The role of glucose metabolites in the activation and translocation of glycogen synthase by insulin in 3T3-L1 adipocytes.

The role of increased glucose transport in the hormonal regulation of glycogen synthase by insulin was investigated in 3T3-L1 adipocytes. Insulin treatment stimulated glycogen synthase activity 4-5-fold in these cells. Cytosolic glycogen synthase levels decreased by 75% in response to insulin, whereas, conversely, the glycogenolytic agent isoproterenol increased cytosolic enzyme levels by 200%. Removal of extracellular glucose reduced glycogen synthase activation by 40% and completely blocked enzymatic translocation. Addition of 5 mM 2-deoxyglucose did not restore glycogen synthase translocation but did augment dephosphorylation of the protein by insulin. The translocation event could be reconstituted in vitro only by the addition of UDP-glucose to basal cell lysates. Amylase pretreatment of the extracts suppressed glycogen synthase translocation, indicating that the enzyme was binding to glycogen. Incubation of 3T3-L1 adipocytes with 10 mM glucosamine induced a state of insulin resistance, blocked the translocation of glycogen synthase, and inhibited insulin-stimulated glycogen synthesis by 50%. Surprisingly, glycogen synthase activation by insulin was enhanced 4-fold, in part due to allosteric activation by a glucosamine metabolite. In vitro, glucosamine 6-phosphate and glucose 6-phosphate stimulated glycogen synthase activity with similar concentration curves. These results indicate that glucose metabolites have an impact on the regulation of glycogen synthase activation and localization by insulin.     

Inhibition of glycogen-synthase kinase 3 stimulates glycogen synthase and glucose transport by distinct mechanisms in 3T3-L1 adipocytes

The role of glycogen-synthase kinase 3 (GSK3) in insulin-stimulated glucose transport and glycogen synthase activation was investigated in 3T3-L1 adipocytes. GSK3 protein was clearly present in adipocytes and was found to be more abundant than in muscle and liver cell lines. The selective GSK3 inhibitor, LiCl, stimulated glucose transport and glycogen synthase activity (20 and 65%, respectively, of the maximal (1 microm) insulin response) and potentiated the responses to a submaximal concentration (1 nm) of insulin. LiCl- and insulin-stimulated glucose transport were abolished by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin; however, LiCl stimulation of glycogen synthase was not. In contrast to the rapid stimulation of glucose transport by insulin, transport stimulated by LiCl increased gradually over 3-5 h reaching 40% of the maximal insulin-stimulated level. Both LiCl- and insulin-stimulated glycogen synthase activity were maximal at 25 min. However, insulin-stimulated glycogen synthase activity returned to basal after 2 h, coincident with reactivation of GSK3. After a 2-h exposure to insulin, glycogen synthase was refractory to restimulation with insulin, indicating selective desensitization of this pathway. However, LiCl could partially stimulate glycogen synthase in desensitized cells. Furthermore, coincubation with LiCl during the 2 h exposure to insulin completely blocked desensitization of glycogen synthase activity. In summary, inhibition of GSK3 by LiCl: 1) stimulated glycogen synthase activity directly and independently of PI3-kinase, 2) stimulated glucose transport at a point upstream of PI3-kinase, 3) stimulated glycogen synthase activity in desensitized cells, and 4) prevented desensitization of glycogen synthase due to chronic insulin treatment. These data are consistent with GSK3 playing a central role in the regulation of glycogen synthase activity and a contributing factor in the regulation of glucose transport in 3T3-L1 adipocytes.     

Activation of glycogen synthase by insulin in 3T3-L1 adipocytes involves c-Cbl-associating protein (CAP)-dependent and CAP-independent signaling pathways

In adipose and muscle, insulin stimulates glucose uptake and glycogen synthase activity. Phosphatidylinositol 3-kinase (PI3K) activation is necessary but not sufficient for these metabolic actions of insulin. The insulin-stimulated translocation of phospho-c-Cbl to lipid rafts, via its association with CAP, comprises a second pathway regulating GLUT4 translocation. In 3T3-L1 adipocytes, overexpression of a dominant negative CAP mutant (CAP Delta SH3) completely blocked the insulin-stimulated glucose transport and glycogen synthesis but only partially inhibited glycogen synthase activation. In contrast, CAP Delta SH3 expression did not affect glycogen synthase activation by insulin in the absence of extracellular glucose. Moreover, CAP Delta SH3 has no effect on the PI3K-dependent activation of protein phosphatase-1 or phosphorylation of glycogen synthase kinase-3. These results indicate blockade of the c-Cbl/CAP pathway directly inhibits insulin-stimulated glucose uptake, which results in secondary inhibition of glycogen synthase activation and glycogen synthesis.     

Role of PDK1 in insulin-signaling pathway for glucose metabolism in 3T3-L1 adipocytes

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the insulin-signaling pathway for glucose metabolism, wild-type (wt), the kinase-dead (kd), or the plecstrin homology (PH) domain deletion (DeltaPH) mutant of PDK1 was expressed using an adenovirus gene transduction system in 3T3-L1 adipocytes. wt-PDK1 and kd-PDK1 were found in both membrane and cytosol fractions, whereas DeltaPH-PDK1, which exhibited PDK1 activity similar to that of wt-PDK1, was detected exclusively in the cytosol fraction. Insulin dose dependently activated protein kinase B (PKB) but did not change atypical protein kinase C (aPKC) activity in control cells. aPKC activity was not affected by expression of wt-, kd-, or DeltaPH-PDK1 in either the presence or the absence of insulin. Overexpression of wt-PDK1 enhanced insulin-induced activation of PKB as well as insulin-induced phosphorylation of glycogen synthase kinase (GSK)3alpha/beta, a direct downstream target of PKB, although insulin-induced glycogen synthesis was not significantly enhanced by wt-PDK1 expression. Neither DeltaPH-PDK1 nor kd-PDK1 expression affected PKB activity, GSK3 phosphorylation, or glycogen synthesis. Thus membrane localization of PDK1 via its PH domain is essential for insulin signaling through the PDK1-PKB-GSK3alpha/beta pathway. Glucose transport activity was unaffected by expression of wt-PDK1, kd-PDK1, or DeltaPH-PDK1 in either the presence or the absence of insulin. These findings suggest the presence of a signaling pathway for insulin-stimulated glucose transport in which PDK1 to PKB or aPKC is not involved.     

Differential effects of constitutively active phosphatidylinositol 3-kinase on glucose transport, glycogen synthase activity, and DNA synthesis in 3T3-L1 adipocytes.

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for many insulin-induced metabolic and mitogenic responses. However, it is unclear whether PI3K activation is sufficient for any of these effects. To address this question we increased PI3K activity in differentiated 3T3-L1 adipocytes by adenovirus-mediated expression of both the inter-SH2 region of the regulatory p85 subunit of PI3K (iSH2) and the catalytic p110 alpha subunit (p110). Coexpression resulted in PI3K activity that exceeded insulin-stimulated activity by two- to fivefold in cytosol, total membranes, and the low density microsome (LDM) fraction, the site of greatest insulin stimulation. While insulin increased glucose transport 15-fold, coexpression of iSH2-p110 increased transport (5.2-) +/- 0.7-fold with a parallel increase in GLUT4 translocation to the plasma membrane. Constitutive activation of PI3K had no effect on maximally insulin-stimulated glucose transport. Neither basal nor insulin-stimulated activity of glycogen synthase or mitogen-activated protein kinase was altered by iSH2-p110 coexpression. DNA synthesis was increased twofold by insulin in control 3T3-L1 adipocytes transduced with beta-galactosidase-encoding recombinant adenovirus, while iSH2-p110 coexpression increased DNA synthesis fivefold. These data indicate that (i) increased PI3K activity is sufficient to activate some but not all metabolic responses to insulin, (ii) activation of PI3K to levels exceeding the effect of insulin in adipocyte LDM results in only a partial stimulation of glucose transport, and (iii) increased PI3K activity in the absence of growth factor or oncoprotein stimulation is a potent stimulus of DNA synthesis.     

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.     

Arsenite stimulated glucose transport in 3T3-L1 adipocytes involves both Glut4 translocation and p38 MAPK activity.

The protein-modifying agent arsenite stimulates glucose uptake in 3T3-L1 adipocytes. In the current study we have analysed the signalling pathways that contribute to this response. By subcellular fractionation we observed that arsenite, like insulin, induces translocation of the GLUT1 and GLUT4 glucose transporters from the low-density membrane fraction to the plasma membrane. Arsenite did not activate early steps of the insulin receptor (IR)-signalling pathway and the response was insensitive to inhibition of phosphatidylinositol-3'-kinase (PI-3') kinase by wortmannin. These findings indicate that the 'classical' IR-IR substrate-PI-3' kinase pathway, that is essential for insulin-induced GLUT4 translocation, is not activated by arsenite. However, arsenite-treatment did induce tyrosine-phosphorylation of c-Cbl. Furthermore, treatment of the cells with the tyrosine kinase inhibitor, tyrphostin A25, abolished arsenite-induced glucose uptake, suggesting that the induction of a tyrosine kinase by arsenite is essential for glucose uptake. Both arsenite and insulin-induced glucose uptake were inhibited partially by the p38 MAP kinase inhibitor, SB203580. This compound had no effect on the magnitude of translocation of glucose transporters indicating that the level of glucose transport is determined by additional factors. Arsenite- and insulin-induced glucose uptake responded in a remarkably similar dose-dependent fashion to a range of pharmacological- and peptide-inhibitors for atypical PKC-lambda, a downstream target of PI-3' kinase signalling in insulin-induced glucose uptake. These data show that in 3T3-L1 adipocytes both arsenite- and insulin-induced signalling pathways project towards a similar cellular response, namely GLUT1 and GLUT4 translocation and glucose uptake. This response to arsenite is not functionally linked to early steps of the IR-IRS-PI-3' kinase pathway, but does coincide with c-Cbl phosphorylation, basal levels of PKC-lambda activity and p38 MAPK activation.     

Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5'-phosphatase catalytic activity

Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5'-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5'-phosphatase-defective SHIP2 (Delta IP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor beta subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or Delta IP-SHIP2. Because WT-SHIP2 possesses the 5'-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of Delta IP-SHIP2, indicating that Delta IP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase C lambda in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase C lambda, whereas these activations were increased by expression of Delta IP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of Delta IP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3beta and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of Delta IP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5'-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.     

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.     

Separation of insulin signaling into distinct GLUT4 translocation and activation steps

GLUT4 (glucose transporter 4) plays a pivotal role in insulin-induced glucose uptake to maintain normal blood glucose levels. Here, we report that a cell-permeable phosphoinositide-binding peptide induced GLUT4 translocation to the plasma membrane without inhibiting IRAP (insulin-responsive aminopeptidase) endocytosis. However, unlike insulin treatment, the peptide treatment did not increase glucose uptake in 3T3-L1 adipocytes, indicating that GLUT4 translocation and activation are separate events. GLUT4 activation can occur at the plasma membrane, since insulin was able to increase glucose uptake with a shorter time lag when inactive GLUT4 was first translocated to the plasma membrane by pretreating the cells with this peptide. Inhibition of phosphatidylinositol (PI) 3-kinase activity failed to inhibit GLUT4 translocation by the peptide but did inhibit glucose uptake when insulin was added following peptide treatment. Insulin, but not the peptide, stimulated GLUT1 translocation. Surprisingly, the peptide pretreatment inhibited insulin-induced GLUT1 translocation, suggesting that the peptide treatment has both a stimulatory effect on GLUT4 translocation and an inhibitory effect on insulin-induced GLUT1 translocation. These results suggest that GLUT4 requires translocation to the plasma membrane, as well as activation at the plasma membrane, to initiate glucose uptake, and both of these steps normally require PI 3-kinase activation.     

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.     

p110beta is up-regulated during differentiation of 3T3-L1 cells and contributes to the highly insulin-responsive glucose transport activity

Activation of p85/p110 type phosphatidylinositol kinase is essential for aspects of insulin-induced glucose metabolism, including translocation of GLUT4 to the cell surface and glycogen synthesis. The enzyme exists as a heterodimer containing a regulatory subunit (e.g. p85alpha) and one of two widely distributed isoforms of the p110 catalytic subunit: p110alpha or p110beta. In the present study, we compared the two isoforms in the regulation of insulin action. During differentiation of 3T3-L1 cells into adipocytes, p110beta was up-regulated approximately 10-fold, whereas expression of p110alpha was unaltered. The effects of the increased p110 expression were further assessed by expressing epitope tagged p110beta and p110alpha in 3T3-L1 cells using adenovirus transduction systems, respectively. In vitro, the basal lipid kinase activity of p110beta was lower than that of p110alpha. When p110alpha and p110beta were overexpressed in 3T3-L1 adipocytes, exposing cells to insulin induced each of the subunits to form complexes with p85alpha and tyrosine-phosphorylated IRS-1 with similar efficiency. However, whereas the kinase activity of p110beta, either endogenous or exogeneous, was markedly enhanced by insulin stimulation, only very small increases of the activity of p110alpha were observed. Interestingly, overexpression of p110beta increased insulin-induced glucose uptake by 3T3-L1 cells without significantly affecting basal glucose transport, whereas overexpression of p110alpha increased both basal and insulin-stimulated glucose uptake. Finally, microinjection of anti-p110beta neutralizing antibody into 3T3-L1 adipocytes abolished insulin-induced translocation of GLUT4 to the cell surface almost completely, whereas anti-p110alpha neutralizing antibody did only slightly. Together, these findings suggest that p110beta plays a crucial role in cellular activities evoked acutely by insulin.     

Protein phosphatase-2C alpha as a positive regulator of insulin sensitivity through direct activation of phosphatidylinositol 3-kinase in 3T3-L1 adipocytes

During differentiation, expression of protein phosphatase-2Calpha (PP2Calpha) is increased in 3T3-L1 adipocytes. To elucidate the role of PP2Calpha in insulin signaling, we overexpressed wild-type (WT) PP2Calpha by adenovirus-mediated gene transfer in 3T3-L1 adipocytes. Overexpression of PP2Calpha-WT enhanced the insulin sensitivity of glucose uptake without any changes in the early steps of insulin signaling. Infection with adenovirus 5 expressing PP2Calpha-WT increased phosphatidylinositol 3-kinase (PI3K) activities in the immunoprecipitate using antibody against the p85 or p110 subunit under both basal and insulin-stimulated conditions, followed by activation of downstream steps in the PI3K pathway, such as phosphorylation of Akt, glycogen synthase kinase-3, and atypical protein kinase C. In contrast, overexpression of the phosphatase-defective mutant PP2Calpha(R174G) did not produce such effects. Furthermore, overexpression of PP2Calpha-WT (but not PP2Calpha(R174G)) decreased the (32)P-labeled phosphorylation state as well as the gel mobility shift of the p85 subunit, suggesting that dephosphorylation of the p85 subunit by PP2Calpha activation might stimulate PI3K catalytic activity. Moreover, knockdown of PP2Calpha by transfection of small interfering RNA led to a significant decrease in Akt phosphorylation. In addition, microinjection of anti-PP2Calpha antibody or PP2Calpha small interfering RNA led to decreased insulin-stimulated GLUT4 translocation. In conclusion, PP2Calpha is a new positive regulator of insulin sensitivity that acts through a direct activation of PI3K in 3T3-L1 adipocytes.     

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

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

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

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

The insulin sensitizing effect of homoisoflavone-enriched fraction in Liriope platyphylla Wang et Tang via PI3-kinase pathway

In the present study, we screened candidates for enhancing insulin action, using glucose uptake as an indicator, from Liriope platyphylla Wang et Tang (LPWT) extract, Liliaceae, in 3T3-L1 adipocytes. The mechanism of insulin sensitizing action in the fractions was also investigated. LPWT extract with 70% MeOH was sequentially separated with Diaion HP-20 and silica gel column chromatography. The 9:1 fraction from silica gel column chromatography increased glucose uptake with 1 ng/mL up to glucose uptake with 50 ng/mL insulin. The 9:1 fraction, determined as homoisoflavone-enriched fraction, worked as an insulin sensitizer. It increased insulin stimulated glucose uptake in 3T3-L1 adipocytes, insulin responsive cells, through increased glucose transporter 4 (GLUT4) contents in the plasma membrane. GLUT4 translocation was increased through insulin receptor substrate 1 (IRS1)-PI3 kinase-Akt signaling mechanism. Thus, homoisoflavone-enriched fraction in LPWT extract played an important role as an insulin sensitizer in adipocytes.     

NOD1 activation induces proinflammatory gene expression and insulin resistance in 3T3-L1 adipocytes

Chronic inflammation is associated with obesity and insulin resistance; however, the underlying mechanisms are not fully understood. Pattern recognition receptors Toll-like receptors and nucleotide-oligomerization domain-containing proteins play critical roles in innate immune response. Here, we report that activation of nucleotide binding oligomerization domain-containing protein-1 (NOD1) in adipocytes induces proinflammatory response and impairs insulin signaling and insulin-induced glucose uptake. NOD1 and NOD2 mRNA are markedly increased in differentiated murine 3T3-L1 adipocytes and human primary adipocyte culture upon adipocyte conversion. Moreover, NOD1 mRNA is markedly increased only in the fat tissues in diet-induced obese mice, but not in genetically obese ob/ob mice. Stimulation of NOD1 with a synthetic ligand Tri-DAP induces proinflammatory chemokine MCP-1, RANTES, and cytokine TNF-α and MIP-2 (human IL-8 homolog) and IL-6 mRNA expression in 3T3-L1 adipocytes in a time- and dose-dependent manner. Similar proinflammatory profiles are observed in human primary adipocyte culture stimulated with Tri-DAP. Furthermore, NOD1 activation suppresses insulin signaling, as revealed by attenuated tyrosine phosphorylation and increased inhibitory serine phosphorylation, of IRS-1 and attenuated phosphorylation of Akt and downstream target GSK3α/3β, resulting in decreased insulin-induced glucose uptake in 3T3-L1 adipocytes. Together, our results suggest that NOD1 may play an important role in adipose inflammation and insulin resistance in diet-induced obesity.     

Mitogen-activated protein kinase (MAPK) phosphatase-1 and -4 attenuate p38 MAPK during dexamethasone-induced insulin resistance in 3T3-L1 adipocytes

Prolonged use of glucocorticoids induces pronounced insulin resistance in vivo. In vitro, treatment of 3T3-L1 adipocytes with dexamethasone for 48 h reduces the maximal level of insulin- and stress (arsenite)-induced glucose uptake by approximately 50%. Although phosphatidylinositol 3-kinase signaling was slightly attenuated, phosphorylation of its downstream effectors such as protein kinase B and protein kinase C-lambda remained intact. Nor was any effect of dexamethasone treatment observed on insulin- or arsenite-induced translocation of glucose transporter 4 (GLUT4) toward the plasma membrane. However, for a maximal response to either arsenite- or insulin-induced glucose uptake in these cells, functional p38 MAPK signaling is required. Dexamethasone treatment markedly attenuated p38 MAPK phosphorylation coincident with an up-regulation of the MAPK phosphatases MKP-1 and MKP-4. Employing lentivirus-mediated ectopic expression in fully differentiated 3T3-L1 adipocytes demonstrated a differential effect of these phosphatases: whereas MKP-1 was a more potent inhibitor of insulin-induced glucose uptake, MKP-4 more efficiently inhibited arsenite-induced glucose uptake. This coincided with the effects of these phosphatases on p38 MAPK phosphorylation, i.e. MKP-1 and MKP-4 attenuated p38 MAPK phosphorylation by insulin and arsenite, respectively. Taken together, these data provide evidence that in 3T3-L1 adipocytes dexamethasone inhibits the activation of the GLUT4 in the plasma membrane by a p38 MAPK-dependent process, rather than in a defect in GLUT4 translocation per se.     

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.     

Defective Akt activation is associated with glucose- but not glucosamine-induced insulin resistance

3T3-L1 adipocytes develop insulin-resistant glucose transport upon preincubation with high glucose or glucosamine, provided insulin (0.6 nM) is present during preincubation. Insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol (PI) 3-kinase activity is unaffected (30). Total cellular IRS-1, PI 3-kinase, or Akt concentrations were unchanged. Akt activation in subcellular fractions was assessed by immunoblotting with two phospho-Akt-specific antibodies. Upon acute 100 nM insulin stimulation, plasma membrane (PM)-associated phospho-Akt was highest in cells preincubated in low glucose with no insulin, less in high glucose with no insulin, even less in low glucose+insulin, and lowest in high glucose+insulin. Only high glucose+insulin caused insulin-resistant glucose transport. Acute insulin stimulation increased total PM-Akt about twofold after preincubation without insulin in low or high glucose. Preincubation with 0.6 nM insulin decreased Akt PM translocation by approximately 25% in low and approximately 50% in high glucose. Preincubation with glucosamine did not affect Akt phosphorylation or translocation. Conclusions: chronic exposure to high glucose or insulin downregulates acute insulin-stimulated Akt activation, acting synergistically distal to PI 3-kinase. Maximal insulin activates more Akt than required for maximal glucose transport stimulation. Insulin resistance may ensue when PM-associated phospho-Akt decreases below a threshold. High glucose and glucosamine cause insulin resistance by different mechanisms in 3T3-L1 adipocytes.