Kinetic Evidence for Unique Regulation of GLUT4 Trafficking by Insulin and AMP-activated Protein Kinase Activators in L6 Myotubes

In L6 myotubes, redistribution of a hemagglutinin (HA) epitope-tagged GLUT4 (HA-GLUT4) to the cell surface occurs rapidly in response to insulin stimulation and AMP-activated protein kinase (AMPK) activation. We have examined whether these separate signaling pathways have a convergent mechanism that leads to GLUT4 mobilization and to changes in GLUT4 recycling. HA antibody uptake on GLUT4 in the basal steady state reached a final equilibrium level that was only 81% of the insulin-stimulated level. AMPK activators (5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and A-769662) led to a similar level of antibody uptake to that found in insulin-stimulated cells. However, the combined responses to insulin stimulation and AMPK activation led to an antibody uptake level of ∼20% above the insulin level. Increases in antibody uptake due to insulin, but not AICAR or A-769662, treatment were reduced by both wortmannin and Akt inhibitor. The GLUT4 internalization rate constant in the basal steady state was very rapid (0.43 min⁻¹) and was decreased during the steady-state responses to insulin (0.18 min⁻¹), AICAR (0.16 min⁻¹), and A-769662 (0.24 min⁻¹). This study has revealed a nonconvergent mobilization of GLUT4 in response to activation of Akt and AMPK signaling. Furthermore, GLUT4 trafficking in L6 muscle cells is very reliant on regulated endocytosis for control of cell surface GLUT4 levels.     

Insulin stimulation of GLUT4 exocytosis, but not its inhibition of endocytosis, is dependent on RabGAP AS160

Insulin maintains whole body blood glucose homeostasis, in part, by regulating the amount of the GLUT4 glucose transporter on the cell surface of fat and muscle cells. Insulin induces the redistribution of GLUT4 from intracellular compartments to the plasma membrane, by stimulating a large increase in exocytosis and a smaller inhibition of endocytosis. A considerable amount is known about the molecular events of insulin signaling and the complex itinerary of GLUT4 trafficking, but less is known about how insulin signaling is transmitted to GLUT4 trafficking. Here, we show that the AS160 RabGAP, a substrate of Akt, is required for insulin stimulation of GLUT4 exocytosis. A dominant-inhibitory mutant of AS160 blocks insulin stimulation of exocytosis at a step before the fusion of GLUT4-containing vesicles with the plasma membrane. This mutant, however, does not block insulin-induced inhibition of GLUT4 endocytosis. These data support a model in which insulin signaling to the exocytosis machinery (AS160 dependent) is distinct from its signaling to the internalization machinery (AS160 independent).     

Inhibition of calpain results in impaired contraction-stimulated GLUT4 translocation in skeletal muscle

It was previously found that transgenic mice that overexpress the calpain inhibitor calpastatin (CsTg) have an approximately 3-fold increase in GLUT4 protein in their skeletal muscles. Despite the increase in GLUT4, which appears to be due to inhibition of its proteolysis by calpain, insulin-stimulated glucose transport is not increased in CsTg muscles. PKB (Akt) protein level is reduced approximately 60% in CsTg muscles, suggesting a possible mechanism for the relative insulin resistance. Muscle contractions stimulate glucose transport by a mechanism that is independent of insulin signaling. The purpose of this study was to test the hypothesis that the threefold increase in GLUT4 in CsTg would result in a large increase in contraction-stimulated glucose transport. CAMKII and AMPK mediate steps in the contraction-stimulated pathway. The protein levels of AMPK and CAMKII were increased three- to fourfold in CsTg muscles, suggesting that these proteins are also calpain substrates. Despite the large increases in GLUT4, AMPK, and CAMKII, contraction-stimulated GLUT4 translocation and glucose transport were not increased above wild-type values. These findings suggest that inhibition of calpain results in impairment of a step in the GLUT4 translocation process downstream of the insulin- and contraction-signaling pathways. They also provide evidence that CAMKII and AMPK are calpain substrates.     

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.     

Insulin-responsive aminopeptidase trafficking in 3T3-L1 adipocytes

The insulin-responsive aminopeptidase (IRAP/VP165/gp160) was identified originally in GLUT4-containing vesicles and shown to translocate in response to insulin, much like the glucose transporter 4 (GLUT4). This study characterizes the trafficking and kinetics of IRAP in exocytosis, endocytosis, and recycling to the membrane in 3T3-L1 adipocytes. After exposure of 3T3-L1 adipocytes to insulin, IRAP translocated to the plasma membrane as assessed by either cell fractionation, surface biotinylation, or the plasma membrane sheet assay. The rate of exocytosis closely paralleled that of GLUT4. In the continuous presence of insulin, IRAP was endocytosed with a half-time of about 3-5 min. IRAP endocytosis is inhibited by cytosol acidification, a property of clathrin-mediated endocytosis, but not by the expression of a constitutively active Akt/PKB. Arrival in an LDM fraction derived via subcellular fractionation exhibited a slower time course than disappearance from the cell surface, suggesting additional endocytic intermediates. As assayed by membrane "sheets," GLUT4 and IRAP showed similar internalization rates that are wortmannin-insensitive and occur with a half-time of roughly 5 min. IRAP remaining on the cell surface 10 min following insulin removal was both biotin- and avidin-accessible, implying the absence of thin-necked invaginations. Finally, endocytosed IRAP quickly recycled back to the plasma membrane in a wortmannin-sensitive process. These results demonstrate rapid endocytosis and recycling of IRAP in the presence of insulin and trafficking that matches GLUT4 in rate.     

Muscle cell depolarization induces a gain in surface GLUT4 via reduced endocytosis independently of AMPK

Contracting skeletal muscle increases glucose uptake to sustain energy demand. This is achieved through a gain in GLUT4 at the membrane, but the traffic mechanisms and regulatory signals involved are unknown. Muscle contraction is elicited by membrane depolarization followed by a rise in cytosolic Ca2+ and actomyosin activation, drawing on ATP stores. It is unknown whether one or more of these events triggers the rise in surface GLUT4. Here, we investigate the effect of membrane depolarization on GLUT4 cycling using GLUT4myc-expressing L6 myotubes devoid of sarcomeres and thus unable to contract. K+-induced membrane depolarization elevated surface GLUT4myc, and this effect was additive to that of insulin, was not prevented by inhibiting phosphatidylinositol 3-kinase (PI3K) or actin polymerization, and did not involve Akt activation. Instead, depolarization elevated cytosolic Ca2+, and the surface GLUT4myc elevation was prevented by dantrolene (an inhibitor of Ca2+ release from sarcoplasmic reticulum) and by extracellular Ca2+ chelation. Ca2+-calmodulin-dependent protein kinase-II (CaMKII) was not phosphorylated after 10 min of K+ depolarization, and the CaMK inhibitor KN62 did not prevent the gain in surface GLUT4myc. Interestingly, although 5'-AMP-activated protein kinase (AMPK) was phosphorylated upon depolarization, lowering AMPKalpha via siRNA did not alter the surface GLUT4myc gain. Conversely, the latter response was abolished by the PKC inhibitors bisindolylmaleimide I and calphostin C. Unlike insulin, K+ depolarization caused only a small increase in GLUT4myc exocytosis and a major reduction in its endocytosis. We propose that K+ depolarization reduces GLUT4 internalization through signals and mechanisms distinct from those engaged by insulin. Such a pathway(s) is largely independent of PI3K, Akt, AMPK, and CaMKII but may involve PKC.     

Rapid activation of Akt2 is sufficient to stimulate GLUT4 translocation in 3T3-L1 adipocytes

The serine/threonine kinase Akt2 has been implicated in insulin-regulated glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. However, it remains unclear whether activation of Akt2 is sufficient since a role for alternate signaling pathways has been proposed. Here we have engineered 3T3-L1 adipocytes to express a rapidly inducible Akt2 system based on drug-inducible heterodimerization. Addition of the dimerizer rapalog resulted in activation of Akt2 within 5 min, concomitant with phosphorylation of the Akt substrates AS160 and GSK3. Comparison with insulin stimulation revealed that the level of Akt2 activity observed with rapalog was within the physiological range, reducing the likelihood of off-target effects. Transient activation of Akt2 also increased glucose transport and GLUT4 translocation to the plasma membrane. These results show that activation of Akt2 is sufficient to stimulate GLUT4 translocation in 3T3-L1 adipocytes to an extent similar to insulin.     

Myosin 5a is an insulin-stimulated Akt2 (protein kinase Bbeta) substrate modulating GLUT4 vesicle translocation

Phosphatidylinositol 3-kinase activation of Akt signaling is critical to insulin-stimulated glucose transport and GLUT4 translocation. However, the downstream signaling events following Akt activation which mediate glucose transport stimulation remain relatively unknown. Here we identify an Akt consensus phosphorylation motif in the actin-based motor protein myosin 5a and show that insulin stimulation leads to phosphorylation of myosin 5a at serine 1650. This Akt-mediated phosphorylation event enhances the ability of myosin 5a to interact with the actin cytoskeleton. Small interfering RNA-induced inhibition of myosin 5a and expression of dominant-negative myosin 5a attenuate insulin-stimulated glucose transport and GLUT4 translocation. Furthermore, knockdown of Akt2 or expression of dominant-negative Akt (DN-Akt) abolished insulin-stimulated phosphorylation of myosin 5a, inhibited myosin 5a binding to actin, and blocked insulin-stimulated glucose transport. Taken together, these data indicate that myosin 5a is a newly identified direct substrate of Akt2 and, upon insulin stimulation, phosphorylated myosin 5a facilitates anterograde movement of GLUT4 vesicles along actin to the cell surface.     

How many signals impinge on GLUT4 activation by insulin?

GLUT4 is the main glucose transporter activated by insulin in skeletal muscle cells and adipocytes. GLUT4 storage vesicles (GSVs) traffic in endocytic and exocytic compartments. In the basal state, GLUT4 compartments are preferentially sequestered in perinuclear deposits wherein stimuli including insulin and non-insulin factors can increase GLUT4 vesicle formation, its exocytosis, and fusion to plasma membrane. In addition to well-established effectors of insulin signaling pathway, such as PKCzeta and Akt, the cytoskeletal network is implicated in GLUT4 translocation. This review will discuss the mechanisms and activation of GLUT4 trafficking and incorporating to PM from three aspects: known molecules of the insulin signaling pathway; Rho and Rab family proteins and cytoskeletal molecules.     

Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria

Although diabetes has been identified as a major risk factor for atrial fibrillation, little is known about glucose metabolism in the healthy and diabetic atria. Glucose transport into the cell, the rate-limiting step of glucose utilization, is regulated by the Glucose Transporters (GLUTs). Although GLUT4 is the major isoform in the heart, GLUT8 has recently emerged as a novel cardiac isoform. We hypothesized that GLUT-4 and -8 translocation to the atrial cell surface will be regulated by insulin and impaired during insulin-dependent diabetes. GLUT protein content was measured by Western blotting in healthy cardiac myocytes and type 1 (streptozotocin-induced, T1Dx) diabetic rodents. Active cell surface GLUT content was measured using a biotinylated photolabeled assay in the perfused heart. In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05). Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria. During diabetes, active cell surface GLUT-4 and -8 content was downregulated in the atria (by 70% and 90%, respectively, P<0.05). Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway. This was confirmed by the rescued translocation of GLUT-4 and -8 to the atrial cell surface upon insulin stimulation in the atria of type 1 diabetic subjects. In conclusion, our data suggest that: 1) both GLUT-4 and -8 are insulin-sensitive in the healthy atria through an Akt/AS160 dependent pathway; 2) GLUT-4 and -8 trafficking is impaired in the diabetic atria and rescued by insulin treatment. Alterations in atrial glucose transport may induce perturbations in energy production, which may provide a metabolic substrate for atrial fibrillation during diabetes.     

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.     

Selective Insulin Resistance in Adipocytes

Aside from glucose metabolism, insulin regulates a variety of pathways in peripheral tissues. Under insulin-resistant conditions, it is well known that insulin-stimulated glucose uptake is impaired, and many studies attribute this to a defect in Akt signaling. Here we make use of several insulin resistance models, including insulin-resistant 3T3-L1 adipocytes and fat explants prepared from high fat-fed C57BL/6J and ob/ob mice, to comprehensively distinguish defective from unaffected aspects of insulin signaling and its downstream consequences in adipocytes. Defective regulation of glucose uptake was observed in all models of insulin resistance, whereas other major actions of insulin such as protein synthesis and anti-lipolysis were normal. This defect corresponded to a reduction in the maximum response to insulin. The pattern of change observed for phosphorylation in the Akt pathway was inconsistent with a simple defect at the level of Akt. The only Akt substrate that showed consistently reduced phosphorylation was the RabGAP AS160 that regulates GLUT4 translocation. We conclude that insulin resistance in adipose tissue is highly selective for glucose metabolism and likely involves a defect in one of the components regulating GLUT4 translocation to the cell surface in response to insulin.     

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.     

Galpha i2 enhances in vivo activation of and insulin signaling to GLUT4.

Heterotrimeric G-proteins, including Galpha(i2), have been implicated in modulating glucose disposal and insulin signaling. This cross-talk between G-protein-coupled and tyrosine kinase-coupled signaling pathways is a focal point for the study of integration of cell signaling. Herein we study the role of Galpha(i2) in modulating glucose transport, focusing upon linkages to insulin signaling. Utilizing mice harboring a transgene that directs the expression of a constitutively activated, GTPase-deficient mutant of Galpha(i2) (Q205L) in adipose tissue, skeletal muscle, and liver, we demonstrate that Galpha(i2) regulates the translocation of the insulin-sensitive GLUT4 glucose transporter in skeletal muscle and adipose tissue. The expression of Q205L Galpha(i2) increased glucose transport and translocation of GLUT4 to the plasma membrane in vivo in the absence of insulin stimulation. Adipocytes from the Q205L Galpha(i2) mice displayed enhanced insulin-stimulated glucose transport and GLUT4 translocation to the plasma membrane to levels nearly twice that of those from littermate controls. Phosphatidylinositol 3-kinase and Akt activities were constitutively activated in tissues expressing the Q205L Galpha(i2). Studies of adipocytes from wild-type mice displayed short term activation of phosphatidylinositol 3-kinase, Akt, and GLUT4 translocation in response to activation of Galpha(i2) by lysophosphatidic acid, a response sensitive to pertussis toxin. These data provide an explanation for the marked glucose tolerance of the Q205L Galpha(i2) mice and demonstrate a linkage between Galpha(i2) and GLUT4 translocation.     

Understanding the Mechanism Underlie the Antidiabetic Activity of Oleuropein Using Ex-Vivo Approach

Background:Oleuropein, the main constituent of olive fruit and leaves, has been reported to protect against insulin resistance and diabetes. While many experimental investigations have examined the mechanisms by which oleuropein improves insulin resistance and diabetes, much of these investigations have been carried out in either muscle cell lines or in vivo models two scenarios with many drawbacks. Accordingly, to simplify identification of mechanisms by which oleuropein regulates specific cellular processes, we resort, in the present study, to isolated muscle preparation which enables better metabolic milieu control and permit more detailed analyses.Methods:For this purpose, soleus muscles were incubated for 12 h without or with palmitate (1.5 mM) in the presence or absence of oleuropein (1.5 mM), and compound C. Insulin-stimulated glucose transport, glucose transporter type 4 (GLUT4) translocation, Akt substrate of 160 kDa (AS160) phosphorylation and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation were examined.Results:Palmitate treatment reduced insulin-stimulated glucose transport, GLUT4 translocation and AS160 phosphorylation, but AMPK phosphorylation was not changed. Oleuropein administration (12 h) fully rescued insulin-stimulated glucose transport, but partially restored GLUT4 translocation. However, it fully restored AS160 phosphorylation, raising the possibility that oleuropein may also have contributed to the restoration of glucose transport by increased GLUT4 intrinsic activity. Inhibition of AMPK phosphorylation with compound C (50 µM) prevented oleuropein -induced improvements in insulin-stimulated glucose transport, GLUT4 translocation, and AS160 phosphorylation.Conclusion:Our results clearly indicate that oleuropein alleviates palmitate-induced insulin resistance appears to occur via an AMPK-dependent mechanism involving improvements in the functionality of the AS160-GLUT4 signaling system.Key Words: AMPK, GLUT4, Muscle, Insulin resistance, Oleuropein     

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.     

Chromium enhances insulin responsiveness via AMPK

Trivalent chromium (Cr³⁺) is known to improve glucose homeostasis. Cr³⁺ has been shown to improve plasma membrane-based aspects of glucose transporter GLUT4 regulation and increase activity of the cellular energy sensor 5’ AMP-activated protein kinase (AMPK). However, the mechanism(s) by which Cr³⁺ improves insulin responsiveness and whether AMPK mediates this action is not known. In this study we tested if Cr³⁺ protected against physiological hyperinsulinemia-induced plasma membrane cholesterol accumulation, cortical filamentous actin (F-actin) loss and insulin resistance in L6 skeletal muscle myotubes. In addition, we performed mechanistic studies to test our hypothesis that AMPK mediates the effects of Cr³⁺ on GLUT4 and glucose transport regulation. Hyperinsulinemia-induced insulin-resistant L6 myotubes displayed excess membrane cholesterol and diminished cortical F-actin essential for effective glucose transport regulation. These membrane and cytoskeletal abnormalities were associated with defects in insulin-stimulated GLUT4 translocation and glucose transport. Supplementing the culture medium with pharmacologically relevant doses of Cr³⁺ in the picolinate form (CrPic) protected against membrane cholesterol accumulation, F-actin loss, GLUT4 dysregulation and glucose transport dysfunction. Insulin signaling was neither impaired by hyperinsulinemic conditions nor enhanced by CrPic, whereas CrPic increased AMPK signaling. Mechanistically, siRNA-mediated depletion of AMPK abolished the protective effects of CrPic against GLUT4 and glucose transport dysregulation. Together these findings suggest that the micronutrient Cr³⁺, via increasing AMPK activity, positively impacts skeletal muscle cell insulin sensitivity and glucose transport regulation.     

Inhibition of GLUT4 translocation by Tbc1d1, a Rab GTPase-activating protein abundant in skeletal muscle, is partially relieved by AMP-activated protein kinase activation

Insulin increases glucose transport by stimulating the trafficking of intracellular GLUT4 to the cell surface, a process known as GLUT4 translocation. A key protein in signaling this process is AS160, a Rab GTPase-activating protein (GAP) whose activity appears to be suppressed by Akt phosphorylation. Tbc1d1 is a Rab GAP with a sequence highly similar to that of AS160 and with the same Rab specificity as that of AS160. The role of Tbc1d1 in regulating GLUT4 trafficking has been unclear. Our previous study showed that overexpressed Tbc1d1 inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes, even though insulin caused phosphorylation on its single canonical Akt motif. In the present study, we show in 3T3-L1 adipocytes that Tbc1d1 is only 1/20 as abundant as AS160, that knockdown of Tbc1d1 has no effect on insulin-stimulated GLUT4 translocation, and that overexpressed Tbc1d1 also inhibits GLUT4 translocation elicited by activated Akt expression. These results indicate that endogenous Tbc1d1 does not participate in insulin-regulated GLUT4 translocation in adipocytes and suggest that the GAP activity of Tbc1d1 is not suppressed by Akt phosphorylation. In addition, we discovered that Tbc1d1 is much more highly expressed in skeletal muscle than fat and that the AMP-activated protein kinase (AMPK) activator 5'-aminoimidazole-4-carboxamide ribonucleoside partially reversed the inhibition of insulin-stimulated GLUT4 translocation by overexpressed Tbc1d1 in 3T3-L1 adipocytes. 5'-Aminoimidazole-4-carboxamide ribonucleoside activation of the kinase AMPK is known to cause GLUT4 translocation in muscle. The above findings strongly suggest that Tbc1d1 is a component in the signal transduction pathway leading to AMPK-stimulated GLUT4 translocation in muscle.     

Cold-induced PGC-1alpha expression modulates muscle glucose uptake through an insulin receptor/Akt-independent, AMPK-dependent pathway

Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) participates in control of expression of genes involved in adaptive thermogenesis, muscle fiber type differentiation, and fuel homeostasis. The objective of the present study was to evaluate the participation of cold-induced PGC-1alpha expression in muscle fiber type-specific activity of proteins that belong to the insulin-signaling pathway. Rats were exposed to 4 degrees C for 4 days and acutely treated with insulin in the presence or absence of an antisense oligonucleotide to PGC-1alpha. Cold exposure promoted a significant increase of PGC-1alpha and uncoupling protein-3 protein expression in type I and type II fibers of gastrocnemius muscle. In addition, cold exposure led to higher glucose uptake during a hyperinsulinemic clamp, which was accompanied by higher expression and membrane localization of GLUT4 in both muscle fiber types. Cold exposure promoted significantly lower insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and Ser473 phosphorylation of acute transforming retrovirus thymoma (Akt) and an insulin-independent increase of Thr172 phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). Inhibition of PGC-1alpha expression in cold-exposed rats by antisense oligonucleotide treatment diminished glucose clearance rates during a hyperinsulinemic clamp and reduced expression and membrane localization of GLUT4. Reduction of PGC-1alpha expression resulted in no modification of insulin-induced tyrosine phosphorylation of the IR and Ser473 phosphorylation of Akt. Finally, reduction of PGC-1alpha resulted in lower Thr172 phosphorylation of AMPK. Thus cold-induced hyperexpression of PGC-1alpha participates in control of skeletal muscle glucose uptake through a mechanism that controls GLUT4 expression and subcellular localization independent of the IR and Akt activities but dependent on AMPK.     

Aldolase mediates the association of F-actin with the insulin-responsive glucose transporter GLUT4.

To identify potential proteins interacting with the insulin-responsive glucose transporter (GLUT4), we generated fusion proteins of glutathione S-transferase (GST) and the final 30 amino acids from GLUT4 (GST-G4) or GLUT1 (GST-G1). Incubation of these carboxyl-terminal fusion proteins with adipocyte cell extracts revealed a specific interaction of GLUT4 with fructose 1, 6-bisphosphate aldolase. In the presence of aldolase, GST-G4 but not GST-G1 was able to co-pellet with filamentous (F)-actin. This interaction was prevented by incubation with the aldolase substrates, fructose 1,6-bisphosphate or glyceraldehyde 3-phosphate. Immunofluorescence confocal microscopy demonstrated a significant co-localization of aldolase and GLUT4 in intact 3T3L1 adipocytes, which decreased following insulin stimulation. Introduction into permeabilized 3T3L1 adipocytes of fructose 1,6-bisphosphate or the metabolic inhibitor 2-deoxyglucose, two agents that disrupt the interaction between aldolase and actin, inhibited insulin-stimulated GLUT4 exocytosis without affecting GLUT4 endocytosis. Furthermore, microinjection of an aldolase-specific antibody also inhibited insulin-stimulated GLUT4 translocation. These data suggest that aldolase functions as a scaffolding protein for GLUT4 and that glucose metabolism may provide a negative feedback signal for the regulation of glucose transport by insulin.