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.     

Synip phosphorylation does not regulate insulin-stimulated GLUT4 translocation

Insulin causes the rapid translocation of the glucose transporter GLUT4 from intracellular sites to the plasma membrane in fat and muscle cells. There is considerable evidence that the signaling to this trafficking process is downstream of the insulin-activated protein kinase Akt. One Akt substrate that connects signaling to trafficking is a 160 kDa GTPase activating protein for Rabs. Another potential connecting substrate is the protein Synip, which associates with the SNARE syntaxin4. A recent study presents evidence that Akt phosphorylates Synip on serine 99, at least in vitro, and proposes that this phosphorylation enables GLUT4 translocation by causing the dissociation of Synip from syntaxin4. In the present study we show that marked overexpression of Synip mutant S99A, which lacks this phosphorylation site, has no effect on insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. This finding is strong evidence that phosphorylation of Synip on serine 99 is not required for GLUT4 translocation.     

Syntaxin4 interacting protein (synip) binds phosphatidylinositol (3,4,5) triphosphate

The insulin responsive Glut4 transport vesicles contain the v-SNARE protein Vamp2 that associate with the plasma membrane t-SNARE protein Syntaxin 4 to drive insulin-stimulated Glut4 translocation in skeletal muscle and adipocytes. The syntaxin 4 interacting protein (Synip) binds to syntaxin 4 in the basal state and dissociates in the insulin-stimulated state allowing for the subsequent binding of Vamp2 containing Glut4 vesicles and fusion with the plasma membrane. In this study, we have found that Synip binds phosphatidylinositol 3,4,5-triphosphate (PIP3), but not phosphatidylinositol 3 phosphate (PIP) or phosphatidylinositol 3,4-biphosphate (PIP2) through the Synip WW domain as deletion of this domain (Synip ΔWW) failed to bind PIP3. Over-expressed Synip ΔWW in 3T3L1 adipocytes reduced the basal levels of Glut4 at the plasma membrane with no effect on the binding to syntaxin 4 in vitro. Subcellular fractionation demonstrated that the amount of Synip ΔWW at the PM was decreased in response to insulin in 3T3L1 adipocytes whereas the amount of Synip WT increased. These data suggest that in the presence of insulin, the dissociated Synip remains anchored to the plasma membrane by binding to PIP3.     

The Association of ClipR-59 Protein with AS160 Modulates AS160 Protein Phosphorylation and Adipocyte Glut4 Protein Membrane Translocation

ClipR-59 is a membrane-associated protein and has been implicated in membrane signaling and vesicle trafficking. Recently, we have identified ClipR-59 as an Akt-interacting protein, and we have found that, by interacting with Akt, ClipR-59 modulates Akt subcellular compartmentalization and Akt substrate AS160 phosphorylation, thereby promoting Glut4 membrane translocation. Here, we have further investigated the regulatory effects of ClipR-59 on AS160 phosphorylation and subsequent adipocyte glucose transport. Our data showed that ClipR-59 interacted with AS160, which was mediated by the ankyrin repeats of ClipR-59 and regulated by insulin signaling. Moreover, the data also demonstrated that the interaction of ClipR-59 with AS160 was required for ClipR-59 to modulate Glut4 membrane translocation as ΔANK-ClipR-59, an AS160 interaction-defective mutant, failed to promote AS160 phosphorylation, Glut4 membrane translocation, and glucose transport induced by insulin in 3T3-L1 adipocytes. Because ClipR-59 also interacts with Akt and enhances the interaction between Akt and AS160, we suggest that ClipR-59 functions as a scaffold protein to facilitate Akt-mediated AS160 phosphorylation, thereby regulating glucose transport.     

The interaction of Akt with APPL1 is required for insulin-stimulated Glut4 translocation

APPL1 (adaptor protein containing PH domain, PTB domain, and leucine zipper motif 1) is an Akt/protein kinase B-binding protein involved in signal transduction and membrane trafficking pathways for various receptors, including receptor tyrosine kinases. Here, we establish a role for APPL1 in insulin signaling in which we demonstrate its interaction with Akt2 by co-immunoprecipitation and pulldown assays. In primary rat adipocytes and skeletal muscle, APPL1 and Akt2 formed a complex that was dissociated upon insulin stimulation in both tissues. To investigate possible APPL1 function in adipocytes, we analyzed Akt phosphorylation, 2-deoxyglucose uptake, and Glut4 translocation by immunofluorescence following APPL1 knockdown by small interfering and short hairpin RNAs. We show that APPL1 knockdown suppressed Akt phosphorylation, glucose uptake, and Glut4 translocation. We also tested the effect in 3T3-L1 adipocytes of expressing full-length APPL1 or an N- or a C-terminal APPL1 construct. Interestingly, expression of full-length APPL1 and its N terminus suppressed insulin-stimulated 2-deoxyglucose uptake and Glut4 translocation to roughly the same extent (40-60%). We confirmed by cellular fractionation that Glut4 translocation was substantially blocked in 3T3-L1 adipocytes transfected with full-length APPL1. By cellular fractionation, APPL1 was localized mainly in the cytosol, and it showed a small degree of re-localization to the light microsomes and nucleus in response to insulin. By immunofluorescence, we also show that APPL1 partially co-localized with Glut4. These data suggest that APPL1 plays an important role in insulin-stimulated Glut4 translocation in muscle and adipose tissues and that its N-terminal portion may be critical for APPL1 function.     

Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation

Insulin stimulates the rapid translocation of intracellular glucose transporters of the GLUT4 isotype to the plasma membrane in fat and muscle cells. The connections between known insulin signaling pathways and the protein machinery of this membrane-trafficking process have not been fully defined. Recently, we identified a 160-kDa protein in adipocytes, designated AS160, that is phosphorylated by the insulin-activated kinase Akt. This protein contains a GTPase-activating domain (GAP) for Rabs, which are small G proteins required for membrane trafficking. In the present study we have identified six sites of in vivo phosphorylation on AS160. These sites lie in the motif characteristic of Akt phosphorylation, and insulin treatment increased phosphorylation at five of the sites. Expression of AS160 with two or more of these sites mutated to alanine markedly inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Moreover, this inhibition did not occur when the GAP function in the phosphorylation site mutant was inactivated by a point mutation. These findings strongly indicate that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation and that this phosphorylation signals translocation through inactivation of the Rab GAP function.     

Myosin 5a is an insulin-stimulated Akt2 (protein kinase Bbeta) substrate modulating GLUT4 vesicle translocation

Phosphatidylinositol 3-kinase activation of Akt signaling is critical to insulin-stimulated glucose transport and GLUT4 translocation. However, the downstream signaling events following Akt activation which mediate glucose transport stimulation remain relatively unknown. Here we identify an Akt consensus phosphorylation motif in the actin-based motor protein myosin 5a and show that insulin stimulation leads to phosphorylation of myosin 5a at serine 1650. This Akt-mediated phosphorylation event enhances the ability of myosin 5a to interact with the actin cytoskeleton. Small interfering RNA-induced inhibition of myosin 5a and expression of dominant-negative myosin 5a attenuate insulin-stimulated glucose transport and GLUT4 translocation. Furthermore, knockdown of Akt2 or expression of dominant-negative Akt (DN-Akt) abolished insulin-stimulated phosphorylation of myosin 5a, inhibited myosin 5a binding to actin, and blocked insulin-stimulated glucose transport. Taken together, these data indicate that myosin 5a is a newly identified direct substrate of Akt2 and, upon insulin stimulation, phosphorylated myosin 5a facilitates anterograde movement of GLUT4 vesicles along actin to the cell surface.     

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.     

Identification of amino acid residues within the C terminus of the Glut4 glucose transporter that are essential for insulin-stimulated redistribution to the plasma membrane

The Glut4 glucose transporter undergoes complex insulin-regulated subcellular trafficking in adipocytes. Much effort has been expended in an attempt to identify targeting motifs within Glut4 that direct its subcellular trafficking, but an amino acid motif responsible for the targeting of the transporter to insulin-responsive intracellular compartments in the basal state or that is directly responsible for its insulin-stimulated redistribution to the plasma membrane has not yet been delineated. In this study we define amino acid residues within the C-terminal cytoplasmic tail of Glut4 that are essential for its insulin-stimulated translocation to the plasma membrane. The residues were identified based on sequence similarity (LXXLXPDEXD) between cytoplasmic domains of Glut4 and the insulin-responsive aminopeptidase (IRAP). Alteration of this putative targeting motif (IRM, insulin-responsive motif) resulted in the targeting of the bulk of the mutant Glut4 molecules to dispersed membrane vesicles that lacked detectable levels of wild-type Glut4 in either the basal or insulin-stimulated states and completely abolished the insulin-stimulated translocation of the mutant Glut4 to the plasma membrane in 3T3L1 adipocytes. The bulk of the dispersed membrane vesicles containing the IRM mutant did not contain detectable levels of any subcellular marker tested. A fraction of the total IRM mutant was also detected in a wild-type Glut4/Syntaxin 6-containing perinuclear compartment. Interestingly, mutation of the IRM sequence did not appreciably alter the subcellular trafficking of IRAP. We conclude that residues within the IRM are critical for the targeting of Glut4, but not of IRAP, to insulin-responsive intracellular membrane compartments in 3T3-L1 adipocytes.     

Regulation of PIKfyve phosphorylation by insulin and osmotic stress

PIKfyve is a protein and lipid kinase that plays an important role in membrane trafficking, including TGN to endosome retrograde sorting and in insulin-stimulated translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the plasma membrane. We have previously demonstrated that PIKfyve is phosphorylated in response to insulin in a PI3-kinase and protein kinase B (PKB)-dependent manner. However, it has been implied that this was not due to direct phosphorylation of PIKfyve by PKB, but as a result of an insulin-induced PIKfyve autophosphorylation event. Here we demonstrate that purified PIKfyve is phosphorylated in vitro by a recombinant active PKB on two separate serine residues, S318 and S105, which flank the N-terminal FYVE domain of the protein. Only S318, however, becomes phosphorylated in intact cells stimulated with insulin. We further demonstrate that S318 is phosphorylated in response to hyperosmotic stress in a PI3-kinase- and PKB-independent manner. Importantly, the effects of insulin and sorbitol were not prevented by the presence of an ATP-competitive PIKfyve inhibitor (YM20163) or in a mutant PIKfyve lacking both lipid and protein kinase activity. Our results confirm, therefore, that PIKfyve is directly phosphorylated by PKB on a single serine residue in response to insulin and are not due to autophosphorylation of the enzyme. We further reveal that two stimuli known to promote glucose uptake in cells, both stimulate phosphorylation of PIKfyve on S318 but via distinct signal transduction pathways.     

Essential role of insulin receptor substrate-2 in insulin stimulation of Glut4 translocation and glucose uptake in brown adipocytes

Insulin and insulin-like growth factor I signals are mediated via phosphorylation of a family of insulin receptor substrate (IRS) proteins, which may serve both complementary and overlapping functions in the cell. To study the metabolic effects of these proteins in more detail, we established brown adipocyte cell lines from wild type and various IRS knockout (KO) animals and characterized insulin action in these cells in vitro. Preadipocytes derived from both wild type and IRS-2 KO mice could be fully differentiated into mature brown adipocytes. In differentiated IRS-2 KO adipocytes, insulin-induced glucose uptake was decreased by 50% compared with their wild type counterparts. This was the result of a decrease in insulin-stimulated Glut4 translocation to the plasma membrane. This decrease in insulin-induced glucose uptake could be partially reconstituted in these cells by retrovirus-mediated re-expression of IRS-2, but not overexpression of IRS-1. Insulin signaling studies revealed a total loss of IRS-2-associated phosphatidylinositol (PI) 3-kinase activity and a reduction in phosphotyrosine-associated PI 3-kinase by 30% (p < 0.05) in the KO cells. The phosphorylation and activity of Akt, a major downstream effector of PI 3-kinase, as well as Akt-dependent phosphorylation of glycogen synthase kinase-3 and p70S6 kinase were not affected by the lack of IRS-2; however, there was a decrease in insulin stimulation of Akt associated with the plasma membrane. These results provide evidence for a critical role of IRS-2 as a mediator of insulin-stimulated Glut4 translocation and glucose uptake in adipocytes. This occurs without effects in differentiation, total activation of Akt and its downstream effectors, but may be caused by alterations in compartmentalization of these downstream signals.     

GRK2 is an endogenous protein inhibitor of the insulin signaling pathway for glucose transport stimulation

G protein-coupled receptor kinases (GRKs) represent a class of proteins that classically phosphorylate agonist-activated G protein-coupled receptors, leading to uncoupling of the receptor from further G protein activation. Recently, we have reported that the heterotrimeric G protein alpha-subunit, Galphaq/11, can mediate insulin-stimulated glucose transport. GRK2 contains a regulator of G protein signaling (RGS) domain with specificity for Galphaq/11. Therefore, we postulated that GRK2 could be an inhibitor of the insulin signaling cascade leading to glucose transport in 3T3-L1 adipocytes. In this study, we demonstrate that microinjection of anti-GRK2 antibody or siRNA against GRK2 increased insulin-stimulated insulin-responsive glucose transporter 4 (GLUT4) translocation, while adenovirus-mediated overexpression of wild-type or kinase-deficient GRK2 inhibited insulin-stimulated GLUT4 translocation as well as 2-deoxyglucose uptake. Importantly, a mutant GRK2 lacking the RGS domain was without effect. Taken together, these results indicate that through its RGS domain endogenous GRK2 functions as a negative regulator of insulin-stimulated glucose transport by interfering with Galphaq/11 signaling to GLUT4 translocation. Furthermore, inhibitors of GRK2 can lead to enhanced insulin sensitivity.     

Inhibition of GLUT4 translocation by Tbc1d1, a Rab GTPase-activating protein abundant in skeletal muscle, is partially relieved by AMP-activated protein kinase activation

Insulin increases glucose transport by stimulating the trafficking of intracellular GLUT4 to the cell surface, a process known as GLUT4 translocation. A key protein in signaling this process is AS160, a Rab GTPase-activating protein (GAP) whose activity appears to be suppressed by Akt phosphorylation. Tbc1d1 is a Rab GAP with a sequence highly similar to that of AS160 and with the same Rab specificity as that of AS160. The role of Tbc1d1 in regulating GLUT4 trafficking has been unclear. Our previous study showed that overexpressed Tbc1d1 inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes, even though insulin caused phosphorylation on its single canonical Akt motif. In the present study, we show in 3T3-L1 adipocytes that Tbc1d1 is only 1/20 as abundant as AS160, that knockdown of Tbc1d1 has no effect on insulin-stimulated GLUT4 translocation, and that overexpressed Tbc1d1 also inhibits GLUT4 translocation elicited by activated Akt expression. These results indicate that endogenous Tbc1d1 does not participate in insulin-regulated GLUT4 translocation in adipocytes and suggest that the GAP activity of Tbc1d1 is not suppressed by Akt phosphorylation. In addition, we discovered that Tbc1d1 is much more highly expressed in skeletal muscle than fat and that the AMP-activated protein kinase (AMPK) activator 5'-aminoimidazole-4-carboxamide ribonucleoside partially reversed the inhibition of insulin-stimulated GLUT4 translocation by overexpressed Tbc1d1 in 3T3-L1 adipocytes. 5'-Aminoimidazole-4-carboxamide ribonucleoside activation of the kinase AMPK is known to cause GLUT4 translocation in muscle. The above findings strongly suggest that Tbc1d1 is a component in the signal transduction pathway leading to AMPK-stimulated GLUT4 translocation in muscle.     

Neomycin prevents the wortmannin inhibition of insulin-stimulated Glut4 translocation and glucose transport in 3T3-L1 adipocytes

Insulin stimulates the movement of the facilitative glucose transporter glucose transporter-4 (Glut4) from an intracellular compartment to the plasma membrane in adipocytes and muscle cells, resulting in an increased rate of glucose uptake. Insulin-stimulated Glut4 translocation and glucose transport are abolished by wortmannin, a specific inhibitor of phosphatidylinositol 3'-kinase (PI3K). Here, we demonstrate that neomycin, a drug that masks the cellular substrate of PI3K, phosphatidylinositol 4,5-bisphosphate (PIP), prevents wortmannin inhibition of insulin-stimulated (2)Glut4 translocation and glucose transport without activating protein kinase B, a downstream effector of PI3K. These results suggest that PIP(2) may have an important regulatory function in insulin-stimulated Glut4 translocation and glucose transport.     

A Novel Pleckstrin Homology Domain-containing Protein Enhances Insulin-stimulated Akt Phosphorylation and GLUT4 Translocation in Adipocytes

Protein kinase B/Akt protein kinases control an array of diverse functions, including cell growth, survival, proliferation, and metabolism. We report here the identification of pleckstrin homology-like domain family B member 1 (PHLDB1) as an insulin-responsive protein that enhances Akt activation. PHLDB1 contains a pleckstrin homology domain, which we show binds phosphatidylinositol PI(3,4)P₂, PI(3,5)P₂, and PI(3,4,5)P₃, as well as a Forkhead-associated domain and coiled coil regions. PHLDB1 expression is increased during adipocyte differentiation, and it is abundant in many mouse tissues. Both endogenous and HA- or GFP-tagged PHLDB1 displayed a cytoplasmic disposition in unstimulated cultured adipocytes but translocated to the plasma membrane in response to insulin. Depletion of PHLDB1 by siRNA inhibited insulin stimulation of Akt phosphorylation but not tyrosine phosphorylation of IRS-1. RNAi-based silencing of PHLDB1 in cultured adipocytes also attenuated insulin-stimulated deoxyglucose transport and Myc-GLUT4-EGFP translocation to the plasma membrane, whereas knockdown of the PHLDB1 isoform PHLDB2 failed to attenuate insulin-stimulated deoxyglucose transport. Furthermore, adenovirus-mediated expression of PHLDB1 in adipocytes enhanced insulin-stimulated Akt and p70 S6 kinase phosphorylation, as well as GLUT4 translocation. These results indicate that PHLDB1 is a novel modulator of Akt protein kinase activation by insulin.     

A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes

Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.     

Arsenite-induced phosphorylation of histone H3 at serine 10 is mediated by Akt1, extracellular signal-regulated kinase 2, and p90 ribosomal S6 kinase 2 but not mitogen- and stress-activated protein kinase 1

Arsenite is known to be an environmental human carcinogen. However, the mechanism of action of this compound in skin carcinogenesis is not completely clear. Here, we provide evidence that arsenite can induce phosphorylation of histone H3 at serine 10 in a time- and dose-dependent manner in JB6 Cl 41 cells. Arsenite induces phosphorylation of Akt1 at serine 473 and increases Akt1 activity. A dominant-negative mutant of Akt1 inhibits the arsenite-induced phosphorylation of histone H3 at serine 10. Additionally, active Akt1 kinase strongly phosphorylates histone H3 at serine 10 in vitro. The arsenite-induced phosphorylation of histone H3 at serine 10 was almost completely blocked by a dominant-negative mutant of extracellular signal-regulated kinase 2 and the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor PD98059. N- or C-terminal mutant mitogen- and stress-activated protein kinase 1 or its inhibitor H89 had no effect on arsenite-induced phosphorylation of histone H3 at serine 10 in JB6 Cl 41 cells. However, cells deficient in p90 ribosomal S6 kinase 2 (Rsk2(-/-)) totally block this phosphorylation in a dose- and time-dependent manner. Taken together, these results suggested that arsenite-induced phosphorylation of histone H3 at serine 10 is mediated by Akt1, extracellular signal-regulated kinase 2 and p90 ribosomal S6 kinase 2 but not mitogen- and stress-activated protein kinase 1.     

C2 domain-containing phosphoprotein CDP138 regulates GLUT4 insertion into the plasma membrane

The protein kinase B(β) (Akt2) pathway is known to?mediate insulin-stimulated glucose transport through increasing glucose transporter GLUT4 translocation from intracellular stores to the plasma membrane (PM). Combining quantitative phosphoproteomics with RNAi-based functional analyses, we show that a previously uncharacterized 138?kDa C2 domain-containing phosphoprotein (CDP138) is a substrate for Akt2, and is required for optimal insulin-stimulated glucose transport, GLUT4 translocation, and fusion of GLUT4 vesicles with the PM in live adipocytes. The purified C2 domain is capable of binding Ca(2+) and lipid membranes. CDP138 mutants lacking the Ca(2+)-binding sites in the C2 domain or Akt2 phosphorylation site S197 inhibit insulin-stimulated GLUT4 insertion into the PM, a rate-limiting step of GLUT4 translocation. Interestingly, CDP138 is dynamically associated with the PM and GLUT4-containing vesicles in response to insulin stimulation. Together, these results suggest that CDP138 is a key molecule linking the Akt2 pathway to the regulation of GLUT4 vesicle-PM fusion.     

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.