Nitric oxide stimulates glucose transport through insulin-independent GLUT4 translocation in 3T3-L1 adipocytes

OBJECTIVE: It is well known that nitric oxide synthase (NOS) is expressed and that it modulates glucose transport in skeletal muscles. Recent studies have shown that adipose tIssues also express inducible and endothelial nitric oxide synthase (eNOS). In the present study, we investigated whether nitric oxide (NO) induces glucose uptake in adipocytes, and the signaling pathway involved in the NO-stimulated glucose uptake in 3T3-L1 adipocytes. METHODS: First, we determined the expression of eNOS in 3T3-L1 adipocytes, and then these cells were treated with the NO donor sodium nitroprusside (SNP) and/or insulin, and glucose uptake and phosphorylation of insulin receptor substrate (IRS)-1 and Akt were evaluated. Moreover, we examined the effects of a NO scavenger, a guanylate cyclase inhibitor or dexamethasone on SNP-stimulated glucose uptake and GLUT4 translocation. RESULTS: SNP at a concentration of 50 mmol/l increased 2-deoxyglucose uptake (1.8-fold) without phosphorylation of IRS-1 and Akt. Treatment with the NO scavenger or guanylate cyclase inhibitor decreased SNP-stimulated glucose uptake to the basal level. Dexamethasone reduced both insulin- and SNP-stimulated glucose uptake with impairment of GLUT4 translocation. CONCLUSION: NO is capable of stimulating glucose transport through GLUT4 translocation in 3T3-L1 adipocytes, via a mechanism different from the insulin signaling pathway.     

Ginsenoside Rb1 stimulates glucose uptake through insulin-like signaling pathway in 3T3-L1 adipocytes

A series of clinical trials and animal experiments have demonstrated that ginseng and its major active constituent, ginsenosides, possess glucose-lowering action. In our previous study, ginsenoside Rb(1) has been shown to regulate peroxisome proliferator-activated receptor gamma activity to facilitate adipogenesis of 3T3-L1 cells. However, the effect of Rb(1) on glucose transport in insulin-sensitive cells and its molecular mechanism need further elucidation. In this study, Rb(1) significantly stimulated basal and insulin-mediated glucose uptake in a time- and dose-dependent manner in 3T3-L1 adipocytes and C2C12 myotubes; the maximal effect was achieved at a concentration of 1 microM and a time of 3 h. In adipocytes, Rb(1) promoted GLUT1 and GLUT4 translocations to the cell surface, which was examined by analyzing their distribution in subcellular membrane fractions, and enhanced translocation of GLUT4 was confirmed using the transfection of GLUT4-green fluorescence protein in Chinese Hamster Ovary cells. Meanwhile, Rb(1) increased the phosphorylation of insulin receptor substrate-1 and protein kinase B (PKB), and stimulated phosphatidylinositol 3-kinase (PI3K) activity in the absence of the activation of the insulin receptor. Rb(1)-induced glucose uptake as well as GLUT1 and GLUT4 translocations was inhibited by the PI3K inhibitor. These results suggest that ginsenoside Rb(1) stimulates glucose transport in insulin-sensitive cells by promoting translocations of GLUT1 and GLUT4 by partially activating the insulin signaling pathway. These findings are useful in understanding the hypoglycemic and anti-diabetic properties of ginseng and ginsenosides.     

Activation of the mammalian target of rapamycin pathway acutely inhibits insulin signaling to Akt and glucose transport in 3T3-L1 and human adipocytes

The mammalian target of rapamycin (mTOR) pathway has recently emerged as a chronic modulator of insulin-mediated glucose metabolism. In this study, we evaluated the involvement of this pathway in the acute regulation of insulin action in both 3T3-L1 and human adipocytes. Insulin rapidly (t(1/2) = 5 min) stimulated the mTOR pathway, as reflected by a 10-fold stimulation of 70-kDa ribosomal S6 kinase 1 (S6K1) activity in 3T3-L1 adipocytes. Inhibition of mTOR/S6K1 by rapamycin increased insulin-stimulated glucose transport by as much as 45% in 3T3-L1 adipocytes. Activation of mTOR/S6K1 by insulin was associated with a rapamycin-sensitive increase in Ser636/639 phosphorylation of insulin receptor substrate (IRS)-1 but, surprisingly, did not result in impaired IRS-1-associated phosphatidylinositol (PI) 3-kinase activity. However, insulin-induced activation of Akt was increased by rapamycin. Insulin also activated S6K1 and increased phosphorylation of IRS-1 on Ser636/639 in human adipocytes. As in murine cells, rapamycin treatment of human adipocytes inhibited S6K1, blunted Ser636/639 phosphorylation of IRS-1, leading to increased Akt activation and glucose uptake by insulin. Further studies in 3T3-L1 adipocytes revealed that rapamycin prevented the relocalization of IRS-1 from the low-density membranes to the cytosol in response to insulin. Furthermore, inhibition of mTOR markedly potentiated the ability of insulin to increase PI 3,4,5-triphosphate levels concomitantly with an increased phosphorylation of Akt at the plasma membrane, low-density membranes, and cytosol. However, neither GLUT4 nor GLUT1 translocation induced by insulin were increased by rapamycin treatment. Taken together, these results indicate that the mTOR pathway is an important modulator of the signals involved in the acute regulation of insulin-stimulated glucose transport in 3T3-L1 and human adipocytes.     

The p38 mitogen-activated protein kinase inhibitor SB203580 reduces glucose turnover by the glucose transporter-4 of 3T3-L1 adipocytes in the insulin-stimulated state

Insulin induces a profound increase in glucose uptake in 3T3-L1 adipocytes through the activity of the glucose transporter-4 (GLUT4). Apart from GLUT4 translocation toward the plasma membrane, there is also an insulin-induced p38 MAPK-dependent step involved in the regulation of glucose uptake. Consequently, treatment with the p38 MAPK inhibitor SB203580 reduces insulin-induced glucose uptake by approximately 30%. Pretreatment with SB203580 does not alter the apparent K(m) of GLUT4-mediated glucose uptake but reduces the maximum velocity by approximately 30%. Insulin-induced GLUT4 translocation and exposure of the transporter to the extracellular environment was not altered by pretreatment with SB203580, as evidenced by a lack of effect of the inhibitor on the amount of GLUT4 present in the plasma membrane, as assessed by subcellular fractionation, the amount of GLUT4 that is able to undergo biotinylation on intact adipocytes and the level of extracellular exposure of an ectopically expressed GLUT-green fluorescence protein construct with a hemagglutinin tag in its first extracellular loop. In contrast, labeling of GLUT4 after insulin stimulation by a membrane-impermeable, mannose moiety-containing, photoaffinity-labeling agent [2-N-4(1-azido-2,2,2-trifluoroethyl)benzoyl-1,3-bis(d-mannose-4-yloxy)-2-propylamine] that binds to the extracellular glucose acceptor domain was markedly reduced by SB203580, although photolabeling with this compound in the absence of insulin was unaffected by SB203580. These data suggest that SB203580 affects glucose turnover by the insulin-responsive GLUT4 transporter in 3T3-L1 adipocytes.     

Requirement for PIKfyve enzymatic activity in acute and long-term insulin cellular effects

PIKfyve is a phosphoinositide 5-kinase that can also act as a protein kinase. PIKfyve's role in acute insulin action has been suggested on the basis of its association with the insulin stimulatable phosphatidylinositol-3-kinase and the ability of acute insulin to recruit and phosphorylate PIKfyve on intracellular membranes of 3T3-L1 adipocytes. Here we have examined several classical insulin-regulated long- and short-term responses in insulin-sensitive cells expressing high levels of either active PIKfyve or kinase-dead mutants with a dominant-negative effect. Up-regulation of PIKfyve protein expression was documented in the early stages of differentiation of cultured 3T3-L1 fibroblasts into adipocytes and a kinase-dead mutant, PIKfyveDeltaK, introduced into the preadipocyte stage profoundly delayed the hormone-induced adipogenesis. Next, insulin-induced mitogenesis was markedly inhibited in HEK293 stable cell lines, inducibly expressing the dominant-negative kinase-dead PIKfyve(K1831E) mutant but not in cells expressing PIKfyve(WT). Similarly, expression of the dominant negative mutants PIKfyve(K1831E) or PIKfyveDeltaK strongly inhibited insulin-stimulated translocation of GLUT4 in 3T3-L1 adipocytes, or GLUT1-mediated glucose uptake in Chinese hamster ovary T cells expressing the human insulin receptor. Expression of PIKfyveDeltaK and PIKfyve(WT) in Chinese hamster ovary T cells decreased or increased, respectively, insulin-stimulated Akt phosphorylation at Ser473 but not at Thr308. Furthermore, a powerful inhibition of PIKfyve was documented at a very low concentration (ID(50) = 6 micro M) of the cell-permeable kinase inhibitor curcumin. When introduced into 3T3-L1 adipocytes, curcumin markedly inhibited insulin-induced GLUT4 translocation and glucose transport. Together these data indicate that PIKfyve enzymatic activity functions as a positive regulatory intermediate in insulin acute and long-term biological responses and identify Ser473 in Akt as one potential PIKfyve downstream target.     

Indinavir uncovers different contributions of GLUT4 and GLUT1 towards glucose uptake in muscle and fat cells and tissues

AIMS/HYPOTHESIS: Insulin-dependent glucose influx in skeletal muscle and adipocytes is believed to rely largely on GLUT4, but this has not been confirmed directly. We assessed the relative functional contribution of GLUT4 in experimental models of skeletal muscle and adipocytes using the HIV-1 protease inhibitor indinavir. METHODS: Indinavir (up to 100 micro mol/l) was added to the glucose transport solution after insulin stimulation of wild-type L6 muscle cells, L6 cells over-expressing either GLUT4myc or GLUT1myc, 3T3-L1 adipocytes, isolated mouse brown or white adipocytes, and isolated mouse muscle preparations. RESULTS: 100 micro mol/l indinavir inhibited 80% of both basal and insulin-stimulated 2-deoxyglucose uptake in L6GLUT4myc myotubes and myoblasts, but only 25% in L6GLUT1myc cells. Cell-surface density of glucose transporters was not affected. In isolated soleus and extensor digitorum longus muscles, primary white and brown adipocytes, insulin-stimulated glucose uptake was inhibited 70 to 80% by indinavir. The effect of indinavir on glucose uptake was variable in 3T3-L1 adipocytes, averaging 45% and 67% inhibition of basal and maximally insulin-stimulated glucose uptake, respectively. In this cell, fractional inhibition of glucose uptake by indinavir correlated positively with the fold-stimulation of glucose uptake by insulin, and was higher with sub-maximal insulin concentrations. The latter finding coincided with an increase only in GLUT4, but not GLUT1, in plasma membrane lawns. CONCLUSION/INTERPRETATION: Indinavir is a useful tool to assess different functional contributions of GLUT4 to glucose uptake in common models of skeletal muscle and adipocytes.     

Insulin-mediated cellular insulin resistance decreases osmotic shock-induced glucose transport in 3T3-L1 adipocytes

Similar to insulin, osmotic shock treatment of 3T3-L1 adipocytes causes translocation of GLUT4 protein to the plasma membrane and an increase in glucose transport activity. In our study, we evaluated the effect of chronic insulin treatment on the osmotic shock signaling pathway leading to GLUT4 translocation and glucose uptake. We found that chronic administration of insulin to the adipocytes induced cellular resistance to osmotic shock-stimulated GLUT4 translocation and glucose transport. We found that chronic insulin treatment attenuated shock-induced Gab-1 tyrosine phosphorylation. Furthermore, chronic insulin exposure led to a marked impairment in the ability of Gab-1 to associate with p85 subunit of PI 3-kinase in response to acute shock and insulin stimulation. Cells that were chronically treated with insulin showed a 70% and a 61% decrease in Gab-1 associated PI 3-kinase activity in shock- vs. insulin-treated cells, respectively. In addition, we found that chronic insulin treatment inhibited both insulin- and osmotic shock-induced membrane ruffling, indicating that two PI 3-kinase dependent effects, GLUT4 translocation and membrane ruffling are decreased in chronically insulin-treated cells. The results described above clearly demonstrate that chronic insulin treatment induces a state of cellular resistance to osmotic shock signal transduction.     

Suppressed intrinsic catalytic activity of GLUT1 glucose transporters in insulin-sensitive 3T3-L1 adipocytes.

Previous studies indicated that the erythroidtype (GLUT1) glucose transporter isoform contributes to basal but not insulin-stimulated hexose transport in mouse 3T3-L1 adipocytes. In the present studies it was found that basal hexose uptake in 3T3-L1 adipocytes was about 50% lower than that in 3T3-L1 or CHO-K1 fibroblasts. Intrinsic catalytic activities of GLUT1 transporters in CHO-K1 and 3T3-L1 cells were compared by normalizing these hexose transport rates to GLUT1 content on the cell surface, as measured by two independent methods. Cell surface GLUT1 levels in 3T3-L1 fibroblasts and adipocytes were about 10- and 25-fold higher, respectively, than in CHO-K1 fibroblasts, as assessed with an anti-GLUT1 exofacial domain antiserum, delta. The large excess of cell surface GLUT1 transporters in 3T3-L1 adipocytes relative to CHO-K1 fibroblasts was confirmed by GLUT1 protein immunoblot analysis and by photoaffinity labelling (with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n) of glucose transporters in isolated plasma membranes. Thus, GLUT1 intrinsic activity is markedly reduced in 3T3-L1 fibroblasts compared with the CHO-K1 fibroblasts, and further reduction occurs upon differentiation to adipocytes. Intrinsic catalytic activities specifically associated with heterologously expressed human GLUT1 protein in transfected CHO-K1 versus 3T3-L1 cells were determined by subtracting appropriate control cell values for hexose transport and delta-antibody binding from those determined in the transfected cells expressing high levels of human GLUT1. The results confirmed a greater than 90% inhibition of the intrinsic catalytic activity of human GLUT1 transporters on the surface of mouse 3T3-L1 adipocytes relative to CHO-K1 fibroblasts. We conclude that a mechanism that markedly suppresses basal hexose transport catalyzed by GLUT1 is a major contributor to the dramatic insulin sensitivity of glucose uptake in 3T3-L1 adipocytes.     

Lentiviral short hairpin ribonucleic acid-mediated knockdown of GLUT4 in 3T3-L1 adipocytes

Adipose tissue is an important insulin target organ, and 3T3-L1 cells are a model cell line for adipocytes. In this study, we have used lentivirus-mediated short hairpin RNA (shRNA) for functional gene knockdown in 3T3-L1 adipocytes to assess the molecular mechanisms of insulin signaling. We chose to target GLUT4 to validate this approach. We showed that lentiviruses efficiently delivered transgenes and small interfering RNA (siRNA) into fully differentiated 3T3-L1 adipocytes. We established a strategy for identifying efficient siRNA sequences for gene knockdown by transfecting 293 cells with the target gene fluorescent fusion protein plasmid along with a plasmid that expresses shRNA. Using these methods, we identified highly efficient siGLUT4 sequences. We demonstrated that lentivirus-mediated shRNA against GLUT4 reduced endogenous GLUT4 expression to almost undetectable levels in 3T3-L1 adipocytes. Interestingly, insulin-stimulated glucose uptake was only reduced by 50-60%, suggesting that another glucose transporter mediates part of this effect. When siGLUT1 was introduced into GLUT4-deficient adipocytes, insulin-stimulated glucose uptake was essentially abolished, indicating that both GLUT4 and GLUT1 contribute to insulin-stimulated glucose transport in 3T3-L1 adipocytes. We also found that GLUT4 knockdown led to impaired insulin-responsive aminopeptidase protein expression that was dependent on whether GLUT4 was knocked down in the differentiating or differentiated stage. We further found that GLUT4 expression was not required for adipogenic differentiation but was necessary for full lipogenic capacity of differentiated adipocytes. These studies indicate that lentiviral shRNA constructs provide an excellent approach to deliver functional siRNAs into 3T3-L1 adipocytes for studying insulin signaling and adipocyte biology.     

Annexin II is a thiazolidinedione-responsive gene involved in insulin-induced glucose transporter isoform 4 translocation in 3T3-L1 adipocytes

The target genes of peroxisomal proliferator-activated receptor-gamma ligands that lead to insulin sensitization are not fully understood. In this study, we have found that the thiazolidinedione, troglitazone, increases expression of annexin II at both the mRNA and protein levels, raising the possibility that annexin II plays a role in insulin-stimulated glucose transporter isoform 4 (GLUT4) translocation and glucose transport. To assess this, we microinjected annexin II antibody or annexin II small interfering RNA into 3T3-L1 adipocytes and found that insulin-stimulated GLUT4 translocation was inhibited by 54 and 60%, respectively. Furthermore, microinjection of annexin II antibody inhibited constitutively active Galphaq (Q209L-Galphaq)-induced but not osmotic shock-induced GLUT4 translocation. When cells were cotransfected with wild-type annexin II, along with an enhanced green fluorescent protein-cmyc-GLUT4 construct, and the percentage of cells expressing cmyc-GLUT4 at the cell surface was measured by immunofluorescence microscopy, there was a marked increase in the ability of insulin to stimulate recruitment of cmyc-GLUT4 protein to the cell surface. In summary, our results show that annexin II is a newly described thiazolidinedione response gene involved in insulin-induced GLUT4 translocation in 3T3-L1 adipocytes.     

游离脂肪酸诱导3T3-L1脂肪细胞胰岛素抵抗的分子机制

目的研究游离脂肪酸（FFA）对3T3-L1脂肪细胞IKKβ及胰岛素信号转导蛋白的影响,探讨FFA诱导胰岛素抵抗（IR）的分子机制。方法诱导成熟的3T3-L1脂肪细胞与0.3-1.0mmol/L的软脂酸（PA）培养6-24h,以2-脱氧-〔^3H〕-D-葡萄糖摄入法观察葡萄糖的转运率,用Western blot检测IKKβ蛋白、IKKβ Ser181磷酸化、IRS-1蛋白、IRS-1 Ser307磷酸化、PI3Kp85蛋白及GluT4蛋白的表达。结果0.3-1.0mmol/LPA作用6-24h后,3T3-L1脂肪细胞的葡萄糖消耗明显减少,同时,Western blot显示,PA对IKKβ及GluT4蛋白的表达无明显影响,却能明显增加IKKβ Ser181及IRS-1 Ser307磷酸化,同时减少IRS-1蛋白和PI3Kp85蛋白的表达。结论FFA可以诱导IR,其分子机制可能与FFA激活IKKβ,使IRS-1丝氨酸残基磷酸化增加而酪氨酸残基磷酸化减少,进而使其下游的PI-3Kp85蛋白表达减少抑制葡萄糖转运有关。     

Alterations in insulin signalling pathway induced by prolonged insulin treatment of 3T3-L1 adipocytes

Summary Insulin-induced glucose transport stimulation, which results from the translocation of glucose transporter 4 (GLUT 4)-containing vesicles, is completely blocked after prolonged insulin treatment of 3T3-L1 adipocytes. Since GLUT 4 expression was reduced by only 30%, we looked at the insulin signalling pathway in this insulin-resistant model. Insulin-induced tyrosine phosphorylation of the major insulin receptor substrate IRS 1 was reduced by 50±7%, while its expression was decreased by 70±4%. When cells were treated with wortmannin (a PI3-kinase inhibitor) together with insulin, the expression of IRS 1 diminished to a much lower extent. Associated with the decrease in IRS 1 expression and phosphorylation, the activation by insulin of antiphosphotyrosine immunoprecipitable PI3-kinase activity and of p44^mapk and p42^mapk activities was altered. However, the expression of these proteins was normal and p44^mapk activity remained responsive to the tumour promoter TPA. Those results indicate that prolonged insulin treatment of 3T3-L1 adipocytes induces an insulin-resistant state with a reduced ability of insulin to stimulate the PI3-kinase and the MAP-kinases and a blockade of glucose transporter translocation.Abbreviations GLUT Glucose transporter - TPA tumour promoter - MAPK mitogen-activated protein kinase - IRS insulin receptor substrate - SH2 src homology 2 - GRB GRB: Growth factor Receptor bound protein - PVDF polyvinyliden difluoride - HDM/LDM high density/low density microsomes - MBP myelin basic protein - DMEM Dulbecco's modified Eagle's medium - PMSF phenylmethanesulphonyl fluoride - PI3-kinase phosphatidylinositol 3-kinase     

Stimulation of glucose transport by thyroid hormone in 3T3-L1 adipocytes: increased abundance of GLUT1 and GLUT4 glucose transporter proteins

In 3T3-L1 adipocytes we have examined the effect of tri-iodothyronine (T(3)) on glucose transport, total protein content and subcellular distribution of GLUT1 and GLUT4 glucose transporters. Cells incubated in T(3)-depleted serum were used as controls. Cells treated with T(3) (50 nM) for three days had a 3.6-fold increase in glucose uptake (P<0.05), and also presented a higher insulin sensitivity, without changes in insulin binding. The two glucose carriers, GLUT1 and GLUT4, increased by 87% (P<0.05) and 90% (P<0. 05), respectively, in cells treated with T(3). Under non-insulin-stimulated conditions, plasma membrane fractions obtained from cells exposed to T(3) were enriched with both GLUT1 (3. 29+/-0.69 vs 1.20+/-0.29 arbitrary units (A.U.)/5 microg protein, P<0.05) and GLUT4 (3.50+/-1.16 vs 0.82+/-0.28 A.U./5 microg protein, P<0.03). The incubation of cells with insulin produced the translocation of both glucose transporters to plasma membranes, and again cells treated with T(3) presented a higher amount of GLUT1 and GLUT4 in the plasma membrane fractions (P<0.05 and P<0.03 respectively). These data indicate that T(3) has a direct stimulatory effect on glucose transport in 3T3-L1 adipocytes due to an increase in GLUT1 and GLUT4, and by favouring their partitioning to plasma membranes. The effect of T(3) on glucose uptake induced by insulin can also be explained by the high expression of both glucose transporters.     

Agent and cell-type specificity in the induction of insulin resistance by HIV protease inhibitors

OBJECTIVE: To test agent and cell-type specificity in insulin resistance induced by prolonged exposure to HIV protease inhibitors (HPI), and to assess its relation to the direct, short-term inhibition of insulin-stimulated glucose uptake. METHODS: Following prolonged (18 h) and short (5-10 min) exposure to HPI, insulin-stimulated glucose transport, protein kinase B (PKB) phosphorylation, and GLUT4 translocation were evaluated in 3T3-L1 adipocytes, fibroblasts, L6 myotubes, and L6 cells overexpressing a myc tag on the first exofacial loop of GLUT4 or GLUT1. RESULTS: Prolonged exposure of 3T3-L1 adipocytes to nelfinavir, but not to indinavir or saquinavir, resulted in increased basal lipolysis but decreased insulin-stimulated glucose transport and PKB phosphorylation. In addition, impaired insulin-stimulated glucose uptake and PKB phosphorylation were also observed in the skeletal muscle cell line L6, and in 3T3-L1 fibroblasts. Interestingly, this coincided with increased basal glucose uptake as well as with elevated total-membrane glucose transporter GLUT1 protein content. In contrast to these unique effects of nelfinavir, the mere presence of any of the agents in the 5 min transport assay inhibited insulin-stimulated glucose-uptake activity. This appeared to be caused by direct and specific interaction of the drugs with GLUT4 fully assembled at the plasma membrane, since insulin-stimulated cell-surface exposure of an exofacial myc epitope on GLUT4 was normal. CONCLUSIONS: Independent mechanisms for HPI-induced insulin resistance exist: prolonged exposure to nelfinavir interferes with insulin signaling and alters cellular metabolism of adipocytes and muscle cells, whereas a direct inhibitory effect on insulin-stimulated glucose uptake may occurs through specific interaction of HPI with GLUT4.     

Regulation of insulin-stimulated glucose transport by chronic glucose exposure in 3T3-L1 adipocytes.

Chronic hyperglycemia causes insulin resistance, termed glucose toxicity. Herein we studied chronic glucose-dependent regulation of the glucose transport system in adipocytes. 3T3-L1 adipocytes were incubated for up to 24 h with low (1 mM) or high (25 mM) glucose, and glucose transport was subsequently analyzed. 100 nM insulin was present throughout the experiments. 24 h incubation with 1 mM glucose caused a 2.3+/-0.4 fold increase in glucose transport activity, compared to the values obtained with 25 mM glucose. This difference was not observed when 24 h incubation was carried out without insulin. Glucose transport activity was not increased at 3 or 6 h incubation with 1 mM glucose, but was increased at 12 h, which closely paralleled increased expression of GLUT1. In addition to increased GLUT1 expression, more efficient translocation of GLUT1 to the plasma membrane was observed when incubated with 1 mM glucose compared to 25 mM glucose. The addition of azaserin or deprivation of glutamine at 25 mM glucose did not increase the glucose transport activity to the level obtained with 1 mM glucose. PD98059 did not affect glucose transport activity when incubated with 1 mM or 25 mM glucose. In conclusion, the present study is the first to show that, in 3T3-L1 adipocytes, chronic exposure to low (1 mM) and high (25 mM) glucose leads to different insulin-stimulated glucose transport activities. These differences result from the difference in the expression and plasma membrane distribution of GLUT1, but not of GLUT4, and the hexosamine biosynthesis pathway or extracellular signal-regulated protein kinase is not involved.     

Neuropeptide Y impairs insulin-stimulated translocation of glucose transporter 4 in 3T3-L1 adipocytes through the Y1 receptor

Neuropeptide Y (NPY) is expressed in adipose tissue and is involved in adipocyte metabolism. Although NPY impacts on glucose utilization in vivo, the underlying cellular mechanism is yet to be fully elucidated.In this study we investigated the effect of NPY on the insulin-stimulated translocation of glucose transporter 4 (GLUT4) from intracellular stores to the cell surface in vitro. Using cellular fractionation and immunofluorescence we analyzed the cellular localization and content of GLUT4 in 3T3-L1 adipocytes. Additionally we investigated the effect of NPY on insulin action in adipocyte cultures by assessing the phosphorylation of Akt and [³H]-deoxyglucose uptake.Our data suggest that in 3T3-L1 adipocytes NPY inhibits insulin-stimulated glucose uptake in a GLUT4-dependent manner. The insulin induced translocation of GLUT4 was attenuated by the Y1 receptor agonist [Phe(7),Pro(34)] pNPY, demonstrating an essential role of the Y1 receptor in GLUT4 translocation. Additionally, we observed an NPY dose-dependent impairment of Akt phosphorylation.This study provides evidence that NPY impairs the insulin sensitivity of adipocytes and suggests that the Y1 receptor could be a potential therapeutic target for type 2 diabetes.     

Platelet-derived growth factor triggers translocation of the insulin-regulatable glucose transporter (type 4) predominantly through phosphatidylinositol 3-kinase binding sites on the receptor.

Insulin is the only known hormone which rapidly stimulates glucose uptake in target tissues, mainly by translocation to the cell surface of the intracellular insulin-regulatable glucose transporter (glucose transporter type 4, GLUT4). We have developed a cell line for direct, sensitive detection of GLUT4 on the cell surface. We have suggested that insulin-activated phosphatidylinositol (PI) 3-kinase may be involved in the signaling pathway of insulin-stimulated GLUT4 translocation. We report that platelet-derived growth factor (PDGF), which stimulates PI 3-kinase activity, triggers GLUT4 translocation in Chinese hamster ovary (CHO) cells stably overexpressing the PDGF receptor and in 3T3-L1 mouse adipocytes. Using mutant PDGF receptors that cannot bind to Ras-GTPase-activating protein, phospholipase C-gamma, and PI 3-kinase, respectively, we obtained evidence that PI 3-kinase binding sites play a key role in the signaling pathway of PDGF-stimulated GLUT4 translocation in the CHO cell system.