Selective regulation of the perinuclear distribution of glucose transporter 4 (GLUT4) by insulin signals in muscle cells

Insulin regulates glucose transporter 4 (GLUT4) availability at the surface of muscle and adipose cells. In L6 myoblasts, stably expressed GLUT4myc is detected mostly in a perinuclear region. In unstimulated cells, about half of perinuclear GLUT4myc colocalizes with the transferrin receptor (TfR). Insulin stimulation selectively decreased the perinuclear colocalization of GLUT4myc with TfR determined by 3D-reconstruction of fluorescence images. Perinuclear GLUT4myc adopted two main distributions defined morphometrically as 'conical' and 'concentric'. Insulin rapidly reduced the proportion of cells with conical in favor of concentric perinuclear GLUT4myc distributions in association with the gain in surface GLUT4myc. Upon removal of insulin, the GLUT4myc perinuclear distribution and surface levels reversed in parallel. In contrast, hypertonicity (which like insulin elevates surface GLUT4myc) did not elicit perinuclear GLUT4myc redistribution. Insulin also caused redistribution of perinuclear vesicle-associated membrane protein-2 (VAMP2), without alteration of perinuclear TfR and VAMP3. Inhibitory mutants of phosphatidylinositol-3 kinase (Deltap85) or Akt substrate AS160 (AS160-4P) prevented insulin-mediated perinuclear GLUT4myc redistribution. Tetanus toxin expression did not prevent the perinuclear GLUT4myc redistribution, suggesting that redistribution is independent of GLUT4myc fusion with the plasma membrane. We propose that insulin causes selective, dynamic relocalization of perinuclear GLUT4myc and VAMP2 and perinuclear GLUT4myc redistribution is a direct target of insulin-derived signals.     

GLUT4 vesicle recruitment and fusion are differentially regulated by Rac, AS160, and Rab8A in muscle cells

Insulin increases glucose uptake into muscle by enhancing the surface recycling of GLUT4 transporters. In myoblasts, insulin signals bifurcate downstream of phosphatidylinositol 3-kinase into separate Akt and Rac/actin arms. Akt-mediated Rab-GAP AS160 phosphorylation and Rac/actin are required for net insulin gain of GLUT4, but the specific steps (vesicle recruitment, docking or fusion) regulated by Rac, actin dynamics, and AS160 target Rab8A are unknown. In L6 myoblasts expressing GLUT4myc, blocking vesicle fusion by tetanus toxin cleavage of VAMP2 impeded GLUT4myc membrane insertion without diminishing its build-up at the cell periphery. Conversely, actin disruption by dominant negative Rac or Latrunculin B abolished insulin-induced surface and submembrane GLUT4myc accumulation. Expression of non-phosphorylatable AS160 (AS160-4P) abrogated membrane insertion of GLUT4myc and partially reduced its cortical build-up, an effect magnified by selective Rab8A knockdown. We propose that insulin-induced actin dynamics participates in GLUT4myc vesicle retention beneath the membrane, whereas AS160 phosphorylation is essential for GLUT4myc vesicle-membrane docking/fusion and also contributes to GLUT4myc cortical availability through Rab8A.     

Protein Kinase B/Akt Participates in GLUT4 Translocation by Insulin in L6 Myoblasts

L6 myoblasts stably transfected with a GLUT4 cDNA harboring an exofacial myc epitope tag (L6-GLUT4myc myoblasts) were used to study the role of protein kinase B alpha (PKBalpha)/Akt1 in the insulin-induced translocation of GLUT4 to the cell surface. Surface GLUT4myc was detected by immunofluorescent labeling of the myc epitope in nonpermeabilized cells. Insulin induced a marked translocation of GLUT4myc to the plasma membrane within 20 min. This was prevented by transient transfection of a dominant inhibitory construct of phosphatidylinositol (PI) 3-kinase (Deltap85alpha). Transiently transfected cells were identified by cotransfection of green fluorescent protein. A constitutively active PKBalpha, created by fusion of a viral Gag protein at its N terminus (GagPKB), increased the cell surface density of GLUT4myc compared to that of neighboring nontransfected cells. A kinase-inactive, phosphorylation-deficient PKBalpha/Akt1 construct with the mutations K179A (substitution of alanine for the lysine at position 179), T308A, and S473A (AAA-PKB) behaved as a dominant-negative inhibitor of insulin-dependent activation of cotransfected wild-type hemagglutinin (HA)-tagged PKB. Furthermore, AAA-PKB markedly inhibited the insulin-induced phosphorylation of cotransfected BAD, demonstrating inhibition of the endogenous PKB/Akt. Under the same conditions, AAA-PKB almost entirely blocked the insulin-dependent increase in surface GLUT4myc. PKBalpha with alanine substitutions T308A and S473A (AA-PKB) or K179A (A-PKB) alone was a less potent inhibitor of insulin-dependent activation of wild-type HA-PKB or GLUT4myc translocation than was AAA-PKB. Cotransfection of AAA-PKB with a fourfold DNA excess of HA-PKB rescued insulin-stimulated GLUT4myc translocation. AAA-PKB did not prevent actin bundling (membrane ruffling), though this response was PI 3-kinase dependent. Therefore, it is unlikely that AAA-PKB acted by inhibiting PI 3-kinase signaling. These results outline an important role for PKBalpha/Akt1 in the stimulation of glucose transport by insulin in muscle cells in culture.     

Glucose transporter 4 can be inserted in the membrane without exposing its catalytic site for photolabeling from the medium

Insulin stimulates the production of PI(3,4,5)P3 in muscle cells, and this is required to stimulate GLUT4fusion with the plasma membrane. Introduction of exogenous PI(3,4,5)P3 to muscle cells recapitulates insulin's effects on GLUT4 fusion with the plasma membrane, but not glucose uptake. This study aims to explore the mechanism behind this difference. In L6-GLUT4myc muscle cells, the availability of the GLUT4 intracellular C-terminus and extracellular myc epitopes for immunoreactivity on plasma membrane lawns was detected with the corresponding antibody. The availability of the active site of GLUT4from extracellular medium was assessed by affinity photolabeling with the cell impermeant compound Bio-LC-ATB-BMPA. 100 nmol/L insulin and 10 μmol/L PI(3,4,5)P3 caused myc signal gain on the plasma membrane lawns by 1.64-fold and 1.58-fold over basal, respectively. Insulin, but not PI(3,4,5)P3, increased photolabeling of GLUT4 and immunolabeling with C-terminus antibody by 2.47-fold and 2.04-fold over basal, respectively. Upon insulin stimulation, the C-terminus signal gain was greater than myc signal gain (2.04-fold vs. 1.64-fold over basal, respectively) in plasma membrane lawns. These results indicate that (i) PI(3,4,5)P3 does not make the active site of GLUT4 available from the extracellular surface despite causing GLUT4 fusion with the plasma membrane; (ii) the availability of the active site of GLUT4 from the extracellular medium and availability of the C-terminus from the cytosolic site are correlated; (iii) in addition to stimulating GLUT4 translocation, insulin stimulation displaces a protein which masks the GLUT4 C-terminus. We propose that a protein which masks the C-terminus also prevents the active site from being available for photolabeliing and possibly glucose uptake after treatment with PI(3,4,5)P3.     

Insulin and hypertonicity recruit GLUT4 to the plasma membrane of muscle cells by using N-ethylmaleimide-sensitive factor-dependent SNARE mechanisms but different v-SNAREs: role of TI-VAMP

Insulin and hypertonicity each increase the content of GLUT4 glucose transporters at the surface of muscle cells. Insulin enhances GLUT4 exocytosis without diminishing its endocytosis. The insulin but not the hypertonicity response is reduced by tetanus neurotoxin, which cleaves vesicle-associated membrane protein (VAMP)2 and VAMP3, and is rescued upon introducing tetanus neurotoxin-resistant VAMP2. Here, we show that hypertonicity enhances GLUT4 recycling, compounding its previously shown ability to reduce GLUT4 endocytosis. To examine whether the canonical soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) mechanism is required for the plasma membrane fusion of the tetanus neurotoxin-insensitive GLUT4 vesicles, L6 myoblasts stably expressing myc-tagged GLUT4 (GLUT4myc) were transiently transfected with dominant negative N-ethylmaleimide-sensitive factor (NSF) (DN-NSF) or small-interfering RNA to tetanus neurotoxin-insensitive VAMP (TI-VAMP siRNA). Both strategies markedly reduced the basal level of surface GLUT4myc and the surface gain of GLUT4myc in response to hypertonicity. The insulin effect was abolished by DN-NSF, but only partly reduced by TI-VAMP siRNA. We propose that insulin and hypertonicity recruit GLUT4myc from partly overlapping, but distinct sources defined by VAMP2 and TI-VAMP, respectively.     

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.     

Conditioned medium from hypoxia-treated adipocytes renders muscle cells insulin resistant

Adipose tissue hypoxia is an early phenotype in obesity, associated with macrophage infiltration and local inflammation. Here we test the hypothesis that adipocytes in culture respond to a hypoxic environment with the release of pro-inflammatory factors that stimulate macrophage migration and cause muscle insulin resistance. 3T3-L1 adipocytes cultured in a 1% O2 atmosphere responded with a classic hypoxia response by elevating protein expression of HIF-1α. This was associated with elevated mRNA expression and peptide release of cytokines TNFα, IL-6 and the chemokine monocyte chemoattractant protein-1 (MCP-1). The mRNA and protein expression of the anti-inflammatory adipokine adiponectin was reduced. Conditioned medium from hypoxia-treated adipocytes (CM-H), inhibited insulin-stimulated and raised basal cell surface levels of GLUT4myc stably expressed in C2C12 myotubes. Insulin stimulation of Akt and AS160 phosphorylation, key regulators of GLUT4myc exocytosis, was markedly impaired. CM-H also caused activation of JNK and S6K, and elevated serine phosphorylation of IRS1 in the C2C12 myotubes. These effects were implicated in reducing propagation of insulin signaling to Akt and AS160. Heat inactivation of CM-H reversed its dual effects on GLUT4myc traffic in muscle cells. Interestingly, antibody-mediated neutralization of IL-6 in CM-H lowered its effect on both the basal and insulin-stimulated cell surface GLUT4myc compared to unmodified CM-H. IL-6 may have regulated GLUT4myc traffic through its action on AMPK. Additionally, antibody-mediated neutralization of MCP-1 partly reversed the inhibition of insulin-stimulated GLUT4myc exocytosis caused by unmodified CM-H. In Transwell co-culture, hypoxia-challenged adipocytes attracted RAW 264.7 macrophages, consistent with elevated release of MCP-1 from adipocytes during hypoxia. Neutralization of MCP-1 in adipocyte CM-H prevented macrophage migration towards it and partly reversed the effect of CM-H on insulin response in muscle cells. We conclude that adipose tissue hypoxia may be an important trigger of its inflammatory response observed in obesity, and the elevated chemokine MCP-1 may contribute to increased macrophage migration towards adipose tissue and subsequent decreased insulin responsiveness of glucose uptake in muscle.     

Differential effects of phosphatidylinositol 3-kinase inhibition on intracellular signals regulating GLUT4 translocation and glucose transport

Phosphatidylinositol (PI) 3-kinase is required for insulin-stimulated translocation of GLUT4 to the surface of muscle and fat cells. Recent evidence suggests that the full stimulation of glucose uptake by insulin also requires activation of GLUT4, possibly via a p38 mitogen-activated protein kinase (p38 MAPK)-dependent pathway. Here we used L6 myotubes expressing Myc-tagged GLUT4 to examine at what level the signals regulating GLUT4 translocation and activation bifurcate. We compared the sensitivity of each process, as well as of signals leading to GLUT4 translocation (Akt and atypical protein kinase C) to PI 3-kinase inhibition. Wortmannin inhibited insulin-stimulated glucose uptake with an IC(50) of 3 nm. In contrast, GLUT4myc appearance at the cell surface was less sensitive to inhibition (IC(50) = 43 nm). This dissociation between insulin-stimulated glucose uptake and GLUT4myc translocation was not observed with LY294002 (IC(50) = 8 and 10 microm, respectively). The sensitivity of insulin-stimulated activation of PKC zeta/lambda, Akt1, Akt2, and Akt3 to wortmannin (IC(50) = 24, 30, 35, and 60 nm, respectively) correlated closely with inhibition of GLUT4 translocation. In contrast, insulin-dependent p38 MAPK phosphorylation was efficiently reduced in cells pretreated with wortmannin, with an IC(50) of 7 nm. Insulin-dependent p38 alpha and p38 beta MAPK activities were also markedly reduced by wortmannin (IC(50) = 6 and 2 nm, respectively). LY294002 or transient expression of a dominant inhibitory PI 3-kinase construct (Delta p85), however, did not affect p38 MAPK phosphorylation. These results uncover a striking correlation between PI 3-kinase, Akt, PKC zeta/lambda, and GLUT4 translocation on one hand and their segregation from glucose uptake and p38 MAPK activation on the other, based on their wortmannin sensitivity. We propose that a distinct, high affinity target of wortmannin, other than PI 3-kinase, may be necessary for activation of p38 MAPK and GLUT4 in response to insulin.     

Tomosyn is a novel Akt substrate mediating insulin-dependent GLUT4 exocytosis

Insulin triggers glucose uptake into skeletal muscle and adipose tissues by gaining the available number of glucose transporter 4 (GLUT4) on the cell surface. GLUT4-loaded vesicles are targeted to plasma membrane from the intracellular reservoir through multiple trafficking and fusion processes that are mainly regulated by Akt. However, it is still largely unknown how GLUT4 expression in the cell surface is promoted by insulin. In the present study, we identified tomosyn at Ser-783 as a possible Akt-substrate motif and examined whether the phosphorylation at Ser-783 is involved in the regulation of GLUT4 expression. Both Akt1 and Akt2 phosphorylated the wild-type tomosyn, but not the mutant tomosyn in which Ser-783 was replaced with Ala. Phosphorylation of tomosyn at Ser-783 was also observed in the intact cells by insulin stimulation, which was blocked by PI3K inhibitor, LY294002. In vitro pull-down assay showed that phosphorylation of tomosyn at Ser-783 by Akt inhibited the interaction with syntaxin 4. Insulin stimulation increased GLUT4 in the cell surface of CHO-K1 cells to promote glucose uptake, however exogenous expression of the mutant tomosyn attenuated the increase by insulin. These results suggest that Ser-783 of tomosyn is a target of Akt and is implicated in the interaction with syntaxin 4.     

Artemisia princeps extract promoted glucose uptake in cultured L6 muscle cells via glucose transporter 4 translocation

Artemisia princeps is a familiar plant as a food substance and medicinal herb. In this study, we evaluated the effects of an ethanol extract of A. princeps (APE) on glucose uptake in differentiated L6 muscle cells. Treatment with APE elevated deoxyglucose uptake, and translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane in L6 myotubes occurred. The PI3K inhibitor LY294002 attenuated glucose uptake induced by APE. Phosphorylation of the Ser(473) residue of Akt was not observed, but phosphorylation of PI3K, Akt (Thr(308)), and atypical PKC was. In addition, APE stimulated phosphorylation of AMP-activated protein kinase (AMPK) at a level similar to 5'-amino-5-imidazolecarboxamide-riboside (AICAR). These results indicate that APE stimulates glucose uptake by inducing GLUT4 translocation, which is in part mediated by combination of the PI3K-dependent atypical PKC pathway and AMPK pathways.     

Acetylcholine-induced phosphorylation and membrane translocation of CPI-17 in bronchial smooth muscle of rats

A translocation of protein kinase C (PKC) from cytosol to plasma membrane has been reported as an association with agonist-induced Ca2+ sensitization in smooth muscle contraction. Therefore, it is possible that a downstream target of PKC, CPI-17 [PKC-potentiated inhibitory protein for heterotrimeric myosin light chain (MLC) phosphatase of 17 kDa], might also be translocated to membrane when activated. To confirm this hypothesis, cytosolic and membrane CPI-17 was measured in acetylcholine (ACh)- and high-K+ depolarization-stimulated bronchial smooth muscle of rats. An active form of CPI-17, i.e., Thr38-phosphorylated CPI-17, was also measured in cytosolic and membrane fractions. Immunoblot analyses demonstrated a translocation of CPI-17 from cytosolic to membrane fraction by ACh, but not high-K+ depolarization, stimulation in time- and concentration-dependent manners. Interestingly, phosphorylated CPI-17 was detected only in membrane fractions in the ACh-stimulated tissues. However, in the high-K+ depolarization-stimulated tissues, phosphorylated CPI-17 was not detected both in membrane and cytosolic fraction. To estimate downstream of activated CPI-17, immunoblotting for phosphorylated MLC was performed in ACh- or high-K+ depolarization-stimulated tissues. ACh- and high-K+ depolarization-induced phosphorylation of MLC was observed in its contraction-dependent manner. In conclusion, we, for the first time, suggested that CPI-17 is translocated and phosphorylated by ACh, but not high-K+ depolarization, in rat bronchial smooth muscle. ACh-induced translocation and phosphorylation of CPI-17 might be caused via the activation of muscarinic receptor.     

Hyperosmolarity reduces GLUT4 endocytosis and increases its exocytosis from a VAMP2-independent pool in l6 muscle cells

The intracellular traffic of the glucose transporter 4 (GLUT4) in muscle cells remains largely unexplored. Here we make use of L6 myoblasts stably expressing GLUT4 with an exofacially directed Myc-tag (GLUT4myc) to determine the exocytic and endocytic rates of the transporter. Insulin caused a rapid (t(12) = 4 min) gain, whereas hyperosmolarity (0.45 m sucrose) caused a slow (t(12) = 20 min) gain in surface GLUT4myc molecules. With prior insulin stimulation followed by addition of hypertonic sucrose, the increase in surface GLUT4myc was partly additive. Unlike the effect of insulin, the GLUT4myc gain caused by hyperosmolarity was insensitive to wortmannin or to tetanus toxin cleavage of VAMP2 and VAMP3. Disappearance of GLUT4myc from the cell surface was rapid (t(12) = 1.5 min). Insulin had no effect on the initial rate of GLUT4myc internalization. In contrast, hyperosmolarity almost completely abolished GLUT4myc internalization. Surface GLUT4myc accumulation in response to hyperosmolarity was only partially blocked by inhibition of tyrosine kinases with erbstatin analog (erbstatin A) and genistein. However, neither inhibitor interfered with the ability of hyperosmolarity to block GLUT4myc internalization. We propose that hyperosmolarity increases surface GLUT4myc by preventing GLUT4 endocytosis and stimulating its exocytosis via a pathway independent of phosphatidylinositol 3-kinase activity and of VAMP2 or VAMP3. A tetanus toxin-insensitive v-SNARE such as TI-VAMP detected in these cells, might mediate membrane fusion of the hyperosmolarity-sensitive pool.     

Maturation of the regulation of GLUT4 activity by p38 MAPK during L6 cell myogenesis

Insulin stimulates glucose uptake in skeletal muscle cells and fat cells by promoting the rapid translocation of GLUT4 glucose transporters to the plasma membrane. Recent work from our laboratory supports the concept that insulin also stimulates the intrinsic activity of GLUT4 through a signaling pathway that includes p38 MAPK. Here we show that regulation of GLUT4 activity by insulin develops during maturation of skeletal muscle cells into myotubes in concert with the ability of insulin to stimulate p38 MAPK. In L6 myotubes expressing GLUT4 that carries an exofacial myc-epitope (L6-GLUT4myc), insulin-stimulated GLUT4myc translocation equals in magnitude the glucose uptake response. Inhibition of p38 MAPK with SB203580 reduces insulin-stimulated glucose uptake without affecting GLUT4myc translocation. In contrast, in myoblasts, the magnitude of insulin-stimulated glucose uptake is significantly lower than that of GLUT4myc translocation and is insensitive to SB203580. Activation of p38 MAPK by insulin is considerably higher in myotubes than in myoblasts, as is the activation of upstream kinases MKK3/MKK6. In contrast, the activation of all three Akt isoforms and GLUT4 translocation are similar in myoblasts and myotubes. Furthermore, GLUT4myc translocation and phosphorylation of regulatory sites on Akt in L6-GLUT4myc myotubes are equally sensitive to insulin, whereas glucose uptake and phosphorylation of regulatory sites on p38 MAPK show lower sensitivity to the hormone. These observations draw additional parallels between Akt and GLUT4 translocation and between p38 MAPK and GLUT4 activation. Regulation of GLUT4 activity by insulin develops upon muscle cell differentiation and correlates with p38 MAPK activation by insulin.     

Insulin regulates the membrane arrival, fusion, and C-terminal unmasking of glucose transporter-4 via distinct phosphoinositides

Insulin increases glucose uptake into muscle via glucose transporter-4 (GLUT4) translocation to the cell membrane, but the regulated events in GLUT4 traffic are unknown. Here we focus on the role of class IA phosphatidylinositol (PI) 3-kinase and specific phosphoinositides in the steps of GLUT4 arrival and fusion with the membrane, using L6 muscle cells expressing GLUT4myc. To this end, we detected the availability of the myc epitope at the cell surface or intravesicular spaces and of the cytosol-facing C-terminal epitope, in cells and membrane lawns derived from them. We observed the following: (a) Wortmannin and LY294002 at concentrations that inhibit class IA PI 3-kinase reduced but did not abate the C terminus gain, yet the myc epitope was unavailable for detection unless lawns or cells were permeabilized, suggesting the presence of GLUT4myc in docked, unfused vesicles. Accordingly, GLUT4myc-containing vesicles were detected by immunoelectron microscopy of membranes from cells pretreated with wortmannin and insulin, but not insulin or wortmannin alone. (b) Insulin caused greater immunological availability of the C terminus than myc epitopes, suggesting that C terminus unmasking had occurred. Delivering phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) to intact cells significantly increased lawn-associated myc signal without C terminus gain. Conversely, phosphatidylinositol 3-phosphate (PI3P) increased the detection of C terminus epitope without any myc gain. We propose that insulin regulates GLUT4 membrane arrival, fusion, and C terminus unmasking, through distinct phosphoinositides. PI(3,4,5)P(3) causes arrival and fusion without unmasking, whereas PI3P causes arrival and unmasking without fusion.     

Epigallocatechin-3-O-gallate (EGCG) protects the insulin sensitivity in rat L6 muscle cells exposed to dexamethasone condition

The tea polyphenol epigallocatechin-3-O-gallate (EGCG) displays some antidiabetic effects; however the mechanisms are incompletely understood. In the present study, the investigation of the effects of EGCG on insulin resistance was performed in rat L6 cells treated with dexamethasone. We found that dexamethasone increased Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1) and reduced phosphorylation of AMPK and Akt. Furthermore, glucose uptake and glucose transporter (GLUT4) translocation were inhibited by dexamethasone. However, the treatment of EGCG improved insulin-stimulated glucose uptake by increasing GLUT4 translocation to plasma membrane. Furthermore, we also demonstrated these EGCG effects essentially depended on the AMPK and Akt activation. Together, our data suggested that EGCG inhibited dexamethasone-induced insulin resistance through AMPK and PI3K/Akt pathway.     

Metformin induces Rab4 through AMPK and modulates GLUT4 translocation in skeletal muscle cells

Metformin is a major oral anti‐diabetic drug and is known as an insulin sensitizer. However, the mechanism by which metformin acts is unclear. In this study, we found that AICAR, an AMPK activator, and metformin increased the expression of Rab4 mRNA and protein levels in skeletal muscle C2C12 cells. The promoter activity of Rab4 was increased by metformin in an AMPK‐dependent manner. Metformin stimulated the phosphorylation of AS160, Akt substrate, and Rab GTPase activating protein (GAP), and also increased the phosphorylation of PKC‐zeta, which is a critical molecule for glucose uptake. Knockdown of AMPK blocked the metformin‐induced phosphorylation of AS160/PKC‐zeta. In addition, a colorimetric absorbance assay showed that insulin‐induced translocation of GLUT4 was suppressed in Rab4 knockdown cells. Moreover, Rab4 interacted with PKC‐zeta but not with GLUT4. The C‐terminal‐deleted Rab4 mutant, Rab4ΔCT, showed diffuse sub‐cellular localization, while wild‐type Rab4 localized exclusively to the perinuclear membrane. Unlike Rab4ΔCT, wild‐type Rab4 co‐localized with PKC‐zeta. Together, these results demonstrate that metformin induces Rab4 expression via AMPK‐AS160‐PKC‐zeta and modulates insulin‐mediated GLUT4 translocation. J. Cell. Physiol. 226: 974–981, 2011. © 2010 Wiley‐Liss, Inc.     

Artemisia princeps Extract Promoted Glucose Uptake in Cultured L6 Muscle Cells via Glucose Transporter 4 Translocation

Artemisia princeps is a familiar plant as a food substance and medicinal herb. In this study, we evaluated the effects of an ethanol extract of A. princeps (APE) on glucose uptake in differentiated L6 muscle cells. Treatment with APE elevated deoxyglucose uptake, and translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane in L6 myotubes occurred. The PI3K inhibitor LY294002 attenuated glucose uptake induced by APE. Phosphorylation of the Ser⁴⁷³ residue of Akt was not observed, but phosphorylation of PI3K, Akt (Thr³⁰⁸), and atypical PKC was. In addition, APE stimulated phosphorylation of AMP-activated protein kinase (AMPK) at a level similar to 5′-amino-5-imidazolecarboxamide-riboside (AICAR). These results indicate that APE stimulates glucose uptake by inducing GLUT4 translocation, which is in part mediated by combination of the PI3K-dependent atypical PKC pathway and AMPK pathways.     

Insulin accelerates inter-endosomal GLUT4 traffic via phosphatidylinositol 3-kinase and protein kinase B

Insulin enhances plasmalemmal-directed traffic of glucose transporter-4 (GLUT4), but it is unknown whether insulin regulates GLUT4 traffic through endosomal compartments. In L6 myoblasts expressing Myc-tagged GLUT4, insulin markedly stimulated the rate of GLUT4myc recycling. In myoblasts stimulated with insulin to maximize surface GLUT4myc levels, we followed the rates of surface-labeled GLUT4myc endocytosis and chased its intracellular distribution in space and time using confocal immunofluorescence microscopy. Surface-labeled GLUT4myc internalized rapidly (t(12) 3 min), reaching the early endosome by 2 min and the transferrin receptor-rich, perinuclear recycling endosome by 20 min. Upon re-addition of insulin, the t(12) of GLUT4 disappearance from the plasma membrane was unchanged (3 min), but strikingly, GLUT4myc reached the recycling endosome by 10 and left by 20 min. This effect of insulin was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002 or by transiently transfected dominant-negative phosphatidylinositol 3-kinase and protein kinase B mutants. In contrast, insulin did not alter the rate of arrival of rhodamine-labeled transferrin at the recycling endosome. These results reveal a heretofore unknown effect of insulin to accelerate inter-endosomal travel rates of GLUT4 and identify the recycling endosome as an obligatory stage in insulin-dependent GLUT4 recycling.     

AMP-activated protein kinase mediates carotid body excitation by hypoxia

Early detection of an O2 deficit in the bloodstream is essential to initiate corrective changes in the breathing pattern of mammals. Carotid bodies serve an essential role in this respect; their type I cells depolarize when O2 levels fall, causing voltage-gated Ca2+ entry. Subsequent neurosecretion elicits increased afferent chemosensory fiber discharge to induce appropriate changes in respiratory function (1). Although depolarization of type I cells by hypoxia is known to arise from K+ channel inhibition, the identity of the signaling pathway has been contested, and the coupling mechanism is unknown (2). We tested the hypothesis that AMP-activated protein kinase (AMPK) is the effector of hypoxic chemotransduction. AMPK is co-localized at the plasma membrane of type I cells with O2-sensitive K+ channels. In isolated type I cells, activation of AMPK using 5-aminoimidazole-4-carboxamide riboside (AICAR) inhibited O2-sensitive K+ currents (carried by large conductance Ca2+-activated (BKCa) channels and TASK (tandem pore, acid-sensing potassium channel)-like channels, leading to plasma membrane depolarization, Ca2+ influx, and increased chemosensory fiber discharge. Conversely, the AMPK antagonist compound C reversed the effects of hypoxia and AICAR on type I cell and carotid body activation. These results suggest that AMPK activation is both sufficient and necessary for the effects of hypoxia. Furthermore, AMPK activation inhibited currents carried by recombinant BKCa channels, whereas purified AMPK phosphorylated thealpha subunit of the channel in immunoprecipitates, an effect that was stimulated by AMP and inhibited by compound C. Our findings demonstrate a central role for AMPK in stimulus-response coupling by hypoxia and identify for the first time a link between metabolic stress and ion channel regulation in an O2-sensing system.     

Extracellular ATP causes Ca2(+)-dependent and -independent insulin secretion in RINm5F cells. Phospholipase C mediates Ca2+ mobilization but not Ca2+ influx and membrane depolarization

The mechanism by which extracellular ATP stimulates insulin secretion was investigated in RINm5F cells. ATP depolarized the cells as demonstrated both by using the patch-clamp technique and a fluorescent probe. The depolarization is due to closure of ATP-sensitive K+ channels as shown directly in outside-out membrane patches. ATP also raised cytosolic Ca2+ [( Ca2+]i). At the single cell level the latency of the [Ca2+]i response was inversely related to ATP concentration. The [Ca2+]i rise is due both to inositol trisphosphate mediated Ca2+ mobilization and to Ca2+ influx. The former component, as well as inositol trisphosphate generation, were inhibited by phorbol myristate acetate which uncouples agonist receptors from phospholipase C. This manoeuvre did not block Ca2+ influx or membrane depolarization. Diazoxide, which opens ATP-sensitive K+ channels, attenuated membrane depolarization and part of the Ca2+ influx stimulated by ATP. However, the main Ca2+ influx component was unaffected by L-type channel blockers, suggesting the activation of other Ca2+ conductance pathways. ATP increased the rate of insulin secretion by more than 12-fold but the effect was transient. Prolonged exposure to EGTA dissociated the [Ca2+]i rise from ATP-induced insulin secretion, since the former was abolished and the latter only decreased by about 60%. In contrast, vasopressin-evoked insulin secretion was more sensitive to Ca2+ removal than the accompanying [Ca2+]i rise. Inhibition of phospholipase C stimulation by phorbol myristate acetate abrogated vasopressin but only reduced ATP-induced insulin secretion by 34%. These results suggest that ATP stimulates insulin release by both phospholipase C dependent and distinct mechanisms. The Ca2+)-independent component of insulin secretion points to a direct triggering of exocytosis by ATP.