Methylene blue stimulates 2-deoxyglucose uptake in L929 fibroblast cells

Methylene blue (MB), a common cell stain, has been shown to inhibit nitric oxide synthase and guanylate cyclase, which has led to the recent use of MB in nitric oxide signaling studies. This study documents the effects of MB on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells where uptake is controlled by a single glucose transporter, GLUT 1. MB significantly activates cytochalasin B-inhibitable glucose transport in a dose dependent fashion within 10 min. A maximal stimulation of up to 800% was achieved by 50 microM MB after a 45-min exposure. The Vmax of transport increased without a change in the Km, which was accomplished without a significant change in the GLUT 1 content. The reduced form of MB, did not stimulate 2DG uptake and potassium ferricyanide, an extracellular redox agent, prevented both the staining and stimulatory effects of MB suggesting MB is reduced at the cell surface before it enters L929 cells. Phenylarsine oxide did not block cell staining as noted in other cells lines, but it did inhibit both basal and MB-stimulated 2DG uptake. Likewise, methyl-beta-cyclodextrin, an agent used to remove membrane cholesterol, blocked both the staining and stimulatory effects of MB. The AMP analog, AICAR, inhibited rather than activated basal 2DG uptake, and it did not alter MB-stimulated uptake suggesting that AMP kinase activation is not critical to the MB effect. Wortmannin, an inhibitor of PI kinase, had no effect on MB-stimulated 2DG uptake. These data provide additional insight into the acute regulation of GLUT 1 transport activity in L929 cells.     

Differential regulation of GLUT1 activity in human corneal limbal epithelial cells and fibroblasts

The corneal epithelial tissue is a layer of rapidly growing cells that are highly glycolytic and express GLUT1 as the major glucose transporter. It has been shown that GLUT1 in L929 fibroblast cells and other cell lines can be acutely activated by a variety agents. However, the acute regulation of glucose uptake in corneal cells has not been systematically investigated. Therefore, we examined glucose uptake in an immortalized human corneal–limbal epithelial (HCLE) cell line and compared it to glucose uptake in L929 fibroblast cells, a cell line where glucose uptake has been well characterized. We report that the expression of GLUT1 in HCLE cells is 6.6-fold higher than in L929 fibroblast cells, but the HCLE cells have a 25-fold higher basal rate of glucose uptake. Treatment with agents that interfere with mitochondrial metabolism, such as sodium azide and berberine, activate glucose uptake in L929 cells over 3-fold, but have no effect on glucose uptake HCLE cells. Also, agents known to react with thiols, such cinnamaldehyde, phenyarsine oxide and nitroxyl stimulate glucose uptake in L929 cells 3–4-fold, but actually inhibit glucose uptake in HCLE cells. These data suggest that in the fast growing HCLE cells, GLUT1 is expressed at a higher concentration and is already highly activated at basal conditions. These data support a model for the acute activation of GLUT1 that suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond within GLUT1 itself.     

Role of the nitric oxide pathway in AMPK-mediated glucose uptake and GLUT4 translocation in heart muscle

AMP-activated protein kinase (AMPK) is a serine-threonine kinase that regulates cellular metabolism and has an essential role in activating glucose transport during hypoxia and ischemia. The mechanisms responsible for AMPK stimulation of glucose transport are uncertain, but may involve interaction with other signaling pathways or direct effects on GLUT vesicular trafficking. One potential downstream mediator of AMPK signaling is the nitric oxide pathway. The aim of this study was to examine the extent to which AMPK mediates glucose transport through activation of the nitric oxide (NO)-signaling pathway in isolated heart muscles. Incubation with 1 mM 5-amino-4-imidazole-1-beta-carboxamide ribofuranoside (AICAR) activated AMPK (P < 0.01) and stimulated glucose uptake (P < 0.05) and translocation of the cardiomyocyte glucose transporter GLUT4 to the cell surface (P < 0.05). AICAR treatment increased phosphorylation of endothelial NO synthase (eNOS) approximately 1.8-fold (P < 0.05). eNOS, but not neuronal NOS, coimmunoprecipitated with both the alpha(2) and alpha(1) AMPK catalytic subunits in heart muscle. NO donors also increased glucose uptake and GLUT4 translocation (P < 0.05). Inhibition of NOS with N(omega)-nitro-l-arginine and N(omega)-methyl-l-arginine reduced AICAR-stimulated glucose uptake by 21 +/- 3% (P < 0.05) and 25 +/- 4% (P < 0.05), respectively. Inhibition of guanylate cyclase with ODQ and LY-83583 reduced AICAR-stimulated glucose uptake by 31 +/- 4% (P < 0.05) and 22 +/- 3% (P < 0.05), respectively, as well as GLUT4 translocation to the cell surface (P < 0.05). Taken together, these results indicate that activation of the NO-guanylate cyclase pathway contributes to, but is not the sole mediator of, AMPK stimulation of glucose uptake and GLUT4 translocation in heart muscle.     

Hesperetin impairs glucose uptake and inhibits proliferation of breast cancer cells

The flavanone hesperetin is known to decrease basal glucose uptake, although the inhibitory mechanism is largely unknown. Here, we used MDA‐MB‐231 breast cancer cells to investigate the molecular pathways affected by hesperetin. The results indicate that the suppression of glucose uptake is caused by the down‐regulation of glucose transporter 1 (GLUT1). Hesperetin was also found to inhibit insulin‐induced glucose uptake through impaired cell membrane translocation of glucose transporter 4 (GLUT4). In addition, the phosphorylation of the insulin receptor‐beta subunit (IR‐beta) and Akt was suppressed. Hesperetin also decreased cellular proliferation, which is likely due to the inhibition of glucose uptake. Cancer cells are highly dependent on glucose and hesperetin may, therefore, have potential application as an anticancer agent. Copyright © 2012 John Wiley & Sons, Ltd.     

GLUT2 is a high affinity glucosamine transporter

When expressed in Xenopus oocytes, GLUT1, 2 and 4 transport glucosamine with V(max) values that are three- to four-fold lower than for glucose. The K(m)s for glucosamine and glucose of GLUT1 and GLUT4 were similar. In contrast, GLUT2 had a much higher apparent affinity for glucosamine than for glucose (K(m)=0.8+/-0.1 mM vs. approximately 17-20 mM). Glucosamine transport by GLUT2 was confirmed in mammalian cells and, using hepatocytes from control or GLUT2-null mice, HgCl(2)-inhibitable glucosamine uptake by liver was shown to be exclusively through GLUT2. These data have implications for glucosamine effects on impaired glucose metabolism and for structure-function studies of transporter sugar binding sites.     

Nitroxyl (HNO) acutely activates the glucose uptake activity of GLUT1

Nitroxyl (HNO) is a molecule of significant interest due to its unique pharmacological properties, particularly within the cardiovascular system. A large portion of HNO biological effects can be attributed to its reactivity with protein thiols, where it can generate disulfide bonds. Evidence from studies in erythrocytes suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond. However, there are no reports that document the effects of HNO on glucose uptake. Therefore, we examined the acute effects of Angeli’s salt (AS), a HNO donor, on glucose uptake activity of GLUT1 in L929 fibroblast cells. We report that AS stimulates glucose uptake with a maximum effective concentration of 5.0 mM. An initial 7.2-fold increase occurs within 2 min, which decreases and plateaus to a 4.0-fold activation after 10 min. About 60% of the 4.0-fold activation recovers within 10 min, and 40% remains after an hour. The activation is blocked by the pretreatment of cells with thiol-reactive compounds, iodoacetamide (0.75 mM), cinnamaldehyde (2.0 mM), and phenylarsine oxide (10 μM). The effects of AS are not additive to the stimulatory effects of other acute activators of glucose uptake in L929 cells, such as azide (5 mM), berberine (50 μM), or glucose deprivation. These data suggest that GLUT1 is acutely activated in L929 cells by the formation of a disulfide bond, likely within GLUT1 itself.     

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.     

Glucose transport activation in human hematopoietic cells M07e is modulated by cytosolic calcium and calmodulin

The aim of this work was to investigate the role of cytosolic calcium and calmodulin-dependent systems in the activation of glucose uptake in the human megakaryocytic cell line M07e. Glucose uptake was significantly raised by elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) with thapsigargin, this effect being additive to the activation induced by cytokines (SCF, GM-CSF and TPO) and hydrogen peroxide. Intracellular Ca(2+) chelation by BAPTA decreased basal and activated glucose uptake in a dose-dependent manner. BAPTA reduced the GLUT1 translocation induced by SCF and H(2)O(2), suggesting a major role for Ca(2+) in GLUT1 intracellular trafficking. In the absence of extracellular Ca(2+), 2-aminoethoxydiphenyl-borate (2-APB) abolished the activation of glucose uptake induced by cytokines and H(2)O(2) suggesting an involvement in GLUT1 regulation in responses related to InsP(3)-induced Ca(2+) release. Under our experimental conditions, all the stimuli inducing glucose uptake activation failed to increase [Ca(2+)](c) suggesting that cytosolic Ca(2+) plays a permissive role in the regulation of GLUT1. The calmodulin antagonist W-7 and the inhibitor of Ca(2+)-calmodulin dependent protein kinase II (CAMK II) KN-62 removed the glucose transport activation by all the tested stimuli. These results suggest that in M07e cells calmodulin and CAMKII are involved in GLUT1 stimulation by cytokines and ROS.     

Osthole activates glucose uptake but blocks full activation in L929 fibroblast cells,and inhibits uptake in HCLE cells

Aims

Osthole, a coumarin derivative, has been used in Chinese medicine and studies have suggested a potential use in treatment of diabetes and cancers. Therefore, we investigated the effects of osthole and other coumarins on GLUT1 activity in two cell lines that exclusively express GLUT1.

Main methods

We measured the magnitude and time frame of the effects of osthole and related coumarins on glucose uptake in two cells lines; L929 fibroblast cells which have low GLUT1 expression levels and low basal glucose uptake and HCLE cells which have high GLUT1 concentrations and high basal uptake. We also explored the effects of these coumarins in combination with other GLUT1 activators.

Key findings

Osthole activates glucose uptake in L929 cells with a modest maximum 1.7-fold activation achieved by 50 μM with both activation and recovery occurring within minutes. However, osthole blocks full acute activation of glucose uptake by other, more robust activators. This behavior mimics the effects of other thiol reactive compounds and suggests that osthole is interacting with cysteine residues, possibly within GLUT1 itself. Coumarin, 7-hydroxycoumarin, and 7-methoxycoumarin, do not affect glucose uptake, which is consistent with the notion that the isoprenoid structure in osthole may be important to gain membrane access to GLUT1. In contrast to its effects in L929 cells, osthole inhibits basal glucose uptake in the more active HCLE cells.

Significance

The differential effects of osthole in L929 and HCLE cells indicated that regulation of GLUT1 varies, likely depending on its membrane concentration.     

A novel method to monitor insulin-stimulated GTP-loading of Rab11a in cardiomyocytes

As a member of the Rab small GTPase family, Rab11a has been shown to be involved in different vesicle trafficking processes. In earlier work we identified Rab11a to be present in GLUT4-containing vesicles after insulin stimulation and showed its involvement in insulin-dependent glucose uptake. However, it remained elusive if Rab11a is directly activated by the insulin signalling cascade and at which step a potential activation occurs. To examine the GTP-loading of Rab11a, we introduced a biotinylated GTP-analog into H9c2-hIR cells, transiently overexpressing HA-tagged Rab11a, and measured its binding to the GTPase after insulin stimulation. We observed that Rab11a is transiently GTP-loaded after insulin stimulation with a 2.3 (+/-0.3) fold activation (n=5), reaching its maximum after 4 min and declining back to basal after additional 2 min. The activation of Rab11a is phosphatidylinositol 3-kinase (PI3-kinase) dependent and downstream of Akt, as shown by in vitro knockdown of this kinase. These data show that Rab11a is directly activated by insulin and represents an element of the GLUT4 trafficking machinery.     

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.     

Silybin and dehydrosilybin decrease glucose uptake by inhibiting GLUT proteins

Silybin, the major flavonoid of Silybum marianum, is widely used to treat liver diseases such as hepatocellular carcinoma and cirrhosis-associated insulin resistance. Research so far has focused on its anti-oxidant properties. Here, we demonstrate that silybin and its derivative dehydrosilybin inhibit glucose uptake in several model systems. Both flavonoids dose-dependently reduce basal and insulin-dependent glucose uptake of 3T3-L1 adipocytes, with dehydrosilybin showing significantly stronger inhibition. However, insulin signaling was not impaired, and immunofluorescence and subcellular fractionation showed that insulin-induced translocation of GLUT4 to the plasma membrane is also unchanged. Likewise, hexokinase activity was not affected suggesting that silybin and dehydrosilybin interfere directly with glucose transport across the PM. Expression of GLUT4 in CHO cells counteracted the inhibition of glucose uptake by both flavonoids. Moreover, treatment of CHO cells with silybin and dehydrosilybin reduced cell viability which was partially rescued by GLUT4 expression. Kinetic analysis revealed that silybin and dehydrosilybin inhibit GLUT4-mediated glucose transport in a competitive manner with K(i)=60 and 116 μM, respectively. We conclude that silybin and dehydrosilybin inhibit cellular glucose uptake by directly interacting with GLUT transporters. Glucose starvation offers a novel explanation for the anti-cancer effects of silybin.     

Properties of the human erythrocyte glucose transport protein are determined by cellular context

Human erythrocyte hexose transfer is mediated by the glucose transport protein GLUT1 and is characterized by a complexity that is unexplained by available hypotheses for carrier-mediated sugar transport [Cloherty, E. K., Heard, K. S., and Carruthers, A. (1996) Biochemistry 35, 10411-10421]. The study presented here examines the possibility that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae (RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1), mutant GLUT1 (GLUT1(338)(-)(A3)), or carboxy-terminal hemagglutinin-polyHis-tagged GLUT1 (GLUT1-HA-H6). GLUT1 and GLUT1-HA-H6 are expressed at the yeast cell membrane and restore 2-deoxy-d-glucose, 3-O-methylglucose, and d-glucose transport capacity to RE700A. GLUT1-HA-H6 confers GLUT1-specific sugar transport characteristics to transfected RE700A, including inhibition by cytochalasin B and high-affinity transport of the nonmetabolized sugar 3-O-methylglucose. GLUT1(338)(-)(A3), a catalytically inactive GLUT1 mutant, is expressed but fails to restore RE700A sugar uptake capacity or growth on glucose. In contrast to transport in human red cells, K(m(app)) for 2-deoxy-d-glucose uptake equals K(i(app)) for 2-deoxy-d-glucose inhibition of 3-O-methylglucose uptake. Unlike transport in human red cells or transport in human embryonic kidney cells transfected with GLUT1-HA-H6, unidirectional sugar uptake in RE700A-GLUT1-HA-H6 is not inhibited by reductant and is not stimulated by intracellular sugar. Net uptake of subsaturating 3-O-methylglucose by RE700A-GLUT1-HA-H6 is a simple, first-order process. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific.     

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

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

Improved lectin-mediated immobilization of human red blood cells in superporous agarose beads

A new type of agarose bead, superporous agarose, was used as a gel support for immobilization of human red blood cells (RBCs) mediated by wheat germ lectin. The number of immobilized cells was similar to that obtained with commercial wheat germ lectin-agarose but the cell stability appeared to be superior. This allowed improved frontal affinity chromatographic analyses of cytochalasin B (CB)-binding to the glucose transporter GLUT1 which established a ratio of one CB-binding site per GLUT1 dimer for both plain RBCs or those treated with different poly amino acids. The measured dissociation constants, 70+/-14 nM for CB and 12+/-3 mM for glucose binding to GLUT1, are similar to those reported earlier.     

Does impaired mitochondrial function affect insulin signaling and action in cultured human skeletal muscle cells?

Insulin-resistant type 2 diabetic patients have been reported to have impaired skeletal muscle mitochondrial respiratory function. A key question is whether decreased mitochondrial respiration contributes directly to the decreased insulin action. To address this, a model of impaired cellular respiratory function was established by incubating human skeletal muscle cell cultures with the mitochondrial inhibitor sodium azide and examining the effects on insulin action. Incubation of human skeletal muscle cells with 50 and 75 microM azide resulted in 48 +/- 3% and 56 +/- 1% decreases, respectively, in respiration compared with untreated cells mimicking the level of impairment seen in type 2 diabetes. Under conditions of decreased respiratory chain function, insulin-independent (basal) glucose uptake was significantly increased. Basal glucose uptake was 325 +/- 39 pmol/min/mg (mean +/- SE) in untreated cells. This increased to 669 +/- 69 and 823 +/- 83 pmol/min/mg in cells treated with 50 and 75 microM azide, respectively (vs. untreated, both P < 0.0001). Azide treatment was also accompanied by an increase in basal glycogen synthesis and phosphorylation of AMP-activated protein kinase. However, there was no decrease in glucose uptake following insulin exposure, and insulin-stimulated phosphorylation of Akt was normal under these conditions. GLUT1 mRNA expression remained unchanged, whereas GLUT4 mRNA expression increased following azide treatment. In conclusion, under conditions of impaired mitochondrial respiration there was no evidence of impaired insulin signaling or glucose uptake following insulin exposure in this model system.     

AMP-Activated protein kinase is activated by the stimulations of G(q)-coupled receptors

The AMP-activated protein kinase (AMPK) functions as a metabolic sensor that monitors cellular AMP and ATP levels. Platelet-activating factor (PAF) activates endogeneous AMPKalpha1 in Chinese hamster ovary cells expressing the PAF receptor coupled with both G(i) and G(q), but its activity was not inhibited after treatment with islet-activating protein. Norepinephrine and bradykinin also activated AMPKalpha1 in cells expressing the G(q)-coupled alpha(1b)-adrenergic receptor and bradykinin receptor, respectively. Stimulations of the G(i)-coupled alpha(2A)-adrenergic receptor, fMet-Leu-Phe receptor, prostaglandin EP3alpha receptor, and G(s)-coupled beta(2)-adrenergic receptor did not activate AMPKalpha1. AMPKalpha1 thus is activated specifically by stimulation of G(q)-coupled receptors. G(q)-coupled receptors transmit the signal for GLUT4 translocation and glucose uptake through an insulin-independent pathway. However, direct activation of AMPKalpha1 with treatment of 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside did not trigger GLUT4 translocation nor stimulate glucose uptake in our cells. Thus, activation of AMPKalpha1 via G(q) is not sufficient to trigger GLUT4 translocation or stimulate glucose uptake.     

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.     

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.     

Activation of the small GTPase Rac1 by a specific guanine-nucleotide-exchange factor suffices to induce glucose uptake into skeletal-muscle cells

Background information. Insulin‐stimulated glucose uptake into skeletal muscle is crucial for glucose homoeostasis, and depends on the recruitment of GLUT4 (glucose transporter 4) to the plasma membrane. Mechanisms underlying insulin‐dependent GLUT4 translocation, particularly the role of Rho family GTPases, remain controversial. Results. In the present study, we show that constitutively active Rac1, but not other Rho family GTPases tested, induced GLUT4 translocation in the absence of insulin, suggesting that Rac1 activation is sufficient for GLUT4 translocation in muscle cells. Rac1 activation occurred in dorsal membrane ruffles of insulin‐stimulated cells as revealed by a novel method to visualize activated Rac1 in situ. We further identified FLJ00068 as a GEF (guanine‐nucleotide‐exchange factor) responsible for this Rac1 activation. Indeed, constitutively active FLJ00068 caused Rac1 activation in dorsal membrane ruffles and GLUT4 translocation without insulin stimulation. Down‐regulation of Rac1 or FLJ00068 by RNA interference, on the other hand, abrogated insulin‐induced GLUT4 translocation. Basal, but not insulin‐stimulated, activity of the serine/threonine kinase Akt was required for the induction of GLUT4 translocation by constitutively active Rac1 or FLJ00068. Conclusion. Collectively, Rac1 activation specifically in membrane ruffles by the GEF FLJ00068 is sufficient for insulin induction of glucose uptake into skeletal‐muscle cells.