Disruption of microtubules ablates the specificity of insulin signaling to GLUT4 translocation in 3T3-L1 adipocytes

Although the cytoskeletal network is important for insulin-induced glucose uptake, several studies have assessed the effects of microtubule disruption on glucose transport with divergent results. Here, we investigated the effects of microtubule-depolymerizing reagent, nocodazole and colchicine, on GLUT4 translocation in 3T3-L1 adipocytes. After nocodazole treatment to disrupt microtubules, GLUT4 vesicles were dispersed from the perinuclear region in the basal state, and insulin-induced GLUT4 translocation was partially inhibited by 20-30%, consistent with other reports. We found that platelet-derived growth factor (PDGF), which did not stimulate GLUT4 translocation in intact cells, was surprisingly able to enhance GLUT4 translocation to approximately 50% of the maximal insulin response, in nocodazole-treated cells with disrupted microtubules. This effect of PDGF was blocked by pretreatment with wortmannin and attenuated in cells pretreated with cytochalasin D. Using confocal microscopy, we found an increased co-localization of GLUT4 and F-actin in nocodazole-treated cells upon PDGF stimulation compared with control cells. Furthermore, microinjection of small interfering RNA targeting the actin-based motor Myo1c, but not the microtubule-based motor KIF3, significantly inhibited both insulin- and PDGF-stimulated GLUT4 translocation after nocodazole treatment. In summary, our data suggest that 1) proper perinuclear localization of GLUT4 vesicles is a requirement for insulin-specific stimulation of GLUT4 translocation, and 2) nocodazole treatment disperses GLUT4 vesicles from the perinuclear region allowing them to engage insulin and PDGF-sensitive actin filaments, which can participate in GLUT4 translocation in a phosphatidylinositol 3-kinase-dependent manner.     

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.     

Mechanisms underlying impaired GLUT-4 translocation in glycogen-supercompensated muscles of exercised rats

Exercise training induces an increase in GLUT-4 in muscle. We previously found that feeding rats a high-carbohydrate diet after exercise, with muscle glycogen supercompensation, results in a decrease in insulin responsiveness so severe that it masks the effect of a training-induced twofold increase in GLUT-4 on insulin-stimulated muscle glucose transport. One purpose of this study was to determine whether insulin signaling is impaired. Maximally insulin-stimulated phosphatidylinositol (PI) 3-kinase activity was not significantly reduced, whereas protein kinase B (PKB) phosphorylation was approximately 50% lower (P < 0.01) in muscles of chow-fed, than in those of fasted, exercise-trained rats. Our second purpose was to determine whether contraction-stimulated glucose transport is also impaired. The stimulation of glucose transport and the increase in cell surface GLUT-4 induced by contractions were both decreased by approximately 65% in glycogen-supercompensated muscles of trained rats. The contraction-stimulated increase in AMP kinase activity, which has been implicated in the activation of glucose transport by contractions, was approximately 80% lower in the muscles of the fed compared with the fasted rats 18 h after exercise. These results show that both the insulin- and contraction-stimulated pathways for muscle glucose transport activation are impaired in glycogen-supercompensated muscles and provide insight regarding possible mechanisms.     

Are tyrosine kinases involved in mediating contraction-stimulated muscle glucose transport?

Muscle contractions and insulin stimulate glucose transport into muscle by separate pathways. The contraction-mediated increase in glucose transport is mediated by two mechanisms, one involves the activation of 5'-AMP-activated protein kinase (AMPK) and the other involves the activation of calcium/calmodulin-dependent protein kinase II (CAMKII). The steps leading from the activation of AMPK and CAMKII to the translocation of GLUT4 to the cell surface have not been identified. Studies with the use of the tyrosine kinase inhibitor genistein suggest that one or more tyrosine kinases could be involved in contraction-stimulated glucose transport. The purpose of the present study was to determine the involvement of tyrosine kinases in contraction-stimulated glucose transport in rat soleus and epitrochlearis muscles. Contraction-stimulated glucose transport was completely prevented by pretreatment with genistein (100 microM) and the related compound butein (100 microM). However, the structurally distinct tyrosine kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyridine and herbimycin did not reduce contraction-stimulated glucose transport. Furthermore, genistein and butein inhibited glucose transport even when muscles were exposed to these compounds after being stimulated to contract. Muscle contractions did not result in increases in tyrosine phosphorylation of proteins such as proline-rich tyrosine kinase and SRC. These results provide evidence that tyrosine kinases do not mediate contraction-stimulated glucose transport and that the inhibitory effects of genistein on glucose transport result from direct inhibition of the glucose transporters at the cell surface.     

Insulin-mediated GLUT4 translocation is dependent on the microtubule network

The GLUT4 facilitative glucose transporter is recruited to the plasma membrane by insulin. This process depends primarily on the exocytosis of a specialized pool of vesicles containing GLUT4 in their membranes. The mechanism of GLUT4 vesicle exocytosis in response to insulin is not understood. To determine whether GLUT4 exocytosis is dependent on intact microtubule network, we measured insulin-mediated GLUT4 exocytosis in 3T3-L1 adipocytes in which the microtubule network was depolymerized by pretreatment with nocodazole. Insulin-mediated GLUT4 translocation was inhibited by more than 80% in nocodazole-treated cells. Phosphorylation of insulin receptor substrate 1 (IRS-1), activation of IRS-1 associated phosphatidylinositide 3-kinase, and phosphorylation of protein kinase B/Akt-1 were not inhibited by nocodazole treatment indicating that the microtubule network was not required for proximal insulin signaling. An intact microtubule network is specifically required for insulin-mediated GLUT4 translocation since nocodazole treatment did not affect insulin-mediated GLUT1 translocation or adipsin secretion. By using in vitro microtubule binding, we demonstrated that both GLUT4 vesicles and IRS-1 bind specifically to microtubules, implicating microtubules in both insulin signaling and GLUT4 translocation. Vesicle binding to microtubules was not mediated through direct binding of GLUT4 or insulin-responsive aminopeptidase to microtubules. A model microtubule-dependent translocation of GLUT4 is proposed.     

Nocodazole inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes via a microtubule-independent mechanism

Insulin stimulates glucose transport in adipocytes and muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Recent reports have shown that the microtubule-depolymerizing agent nocodazole inhibits insulin-stimulated glucose transport, implicating an important role for microtubules in this process. In the present study we show that 2 microm nocodazole completely depolymerized microtubules in 3T3-L1 adipocytes, as determined morphologically and biochemically, resulting in dispersal of the perinuclear GLUT4 compartment and the Golgi apparatus. However, 2 microm nocodazole did not significantly effect either the kinetics or magnitude of insulin-stimulated glucose transport. Consistent with previous studies, higher concentrations of nocodazole (10-33 microm) significantly inhibited basal and insulin-stimulated glucose uptake in adipocytes. This effect was not likely the result of microtubule depolymerization because in the presence of taxol, which blocked nocodazole-induced depolymerization of microtubules as well as the dispersal of the perinuclear GLUT4 compartment, the inhibitory effect of 10-33 microm nocodazole on insulin-stimulated glucose uptake prevailed. Despite the decrease in insulin-stimulated glucose transport with 33 microm nocodazole we did not observe inhibition of insulin-stimulated GLUT4 translocation to the cell surface under these conditions. Consistent with a direct effect of nocodazole on glucose transporter function we observed a rapid inhibitory effect of nocodazole on glucose transport activity when added to either 3T3-L1 adipocytes or to Chinese hamster ovary cells at 4 degrees C. These studies reveal a new and unexpected effect of nocodazole in mammalian cells which appears to occur independently of its microtubule-depolymerizing effects.     

Insulin recruits GLUT4 from distinct compartments via distinct traffic pathways with differential microtubule dependence in rat adipocytes

In the present study, we investigated the physiological significance of the microtubules in the subcellular localization and trafficking of GLUT4 in rat primary adipocytes. Morphological and biochemical analyses revealed a dose- and time-dependent disruption of the microtubules by treatment with nocodazole. With nearly complete disruption of the microtubules, the insulin-stimulated glucose transport activity was inhibited by 55%. This inhibition was concomitant with a comparable inhibition of GLUT4 translocation measured by the subcellular fractionation and the cell-surface GLUT4 labeling by trypsin cleavage. In addition, the time-course of insulin stimulation of the glucose transport activity was significantly delayed by microtubule disruption (t(1/2) were 7 and 2.3 min in nocodazole-treated and control cells, respectively), while the rate of GLUT4 endocytosis was little affected. The impaired insulin-stimulated glucose transport activity was not fully restored to the level in control cells by blocking GLUT4 endocytosis, suggesting that the inhibition was due to the existence of a microtubule-dependent subpopulation in the insulin-responsive GLUT4 pool. On the other hand, nocodazole partially inhibited insulin-induced translocation of the insulin-regulated aminopeptidase and the vesicle-associated membrane protein (VAMP)-2 without affecting GLUT1 and VAMP-3. In electrically permeabilized adipocytes, the insulin-stimulated glucose transport was inhibited by 40% by disruption of the microtubules whereas that stimulated with GTP gamma S was not affected. Intriguingly, the two reagents stimulated glucose transport to the comparable level by disruption of the microtubules. These data suggest that insulin recruits GLUT4 to the plasma membrane from at least two distinct intracellular compartments via distinct traffic routes with differential microtubule dependence in rat primary adipocytes.     

AS160 regulates insulin- and contraction-stimulated glucose uptake in mouse skeletal muscle

Insulin and contraction are potent stimulators of GLUT4 translocation and increase skeletal muscle glucose uptake. We recently identified the Rab GTPase-activating protein (GAP) AS160 as a putative point of convergence linking distinct upstream signaling cascades induced by insulin and contraction in mouse skeletal muscle. Here, we studied the functional implications of these AS160 signaling events by using an in vivo electroporation technique to overexpress wild type and three AS160 mutants in mouse tibialis anterior muscles: 1) AS160 mutated to prevent phosphorylation on four regulatory phospho-Akt-substrate sites (4P); 2) AS160 mutated to abolish Rab GTPase activity (R/K); and 3) double mutant AS160 containing both 4P and R/K mutations (2M). One week following gene injection, protein expression for all AS160 isoforms was elevated over 7-fold. To determine the effects of AS160 on insulin- and contraction-stimulated glucose uptake in transfected muscles, we measured [3H]2-deoxyglucose uptake in vivo following intravenous glucose administration and in situ muscle contraction, respectively. Insulin-stimulated glucose uptake was significantly inhibited in muscles overexpressing 4P mutant AS160. However, this inhibition was completely prevented by concomitant disruption of AS160 Rab GAP activity. Transfection with 4P mutant AS160 also significantly impaired contraction-stimulated glucose uptake, as did overexpression of wild type AS160. In contrast, overexpressing mutant AS160 lacking Rab GAP activity resulted in increases in both sham and contraction-stimulated muscles. These data suggest that AS160 regulates both insulin- and contraction-stimulated glucose metabolism in mouse skeletal muscle in vivo and that the effects of mutant AS160 on the actions of insulin and contraction are not identical. Our findings directly implicate AS160 as a critical convergence factor for independent stimulators of skeletal muscle glucose uptake.     

Contraction signaling to glucose transport in skeletal muscle.

Contracting skeletal muscles acutely increases glucose transport in both healthy individuals and in people with Type 2 diabetes, and regular physical exercise is a cornerstone in the treatment of the disease. Glucose transport in skeletal muscle is dependent on the translocation of GLUT4 glucose transporters to the cell surface. It has long been believed that there are two major signaling mechanisms leading to GLUT4 translocation. One mechanism is insulin-activated signaling through insulin receptor substrate-1 and phosphatidylinositol 3-kinase. The other is an insulin-independent signaling mechanism that is activated by contractions, but the mediators of this signal are still unknown. Accumulating evidence suggests that the energy-sensing enzyme AMP-activated protein kinase plays an important role in contraction-stimulated glucose transport. However, more recent studies in transgenic and knockout animals show that AMP-activated protein kinase is not the sole mediator of the signal to GLUT4 translocation and suggest that there may be redundant signaling pathways leading to contraction-stimulated glucose transport. The search for other possible signal intermediates is ongoing, and calcium, nitric oxide, bradykinin, and the Akt substrate AS160 have been suggested as possible candidates. Further research is needed because full elucidation of an insulin-independent signal leading to glucose transport would be a promising pharmacological target for the treatment of Type 2 diabetes.     

How muscle insulin sensitivity is regulated: testing of a hypothesis

Muscle contractions induce an increase in glucose transport. The acute effect of muscle contractions on glucose transport is independent of insulin and reverses rapidly after cessation of exercise. As the acute increase in glucose transport reverses, a marked increase in the sensitivity of muscle to insulin occurs. The mechanism for this phenomenon is unknown. We hypothesize that an increase in insulin sensitivity is a general phenomenon that occurs during reversal of an increase in cell surface GLUT4 induced by any stimulus, not just exercise. To test this hypothesis, epitrochlearis, rat soleus, and flexor digitorum brevis muscles were incubated for 30 min with a maximally effective insulin concentration (1.0 mU/ml). Muscles were allowed to recover for 3 h in the absence of insulin. Muscles were then exposed to 60 microU/ml insulin for 30 min followed by measurement of glucose transport. Preincubation with 1.0 mU/ml insulin resulted in an approximately 2-fold greater increase in glucose transport 3.5 h later in response to 60 microU/ml insulin than that which occurred in control muscles treated with 60 microU/ml insulin. Pretreatment of muscles with combined maximal insulin and exercise stimuli greatly amplified the increase in insulin sensitivity. The increases in glucose transport were paralleled by increases in cell surface GLUT4. We conclude that stimulation of glucose transport by any agent is followed by an increase in sensitivity of glucose transport to activation that is mediated by translocation of more GLUT4 to the cell surface.     

Disruption of microtubules in living cells by tyrphostin AG-1714

Tyrphostin AG-1714 and several related molecules with the general structure of nitro-benzene malononitrile (BMN) disrupt microtubules in a large variety of cultured cells. This process can be inhibited by the stabilization of microtubules with taxol or by pretreatment of the cells with pervanadate, which inhibits tyrosine phosphatases and increases the overall levels of phosphotyrosine in cells. Unlike other microtubule-disrupting drugs such as nocodazole or colchicine, tyrphostin AG-1714 does not interfere with microtubule polymerization or stability in vitro, suggesting that the effect of this tyrphostin on microtubules is indirect. These results imply an involvement of protein tyrosine phosphorylation in the regulation of overall microtubule dynamics. Tyrphostins of AG-1714 type could thus be powerful tools for the identification of such microtubule regulatory pathways.     

Absence of insulin receptor substrate-1 expression does not alter GLUT1 or GLUT4 abundance or contraction-stimulated glucose uptake by mouse skeletal muscle.

The purpose of this study was to determine the influence of insulin receptor substrate-1 (IRS-1) expression on GLUT1 and GLUT4 glucose transporter protein abundance, contraction-stimulated glucose uptake, and contraction-induced glycogen depletion by skeletal muscle. Mice (6 months old) from three genotypes were studied: wild-type (IRS-1(+/+)), heterozygous (IRS-1(+/-)) for the null allele, and IRS-1 knockouts (IRS-1(-/-)) lacking a functional IRS-1 gene. In situ muscle contraction was induced (electrical stimulation of sciatic nerve) in one hindlimb using contralateral muscles as controls. Soleus and extensor digitorum longus were dissected and 2-deoxyglucose uptake was measured in vitro. 2-Deoxyglucose uptake was higher in basal muscles (no contractions) from IRS-1(-/-) vs. both other genotypes. Contraction-stimulated 2-deoxyglucose uptake and glycogen depletion did not differ among genotypes. Muscle IRS-1 protein was undetectable for IRS-1(-/-) mice, and values were approximately 40 % lower in IRS-1(+/-) than in IRS-1(+/+) mice. No difference was found in IRS-1(+/+) compared to IRS-1(-/-) groups regarding muscle abundance of GLUT1 and GLUT4. Substantial reduction or elimination of IRS-1 did not alter the hallmark effects of contractions on muscle carbohydrate metabolism--activation of glucose uptake and glycogen depletion.     

Myo1c regulates glucose uptake in mouse skeletal muscle

Contraction and insulin promote glucose uptake in skeletal muscle through GLUT4 translocation to cell surface membranes. Although the signaling mechanisms leading to GLUT4 translocation have been extensively studied in muscle, the cellular transport machinery is poorly understood. Myo1c is an actin-based motor protein implicated in GLUT4 translocation in adipocytes; however, the expression profile and role of Myo1c in skeletal muscle have not been investigated. Myo1c protein abundance was higher in more oxidative skeletal muscles and heart. Voluntary wheel exercise (4 weeks, 8.2 ± 0.8 km/day), which increased the oxidative profile of the triceps muscle, significantly increased Myo1c protein levels by ～2-fold versus sedentary controls. In contrast, high fat feeding (9 weeks, 60% fat) significantly reduced Myo1c by 17% in tibialis anterior muscle. To study Myo1c regulation of glucose uptake, we expressed wild-type Myo1c or Myo1c mutated at the ATPase catalytic site (K111A-Myo1c) in mouse tibialis anterior muscles in vivo and assessed glucose uptake in vivo in the basal state, in response to 15 min of in situ contraction, and 15 min following maximal insulin injection (16.6 units/kg of body weight). Expression of wild-type Myo1c or K111A-Myo1c had no effect on basal glucose uptake. However, expression of wild-type Myo1c significantly increased contraction- and insulin-stimulated glucose uptake, whereas expression of K111A-Myo1c decreased both contraction-stimulated and insulin-stimulated glucose uptake. Neither wild-type nor K111A-Myo1c expression altered GLUT4 expression, and neither affected contraction- or insulin-stimulated signaling proteins. Myo1c is a novel mediator of both insulin-stimulated and contraction-stimulated glucose uptake in skeletal muscle.     

Wortmannin inhibits both insulin- and contraction-stimulated glucose uptake and transport in rat skeletal muscle

Wojtaszewski, Jørgen F. P., Bo F. Hansen, BirgitteUrsø, and Erik A. Richter. Wortmannin inhibits both insulin-and contraction-stimulated glucose uptake and transport in rat skeletal muscle. J. Appl. Physiol. 81(4):1501-1509, 1996.The role of phosphatidylinositol (PI) 3-kinasefor insulin- and contraction-stimulated muscle glucose transport wasinvestigated in rat skeletal muscle perfused with a cell-freeperfusate. The insulin receptor substrate-1-associated PI 3-kinaseactivity was increased sixfold upon insulin stimulation but wasunaffected by contractions. In addition, the insulin-stimulated PI3-kinase activity and muscle glucose uptake and transport in individualmuscles were dose-dependently inhibited by wortmannin with one-halfmaximal inhibition values of ~10 nM and total inhibition at 1 µM.This concentration of wortmannin also decreased thecontraction-stimulated glucose transport and uptake by ~30-70%without confounding effects on contractility or on muscle ATP andphosphocreatine concentrations. At higher concentrations(3 and 10 µM), wortmannin completely blocked thecontraction-stimulated glucose uptake but also decreased thecontractility. In conclusion, inhibition of PI 3-kinase with wortmanninin skeletal muscle coincides with inhibition of insulin-stimulated glucose uptake and transport. Furthermore, in contrast to recent findings in incubated muscle, wortmannin also inhibitedcontraction-stimulated glucose uptake and transport. The inhibitoryeffect of wortmannin on contraction-stimulated glucose uptake may beindependent of PI 3-kinase activity or due to inhibition of asubfraction of PI 3-kinase with low sensitivity to wortmannin.

     

Contraction- and hypoxia-stimulated glucose transport is mediated by a Ca2+-dependent mechanism in slow-twitch rat soleus muscle

Increases in contraction-stimulated glucose transport in fast-twitch rat epitrochlearis muscle are mediated by AMPK- and Ca2+/calmodulin-dependent protein kinase (CAMK)-dependent signaling pathways. However, recent studies provide evidence suggesting that contraction-stimulated glucose transport in slow-twitch skeletal muscle is mediated through an AMPK-independent pathway. The purpose of the present study was to test the hypothesis that contraction-stimulated glucose transport in rat slow-twitch soleus muscle is mediated by an AMPK-independent/Ca2+-dependent pathway. Caffeine, a sarcoplasmic reticulum (SR) Ca2+-releasing agent, at a concentration that does not cause muscle contractions or decreases in high-energy phosphates, led to an approximately 2-fold increase in 2-deoxyglucose (2-DG) uptake in isolated split soleus muscles. This increase in glucose transport was prevented by the SR calcium channel blocker dantrolene and the CAMK inhibitor KN93. Conversely, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an AMPK activator, had no effect on 2-DG uptake in isolated split soleus muscles yet resulted in an approximately 2-fold increase in the phosphorylation of AMPK and its downstream substrate acetyl-CoA carboxylase. The hypoxia-induced increase in 2-DG uptake was prevented by dantrolene and KN93, whereas hypoxia-stimulated phosphorylation of AMPK was unaltered by these agents. Tetanic muscle contractions resulted in an approximately 3.5-fold increase in 2-DG uptake that was prevented by KN93, which did not prevent AMPK phosphorylation. Taken in concert, our results provide evidence that hypoxia- and contraction-stimulated glucose transport is mediated entirely through a Ca2+-dependent mechanism in rat slow-twitch muscle.     

Insulin receptor substrate-2 is not necessary for insulin- and exercise-stimulated glucose transport in skeletal muscle.

Insulin receptor substrate-2-deficient (IRS2(-/-)) mice develop type 2 diabetes. The purpose of this study was to determine whether there is a defect in basal, insulin-, and exercise-stimulated glucose transport in the skeletal muscle of these animals. IRS2(-/-) and wild-type (WT) mice (male, 8-10 weeks) exercised on a treadmill for 1 h or remained sedentary. 2-Deoxyglucose (2DG) uptake was measured in isolated soleus muscles incubated in vitro in the presence or absence of insulin. Resting blood glucose concentration in IRS2(-/-) mice (10.3 mM) was higher than WT animals (4.1 mM), but there was a wide range among the IRS2(-/-) mice (3-25 mM). Therefore, IRS2(-/-) mice were divided into two subgroups based on blood glucose concentrations (IRS2(-/-)L < 7.2 mM, IRS2(-/-)H > 7.2 mM). Only IRS2(-/-)H had lower basal, exercise-, and submaximally insulin-stimulated 2DG uptake, while maximal insulin-stimulated 2DG uptake was similar among the three groups. The ED(50) for insulin to stimulate 2DG uptake above basal in IRS2(-/-)H was higher than WT and IRS2(-/-)L mice, suggesting insulin resistance in the skeletal muscle from the IRS2(-/-) mice with high blood glucose concentrations. Furthermore, resting blood glucose concentrations from all groups were negatively correlated to submaximally insulin-stimulated 2DG uptake (r(2) = 0.33, p < 0.01). Muscle GLUT4 content was significantly lower in IRS2(-/-)H mice compared with WT and IRS2(-/-)L mice. These results demonstrate that the IRS2 protein in muscle is not necessary for insulin- or exercise-stimulated glucose transport, suggesting that the onset of diabetes in the IRS2(-/-) mice is not due to a defect in skeletal muscle glucose transport; hyperglycemia may cause insulin resistance in the muscle of IRS2(-/-) mice.     

Bi-directional transport of GLUT4 vesicles near the plasma membrane of primary rat adipocytes

Insulin stimulates glucose uptake into adipocytes by mobilizing intracellular membrane vesicles containing GLUT4 proteins to the plasma membrane. Here we applied time-lapse total internal reflection fluorescence microscopy to study moving parameters and characters of exogenously expressed GLUT4 vesicles in basal, insulin and nocodazole treated primary rat adipocytes. Our results showed that microtubules were essential for long-range transport of GLUT4 vesicles but not obligatory for GLUT4 distribution in rat adipocytes. Insulin reduced the mobility of the vesicles, made them tethered/docked to the PM and finally had constitutive exocytosis. Moreover, long-range bi-directional movements of GLUT4 vesicles were visualized for the first time by TIRFM. It is likely that there are interactions between insulin signaling and microtubules, to regulating GLUT4 translocation in rat adipocytes.     

Overexpression of Vesicle-associated Membrane Protein (VAMP) 3, but Not VAMP2, Protects Glucose Transporter (GLUT) 4 Protein Translocation in an in Vitro Model of Cardiac Insulin Resistance

Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.     

A role for kinesin in insulin-stimulated GLUT4 glucose transporter translocation in 3T3-L1 adipocytes

Insulin regulates glucose uptake in adipocytes and muscle by stimulating the movement of sequestered glucose transporter 4 (GLUT4) proteins from intracellular membranes to the cell surface. Here we report that optimal insulin-mediated GLUT4 translocation is dependent upon both microtubule and actin-based cytoskeletal structures in cultured adipocytes. Depolymerization of microtubules and F-actin in 3T3-L1 adipocytes causes the dispersion of perinuclear GLUT4-containing membranes and abolishes insulin action on GLUT4 movements to the plasma membrane. Furthermore, heterologous expression in 3T3-L1 adipocytes of the microtubule-binding protein hTau40, which impairs kinesin motors that move toward the plus ends of microtubules, markedly delayed the appearance of GLUT4 at the plasma membrane in response to insulin. The hTau40 protein had no detectable effect on microtubule structure or perinuclear GLUT4 localization under these conditions. These results are consistent with the hypothesis that both the actin and microtubule-based cytoskeleton, as well as a kinesin motor, direct the translocation of GLUT4 to the plasma membrane in response to insulin.     

Microtubule network is required for insulin signaling through activation of Akt/protein kinase B: evidence that insulin stimulates vesicle docking/fusion but not intracellular mobility

The microtubule network has been shown to be required for insulin-dependent GLUT4 redistribution; however, the precise molecular function has not been elucidated. In this article, we used fluorescence recovery after photobleaching (FRAP) to evaluate the role of microtubules in intracellular GLUT4 vesicle mobility. A comparison of the rate of fluorescence recovery (t((1/2))), and the maximum fluorescence recovered (F(max)) was made between basal and insulin-treated cells with or without nocodazole treatment to disrupt microtubules. We found that intracellular mobility of fluorescently tagged GLUT4 (HA-GLUT4-GFP) was high in basal cells. Mobility was not increased by insulin treatment. Basal mobility was dependent upon an intact microtubule network. Using a constitutively active Akt to signal GLUT4 redistribution, we found that microtubule-based GLUT4 vesicle mobility was not obligatory for GLUT4 plasma membrane insertion. Our findings suggest that microtubules organize the insulin-signaling complex and provide a surface for basal mobility of GLUT4 vesicles. Our data do not support an obligatory requirement for long range microtubule-based movement of GLUT4 vesicles for insulin-mediated GLUT4 redistribution to the cell surface. Taken together, these findings suggest a model in which insulin signaling targets membrane docking and/or fusion rather than GLUT4 trafficking to the cell surface.