 storage vesicles to the plasma membrane takes place along microtubules and that this transport is obligatory for insulin-stimulated GLUT4 translocation.     

Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase

The glucose transporter GLUT4 and the aminopeptidase IRAP (insulin-responsive aminopeptidase) are the major cargo proteins of GSVs (GLUT4 storage vesicles) in adipocytes and myocytes. In the basal state, most GSVs are sequestered in perinuclear and other cytosolic compartments. Following insulin stimulation, GSVs undergo exocytic translocation to insert GLUT4 and IRAP into the plasma membrane. The mechanisms regulating GSV trafficking are not fully defined. In the present study, using 3T3-L1 adipocytes transfected with siRNAs (small interfering RNAs), we show that insulin-stimulated IRAP translocation remained intact despite substantial GLUT4 knockdown. By contrast, insulin-stimulated GLUT4 translocation was impaired upon IRAP knockdown, indicating that IRAP plays a role in GSV trafficking. We also show that knockdown of tankyrase, a Golgi-associated IRAP-binding protein that co-localizes with perinuclear GSVs, attenuated insulin-stimulated GSV translocation and glucose uptake without disrupting insulin-induced phosphorylation cascades. Moreover, iodixanol density gradient analyses revealed that tankyrase knockdown altered the basal-state partitioning of GLUT4 and IRAP within endosomal compartments, apparently by shifting both proteins toward less buoyant compartments. Importantly, the afore-mentioned effects of tankyrase knockdown were reproduced by treating adipocytes with PJ34, a general PARP (poly-ADP-ribose polymerase) inhibitor that abrogated tankyrase-mediated protein modification known as poly-ADP-ribosylation. Collectively, these findings suggest that physiological GSV trafficking depends in part on the presence of IRAP in these vesicles, and that this process is regulated by tankyrase and probably its PARP activity.     

Dimethyl sulfoxide enhances GLUT4 translocation through a reduction in GLUT4 endocytosis in insulin-stimulated 3T3-L1 adipocytes

Insulin increases muscle and fat cell glucose uptake by inducing the translocation of glucose transporter GLUT4 from intracellular compartments to the plasma membrane. Here, we have demonstrated that in 3T3-L1 adipocytes, DMSO at concentrations higher than 7.5% augmented cell surface GLUT4 levels in the absence and presence of insulin, but that at lower concentrations, DMSO only enhanced GLUT4 levels in insulin-stimulated cells. At a 5% concentration, DMSO also increased cell surface levels of the transferrin receptor and GLUT1. Glucose uptake experiments indicated that while DMSO enhanced cell surface glucose transporter levels, it also inhibited glucose transporter activity. Our studies further demonstrated that DMSO did not sensitize the adipocytes for insulin and that its effect on GLUT4 was readily reversible (t1/2∼12 min) and maintained in insulin-resistant adipocytes. An enhancement of insulin-induced GLUT4 translocation was not observed in 3T3-L1 preadipocytes and L6 myotubes, indicating cell specificity. DMSO did not enhance insulin signaling nor exocytosis of GLUT4 vesicles, but inhibited GLUT4 internalization. While other chemical chaperones (glycerol and 4-phenyl butyric acid) also acutely enhanced insulin-induced GLUT4 translocation, these effects were not mediated via changes in GLUT4 endocytosis. We conclude that DMSO is the first molecule to be described that instantaneously enhances insulin-induced increases in cell surface GLUT4 levels in adipocytes, at least in part through a reduction in GLUT4 endocytosis.