The insulin-sensitive glucose transporter, GLUT4, interacts physically with Daxx. Two proteins with capacity to bind Ubc9 and conjugated to SUMO1

In this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxyl-cytoplasmic domain (residues 464-509) of the insulin-sensitive glucose transporter GLUT4 (C-GLUT4). Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-beta (TbetaRII) receptors (1,2 ). The two-hybrid interaction between C-GLUT4 and C-Daxx is validated by the ability of in vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx. C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4. Replacement of alanine and serine for the dileucine pair (Leu(489)-Leu(490)) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx. Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins. Similarly, GLUT4 is recovered with anti-Daxx immunoprecipitates. Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes. Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane. Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4.     

Insulin-induced GLUT4 translocation involves protein kinase C-lambda-mediated functional coupling between Rab4 and the motor protein kinesin

Insulin stimulates glucose transport by promoting translocation of GLUT4 proteins from the perinuclear compartment to the cell surface. It has been previously suggested that the microtubule-associated motor protein kinesin, which transports cargo toward the plus end of microtubules, plays a role in translocating GLUT4 vesicles to the cell surface. In this study, we investigated the role of Rab4, a small GTPase-binding protein, and the motor protein KIF3 (kinesin II in mice) in insulin-induced GLUT4 exocytosis in 3T3-L1 adipocytes. Photoaffinity labeling of Rab4 with [gamma-(32)P]GTP-azidoanilide showed that insulin stimulated Rab4 GTP loading and that this insulin effect was inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 or expression of dominant-negative protein kinase C-lambda (PKC-lambda). Consistent with previous reports, expression of dominant-negative Rab4 (N121I) decreased insulin-induced GLUT4 translocation by 45%. Microinjection of an anti-KIF3 antibody into 3T3-L1 adipocytes decreased insulin-induced GLUT4 exocytosis by 65% but had no effect on endocytosis. Coimmunoprecipitation experiments showed that Rab4, but not Rab5, physically associated with KIF3, and this was confirmed by showing in vitro association using glutathione S-transferase-Rab4. A microtubule capture assay demonstrated that insulin stimulation increased the activity for the binding of KIF3 to microtubules and that this activation was inhibited by pretreatment with the PI3-kinase inhibitor LY294002 or expression of dominant-negative PKC-lambda. Taken together, these data indicate that (i) insulin signaling stimulates Rab4 activity, the association of Rab4 with kinesin, and the interaction of KIF3 with microtubules and (ii) this process is mediated by insulin-induced PI3-kinase-dependent PKC-lambda activation and participates in GLUT4 exocytosis in 3T3-L1 adipocytes.     

A role for kinesin in insulin-stimulated GLUT4 glucose transporter translocation in 3T3-L1 adipocytes

Insulin regulates glucose uptake in adipocytes and muscle by stimulating the movement of sequestered glucose transporter 4 (GLUT4) proteins from intracellular membranes to the cell surface. Here we report that optimal insulin-mediated GLUT4 translocation is dependent upon both microtubule and actin-based cytoskeletal structures in cultured adipocytes. Depolymerization of microtubules and F-actin in 3T3-L1 adipocytes causes the dispersion of perinuclear GLUT4-containing membranes and abolishes insulin action on GLUT4 movements to the plasma membrane. Furthermore, heterologous expression in 3T3-L1 adipocytes of the microtubule-binding protein hTau40, which impairs kinesin motors that move toward the plus ends of microtubules, markedly delayed the appearance of GLUT4 at the plasma membrane in response to insulin. The hTau40 protein had no detectable effect on microtubule structure or perinuclear GLUT4 localization under these conditions. These results are consistent with the hypothesis that both the actin and microtubule-based cytoskeleton, as well as a kinesin motor, direct the translocation of GLUT4 to the plasma membrane in response to insulin.     

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.     

A dominant-negative p38 MAPK mutant and novel selective inhibitors of p38 MAPK reduce insulin-stimulated glucose uptake in 3T3-L1 adipocytes without affecting GLUT4 translocation

Participation of p38 mitogen-activated protein kinase (p38) in insulin-induced glucose uptake was suggested using pyridinylimidazole p38 inhibitors (e.g. SB203580). However, the role of p38 in insulin action remains controversial. We further test p38 participation in glucose uptake using a dominant-negative p38 mutant and two novel pharmacological p38 inhibitors related to but different from SB203580. We present the structures and activities of the azaazulene pharmacophores A291077 and A304000. p38 kinase activity was inhibited in vitro by A291077 and A304000 (IC(50) = 0.6 and 4.7 microm). At higher concentrations A291077 but not A304000 inhibited JNK2alpha (IC(50) = 3.5 microm). Pretreatment of 3T3-L1 adipocytes and L6 myotubes expressing GLUT4myc (L6-GLUT4myc myotubes) with A291077, A304000, SB202190, or SB203580 reduced insulin-stimulated glucose uptake by 50-60%, whereas chemical analogues inert toward p38 were ineffective. Expression of an inducible, dominant-negative p38 mutant in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. GLUT4 translocation to the cell surface, immunodetected on plasma membrane lawns of 3T3-L1 adipocytes or on intact L6-GLUT4myc myotubes, was not altered by chemical or molecular inhibition of p38. We propose that p38 contributes to enhancing GLUT4 activity, thereby increasing glucose uptake. In addition, the azaazulene class of inhibitors described will be useful to decipher cellular actions of p38 and JNK.     

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.     

PYK2 as a mediator of endothelin-1/G alpha 11 signaling to GLUT4 glucose transporters

Endothelin-1 (ET-1) signaling through G alpha(q/11) stimulates translocation of intracellular GLUT4 glucose transporters to the plasma membrane of 3T3-L1 adipocytes by an unknown mechanism that requires protein tyrosine phosphorylation and ADP-ribosylation factor 6 (ARF6) but is independent of phosphatidylinositol 3 (PI3)-kinase. In contrast, insulin action on this process requires PI3-kinase but not ARF6. Here we report the identification of two proteins selectively tyrosine-phosphorylated in response to ET-1 but not insulin: the Ca(2+)-activated tyrosine kinase PYK2 and its physiological substrate, the adhesion scaffold protein paxillin. Endogenous paxillin as well as expressed Myc-tagged PYK2 or a Myc-tagged kinase-deficient PYK2 protein were acutely directed to F-actin-rich adhesion sites from the adipocyte cytoplasm in response to ET-1 but not insulin. CADTK-related non-kinase (CRNK) is a dominant negative form of PYK2 containing the C-terminal portion of the protein, which binds paxillin but lacks the PYK2 autophosphorylation site (Tyr(402)). CRNK expression in 3T3-L1 adipocytes inhibited ET-1-mediated F-actin polymerization and translocation of Myc-tagged GLUT4-enhanced green fluorescent protein (EGFP) to the plasma membrane without disrupting insulin action on these processes. These data reveal the tyrosine kinase PYK2 as a required signaling element in the regulation of GLUT4 recycling in 3T3-L1 adipocytes by ET-1, whereas insulin signaling is directed through a different pathway.     

Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr(590). RNAi (RNA interference)-mediated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxyglucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr(389), a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway.     

Role of SNAP23 in insulin-induced translocation of GLUT4 in 3T3-L1 adipocytes. Mediation of complex formation between syntaxin4 and VAMP2

Both syntaxin4 and VAMP2 are implicated in insulin regulation of glucose transporter-4 (GLUT4) trafficking in adipocytes as target (t) soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and vesicle (v)-SNARE proteins, respectively, which mediate fusion of GLUT4-containing vesicles with the plasma membrane. Synaptosome-associated 23-kDa protein (SNAP23) is a widely expressed isoform of SNAP25, the principal t-SNARE of neuronal cells, and colocalizes with syntaxin4 in the plasma membrane of 3T3-L1 adipocytes. In the present study, two SNAP23 mutants, SNAP23-DeltaC8 (amino acids 1 to 202) and SNAP23-DeltaC49 (amino acids 1 to 161), were generated to determine whether SNAP23 is required for insulin-induced translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. Wild-type SNAP23 (SNAP23-WT) promoted the interaction between syntaxin4 and VAMP2 both in vitro and in vivo. Although SNAP23-DeltaC49 bound to neither syntaxin4 nor VAMP2, the SNAP23-DeltaC8 mutant bound to syntaxin4 but not to VAMP2. In addition, although SNAP23-DeltaC8 bound to syntaxin4, it did not mediate the interaction between syntaxin4 and VAMP2. Moreover, overexpression of SNAP23-DeltaC8 in 3T3-L1 adipocytes by adenovirus-mediated gene transfer inhibited insulin-induced translocation of GLUT4 but not that of GLUT1. In contrast, overexpression of neither SNAP23-WT nor SNAP23-DeltaC49 in 3T3-L1 adipocytes affected the translocation of GLUT4 or GLUT1. Together, these results demonstrate that SNAP23 contributes to insulin-dependent trafficking of GLUT4 to the plasma membrane in 3T3-L1 adipocytes by mediating the interaction between t-SNARE (syntaxin4) and v-SNARE (VAMP2).     

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.     

Perinuclear localization and insulin responsiveness of GLUT4 requires cytoskeletal integrity in 3T3-L1 adipocytes

The GLUT4 glucose transporter resides mostly in perinuclear membranes in unstimulated 3T3-L1 adipocytes and is acutely translocated to the cell surface in response to insulin. Using a novel method to purify intracellular GLUT4-enriched membranes, we identified by mass spectrometry the intermediate filament protein vimentin and the microtubule protein alpha-tubulin as components of these membranes. Immunoelectron microscopy of the GLUT4-containing membranes also revealed their association with these cytoskeletal proteins. Disruption of intermediate filaments and microtubules in 3T3-L1 adipocytes by microinjection of a vimentin-derived peptide of the helix initiation 1A domain caused marked dispersion of perinuclear GLUT4 to peripheral regions of the cells. Inhibition of the microtubule-based motor dynein by brief cytoplasmic acidification of cultured adipocytes also dispersed perinuclear GLUT4 and inhibited insulin-stimulated GLUT4 translocation to the cell surface. Insulin sensitivity was restored as GLUT4 was again concentrated near the nucleus upon recovery of cells in physiological buffer. These data suggest that GLUT4 trafficking to perinuclear membranes of cultured adipocytes is directed by dynein and is required for optimal GLUT4 regulation by insulin.     

Transient effect of platelet-derived growth factor on GLUT4 translocation in 3T3-L1 adipocytes.

We earlier developed a novel method to detect translocation of the glucose transporter (GLUT) directly and simply using c-MYC epitope-tagged GLUT (GLUTMYC). To define the effect of platelet-derived growth factor (PDGF) on glucose transport in 3T3-L1 adipocytes, we investigated the PDGF- and insulin-induced glucose uptake, translocation of glucose transporters, and phosphatidylinositol (PI) 3-kinase activity in 3T3-L1, 3T3-L1GLUT4MYC, and 3T3-L1GLUT1MYC adipocytes. Insulin and PDGF stimulated glucose uptake by 9-10- and 5.5-6.5-fold, respectively, in both 3T3-L1 and 3T3-L1GLUT4MYC adipocytes. Exogenous GLUT4MYC expression led to enhanced PDGF-induced glucose transport. In 3T3-L1GLUT4MYC adipocytes, insulin and PDGF induced an 8- and 5-fold increase in GLUT4MYC translocation, respectively, determined in a cell-surface anti-c-MYC antibody binding assay. This PDGF-induced GLUT4MYC translocation was further demonstrated with fluorescent detection. In contrast, PDGF stimulated a 2-fold increase of GLUT1MYC translocation and 2.5-fold increase of glucose uptake in 3T3-L1GLUT1MYC adipocytes. The PDGF-induced GLUT4MYC translocation, glucose uptake, and PI 3-kinase activity were maximal (100%) at 5-10 min and thereafter rapidly declined to 40, 30, and 12%, respectively, within 60 min, a time when effects of insulin were maximal. Wortmannin (0.1 microM) abolished PDGF-induced GLUT4MYC translocation and glucose uptake in 3T3-L1GLUT4MYC adipocytes. These results suggest that PDGF can transiently trigger the translocation of GLUT4 and stimulate glucose uptake by translocation of both GLUT4 and GLUT1 in a PI 3-kinase-dependent signaling pathway in 3T3-L1 adipocytes.     

Activators of AMP-activated protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1 adipocytes

To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P < 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin- and AMPK-activated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P < 0.05). AMPK- and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P < 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P < 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism.     

High density lipoprotein (HDL) promotes glucose uptake in adipocytes and glycogen synthesis in muscle cells

Background

High density lipoprotein (HDL) was reported to decrease plasma glucose and promote insulin secretion in type 2 diabetes patients. This investigation was designed to determine the effects and mechanisms of HDL on glucose uptake in adipocytes and glycogen synthesis in muscle cells.

Methods and Results

Actions of HDL on glucose uptake and GLUT4 translocation were assessed with 1-[³H]-2-deoxyglucose and plasma membrane lawn, respectively, in 3T3-L1 adipocytes. Glycogen analysis was performed with amyloglucosidase and glucose oxidase-peroxidase methods in normal and palmitate-treated L6 cells. Small interfering RNA was used to observe role of scavenger receptor type I (SR-BI) in glucose uptake of HDL. Corresponding signaling molecules were detected by immunoblotting. HDL stimulated glucose uptake in a time- and concentration-dependent manner in 3T3-L1 adipocytes. GLUT4 translocation was significantly increased by HDL. Glycogen deposition got enhanced in L6 muscle cells paralleling with elevated glycogen synthase kinase3 (GSK3) phosphorylation. Meanwhile, increased phosphorylations of Akt-Ser473 and AMP activated protein kinase (AMPK) α were detected in 3T3-L1 adipocytes. Glucose uptake and Akt-Ser473 activation but not AMPK-α were diminished in SR-BI knock-down 3T3-L1 cells.

Conclusions

HDL stimulates glucose uptake in 3T3-L1 adipocytes through enhancing GLUT4 translocation by mechanisms involving PI3K/Akt via SR-BI and AMPK signaling pathways, and increases glycogen deposition in L6 muscle cells through promoting GSK3 phosphorylation.     

Inhibition of insulin sensitivity by free fatty acids requires activation of multiple serine kinases in 3T3-L1 adipocytes

Insulin receptor substrate (IRS) has been suggested as a molecular target of free fatty acids (FFAs) for insulin resistance. However, the signaling pathways by which FFAs lead to the inhibition of IRS function remain to be established. In this study, we explored the FFA-signaling pathway that contributes to serine phosphorylation and degradation of IRS-1 in adipocytes and in dietary obese mice. Linoleic acid, an FFA used in this study, resulted in a reduction in insulin-induced glucose uptake in 3T3-L1 adipocytes. This mimics insulin resistance induced by high-fat diet in C57BL/6J mice. The reduction in glucose uptake is associated with a decrease in IRS-1, but not IRS-2 or GLUT4 protein abundance. Decrease in IRS-1 protein was proceeded by IRS-1 (serine 307) phosphorylation that was catalyzed by serine kinases inhibitor kappaB kinase (IKK) and c-JUN NH2-terminal kinase (JNK). IKK and JNK were activated by linoleic acid and inhibition of the two kinases led to prevention of IRS-1 reduction. We demonstrate that protein kinase C (PKC) theta is expressed in adipocytes. In 3T3-L1 adipocytes and fat tissue, PKCtheta was activated by fatty acids as indicated by its phosphorylation status, and by its protein level, respectively. Activation of PKCtheta contributes to IKK and JNK activation as inhibition of PKCtheta by calphostin C blocked activation of the latter kinases. Inhibition of either PKCtheta or IKK plus JNK by chemical inhibitors resulted in protection of IRS-1 function and insulin sensitivity in 3T3-L1 adipocytes. These data suggest that: 1) activation of PKCtheta contributes to IKK and JNK activation by FFAs; 2) IKK and JNK mediate PKCtheta signals for IRS-1 serine phosphorylation and degradation; and 3) this molecular mechanism may be responsible for insulin resistance associated with hyperlipidemia.     

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.     

Prolonged Insulin Stimulation Down-regulates GLUT4 through Oxidative Stress-mediated Retromer Inhibition by a Protein Kinase CK2-dependent Mechanism in 3T3-L1 Adipocytes

Although insulin acutely stimulates glucose uptake by promotion of GLUT4 translocation from intracellular compartments to the plasma membrane in adipocytes and muscles, long term insulin stimulation causes GLUT4 depletion that is particularly prominent in the insulin-responsive GLUT4 storage compartment. This effect is caused mainly by accelerated lysosomal degradation of GLUT4, although the mechanism is not fully defined. Here we show that insulin acutely induced dissociation of retromer components from the low density microsomal membranes of 3T3-L1 adipocytes that was accompanied by disruption of the interaction of Vps35 with sortilin. This insulin effect was dependent on the activity of protein kinase CK2 but not phosphatidylinositol 3-kinase or extracellular signal-regulated kinase 1/2. Knockdown of Vps26 decreased GLUT4 to a level comparable with that with insulin stimulation for 4 h. Vps35 with a mutation in the CK2 phosphorylation motif (Vps35-S7A) was resistant to insulin-induced dissociation from the low density microsomal membrane, and its overexpression attenuated GLUT4 down-regulation with insulin. Furthermore, insulin-generated hydrogen peroxide was an upstream mediator of the insulin action on retromer and GLUT4. These results suggested that insulin-generated oxidative stress switches the GLUT4 sorting direction to lysosomes through inhibition of the retromer function in a CK2-dependent manner.     

Role of EHD1 and EHBP1 in perinuclear sorting and insulin-regulated GLUT4 recycling in 3T3-L1 adipocytes

Insulin stimulates glucose transport in muscle and adipose tissues by recruiting intracellular membrane vesicles containing the glucose transporter GLUT4 to the plasma membrane. The mechanisms involved in the biogenesis of these vesicles and their translocation to the cell surface are poorly understood. Here, we report that an Eps15 homology (EH) domain-containing protein, EHD1, controls the normal perinuclear localization of GLUT4-containing membranes and is required for insulin-stimulated recycling of these membranes in cultured adipocytes. EHD1 is a member of a family of four closely related proteins (EHD1, EHD2, EHD3, and EHD4), which also contain a P-loop near the N terminus and a central coiled-coil domain. Analysis of cultured adipocytes stained with anti-GLUT4, anti-EHD1, and anti-EHD2 antibodies revealed that EHD1, but not EHD2, partially co-localizes with perinuclear GLUT4. Expression of a dominant-negative construct of EHD1 missing the EH domain (DeltaEH-EHD1) markedly enlarged endosomes, dispersed perinuclear GLUT4-containing membranes throughout the cytoplasm, and inhibited GLUT4 translocation to the plasma membranes of 3T3-L1 adipocytes stimulated with insulin. Similarly, small interfering RNA-mediated depletion of endogenous EHD1 protein also markedly dispersed perinuclear GLUT4 in cultured adipocytes. Moreover, EHD1 is shown to interact through its EH domain with the protein EHBP1, which is also required for insulin-stimulated GLUT4 movements and hexose transport. In contrast, disruption of EHD2 function was without effect on GLUT4 localization or translocation to the plasma membrane. Taken together, these results show that EHD1 and EHBP1, but not EHD2, are required for perinuclear localization of GLUT4 and reveal that loss of EHBP1 disrupts insulin-regulated GLUT4 recycling in cultured adipocytes.     

灵芝多糖对3T3-L1胰岛素抵抗细胞模型PI-3K p85和GLUT4蛋白表达的影响

目的 研究灵芝多糖对3T3-L1胰岛素抵抗细胞模型PI-3K p85和GLUT4蛋白表达的影响,探讨灵芝多糖改善胰岛素抵抗的分子机制.方法 3T3-L1前脂肪细胞经1-甲基-3-异丁基-黄嘌呤、地塞米松、胰岛素诱导分化成3T3-L1脂肪细胞,以葡萄糖氧化酶法测定培养液中残余的葡萄糖含量.比较二甲双胍组,检测培养液中葡萄糖含量及PI-3K p85和GLUT4蛋白表达变化.结果 地塞米松联合胰岛素诱导3T3-L1脂肪细胞产生胰岛素抵抗,细胞对葡萄糖的摄取量减少.灵芝多糖可改善3T3-L1脂肪细胞胰岛素抵抗.胰岛素抵抗细胞的PI-3K p85和GLUT4蛋白表达明显减少;应用灵芝多糖后,相关蛋白表达增加.结论 灵芝多糖通过提高PI-3K p85和GLUT4蛋白的表达,参与胰岛素抵抗状态下3T3-L1细胞的葡萄糖代谢.     

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.