O-23: Mechanisms of insulin-dependent and insulin-independent GLUT4 translocation

Whether or not the atrophic skeletal muscle induces insulin resistance and its mechanisms are not resolved now. The antigravity soleus muscle showed a progressive atrophy in 1-week, 2-week, and 4-week tail-suspended rats. Hyperinsulinemic-euglycemic clamp showed that the steady-state glucose infusion rate was lower in 4-week tail-suspended rats than that in the control rats. The glucose uptake rates under insulin- or contraction-stimulation were significantly decreased in 4-week unloaded soleus muscle. The key protein expressions of IRS-1, PI3K, and Akt on the insulin-dependent pathway and of AMPK, ERK, and p38 on the insulin-independent pathway were unchanged in unloaded soleus muscle. The unchanged phosphorylation of Akt and p38 suggested that the activity of two signal pathways was not altered in unloaded soleus muscle. The AS160 and GLUT4 expression on the common downstream pathway also was not changed in unloaded soleus muscle. But the GLUT4 translocation to sarcolemma was inhibited during insulin stimulation in unloaded soleus muscle. The above results suggest that hindlimb unloading in tail-suspended rat induces atrophy in antigravity soleus muscle. The impaired GLUT4 translocation to sarcolemma under insulin stimulation may mediate insulin resistance in unloaded soleus muscle and further affect the insulin sensitivity of whole body in tail-suspended rats.

Download Full-text

Akt Activation Is Required at a Late Stage of Insulin-Induced GLUT4 Translocation to the Plasma Membrane

Molecular Endocrinology ◽

10.1210/me.2004-0413 ◽

2005 ◽

Vol 19 (4) ◽

pp. 1067-1077 ◽

Cited By ~ 64

Author(s):

Ellen M. van Dam ◽

Roland Govers ◽

David E. James

Keyword(s):

Plasma Membrane ◽

Glucose Transporter ◽

Cell Cortex ◽

Glut4 Translocation ◽

Akt Activation ◽

Multistep Process ◽

Intracellular Compartments ◽

Insulin Dependent ◽

Glucose Transporter Glut4 ◽

Golgi Network

Abstract Insulin stimulates the translocation of glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (PM). This involves multiple steps as well as multiple intracellular compartments. The Ser/Thr kinase Akt has been implicated in this process, but its precise role is ill defined. To begin to dissect the role of Akt in these different steps, we employed a low-temperature block. Upon incubation of 3T3-L1 adipocytes at 19 C, GLUT4 accumulated in small peripheral vesicles with a slight increase in PM labeling concomitant with reduced trans-Golgi network labeling. Although insulin-dependent translocation of GLUT4 to the PM was impaired at 19 C, we still observed movement of vesicles toward the surface. Strikingly, insulin-stimulated Akt activity, but not phosphatidylinositol 3 kinase activity, was blocked at 19 C. Consistent with a multistep process in GLUT4 trafficking, insulin-stimulated GLUT4 translocation could be primed by treating cells with insulin at 19 C, whereas this was not the case for Akt activation. These data implicate two insulin-regulated steps in GLUT4 translocation: 1) redistribution of GLUT4 vesicles toward the cell cortex—this process is Akt-independent and is not blocked at 19 C; and 2) docking and/or fusion of GLUT4 vesicles with the PM—this process may be the major Akt-dependent step in the insulin regulation of glucose transport.

Download Full-text

A Crucial Role for the Small GTPase Rac1 Downstream of the Protein Kinase Akt2 in Insulin Signaling that Regulates Glucose Uptake in Mouse Adipocytes

International Journal of Molecular Sciences ◽

10.3390/ijms20215443 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5443 ◽

Cited By ~ 5

Author(s):

Takenaka ◽

Nakao ◽

Matsui ◽

Satoh

Keyword(s):

Skeletal Muscle ◽

Protein Kinase ◽

Glucose Uptake ◽

Glucose Transporter ◽

Specific Inhibitor ◽

Small Gtpase ◽

Glut4 Translocation ◽

Insulin Dependent ◽

Mouse Adipocytes ◽

Phosphoinositide 3 Kinase

Insulin-stimulated glucose uptake is mediated by translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes and skeletal muscle cells. In both types of cells, phosphoinositide 3-kinase and the protein kinase Akt2 have been implicated as critical regulators. In skeletal muscle, the small GTPase Rac1 plays an important role downstream of Akt2 in the regulation of insulin-stimulated glucose uptake. However, the role for Rac1 in adipocytes remains controversial. Here, we show that Rac1 is required for insulin-dependent GLUT4 translocation also in adipocytes. A Rac1-specific inhibitor almost completely suppressed GLUT4 translocation induced by insulin or a constitutively activated mutant of phosphoinositide 3-kinase or Akt2. Constitutively activated Rac1 also enhanced GLUT4 translocation. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was abrogated by inhibition of phosphoinositide 3-kinase or Akt2. On the other hand, constitutively activated Akt2 caused Rac1 activation, and insulin-induced Rac1 activation was suppressed by an Akt2-specific inhibitor. Moreover, GLUT4 translocation induced by a constitutively activated mutant of Akt2 or Rac1 was diminished by knockdown of another small GTPase RalA. RalA was activated by a constitutively activated mutant of Akt2 or Rac1, and insulin-induced RalA activation was suppressed by an Akt2- or Rac1-specific inhibitor. Collectively, these results suggest that Rac1 plays an important role in the regulation of insulin-dependent GLUT4 translocation downstream of Akt2, leading to RalA activation in adipocytes.

Download Full-text

Muscle cells engage Rab8A and myosin Vb in insulin-dependent GLUT4 translocation

AJP Cell Physiology ◽

10.1152/ajpcell.00277.2008 ◽

2008 ◽

Vol 295 (4) ◽

pp. C1016-C1025 ◽

Cited By ~ 100

Author(s):

Shuhei Ishikura ◽

Amira Klip

Keyword(s):

Glucose Transporter ◽

Muscle Cells ◽

Rab Gtpase ◽

Glut4 Translocation ◽

Interacting Protein ◽

Akt Activation ◽

Gtpase Activating Proteins ◽

Insulin Dependent ◽

Myosin Vb

Insulin causes translocation of glucose transporter 4 (GLUT4) to the membrane of muscle and fat cells, a process requiring Akt activation. Two Rab-GTPase-activating proteins (Rab-GAP), AS160 and TBC1D1, were identified as Akt substrates. AS160 phosphorylation is required for insulin-stimulated GLUT4 translocation, but the participation of TBC1D1 on muscle cell GLUT4 is unknown. Moreover, there is controversy as to the AS160/TBC1D1 target Rabs in fat and muscle cells, and Rab effectors are unknown. Here we examined the effect of knockdown of AS160, TBC1D1, and Rabs 8A, 8B, 10, and 14 (in vitro substrates of AS160 and TBC1D1 Rab-GAP activities) on insulin-induced GLUT4 translocation in L6 muscle cells. Silencing AS160 or TBC1D1 increased surface GLUT4 in unstimulated cells but did not prevent insulin-induced GLUT4 translocation. Knockdown of Rab8A and Rab14, but not of Rab8B or Rab10, inhibited insulin-induced GLUT4 translocation. Furthermore, silencing Rab8A or Rab14 but not Rab8B or Rab10 restored the basal-state intracellular retention of GLUT4 impaired by AS160 or TBC1D1 knockdown. Lastly, overexpression of a fragment of myosin Vb, a recently identified Rab8A-interacting protein, inhibited insulin-induced GLUT4 translocation and altered the subcellular distribution of GTP-loaded Rab8A. These results support a model whereby AS160, Rab8A, and myosin Vb are required for insulin-induced GLUT4 translocation in muscle cells, potentially as part of a linear signaling cascade.

Download Full-text

VAMP2, but Not VAMP3/Cellubrevin, Mediates Insulin-dependent Incorporation of GLUT4 into the Plasma Membrane of L6 Myoblasts

Molecular Biology of the Cell ◽

10.1091/mbc.11.7.2403 ◽

2000 ◽

Vol 11 (7) ◽

pp. 2403-2417 ◽

Cited By ~ 78

Author(s):

Varinder K. Randhawa ◽

Philip J. Bilan ◽

Zayna A. Khayat ◽

Nicholas Daneman ◽

Zhi Liu ◽

...

Keyword(s):

Plasma Membrane ◽

Tetanus Toxin ◽

Synaptic Vesicles ◽

Transient Transfection ◽

Fat Cells ◽

Glut4 Translocation ◽

Cortical Actin ◽

Functional Involvement ◽

Insulin Dependent ◽

Individual Vesicle

Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.

Download Full-text

Akt2 regulates Rac1 activity in the insulin-dependent signaling pathway leading to GLUT4 translocation to the plasma membrane in skeletal muscle cells

Cellular Signalling ◽

10.1016/j.cellsig.2013.02.023 ◽

2013 ◽

Vol 25 (6) ◽

pp. 1361-1371 ◽

Cited By ~ 28

Author(s):

Shinsuke Nozaki ◽

Tomoya Takeda ◽

Takuya Kitaura ◽

Nobuyuki Takenaka ◽

Tohru Kataoka ◽

...

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Signaling Pathway ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Glut4 Translocation ◽

Rac1 Activity ◽

Insulin Dependent ◽

Dependent Signaling Pathway

Download Full-text

A complex of Rab13 with MICAL-L2 and α-actinin-4 is essential for insulin-dependent GLUT4 exocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e15-05-0319 ◽

2016 ◽

Vol 27 (1) ◽

pp. 75-89 ◽

Cited By ~ 26

Author(s):

Yi Sun ◽

Javier Jaldin-Fincati ◽

Zhi Liu ◽

Philip J. Bilan ◽

Amira Klip

Keyword(s):

Glucose Transporter ◽

Cell Periphery ◽

Rab Gtpases ◽

Structured Illumination ◽

Glut4 Translocation ◽

Confocal Fluorescence ◽

Impaired Insulin ◽

Insulin Dependent ◽

Intracellular Vesicles ◽

Confocal Fluorescence Imaging

Insulin promotes glucose uptake into skeletal muscle through recruitment of glucose transporter 4 (GLUT4) to the plasma membrane. Rab GTPases are molecular switches mobilizing intracellular vesicles, and Rab13 is necessary for insulin-regulated GLUT4–vesicle exocytic translocation in muscle cells. We show that Rab13 engages the scaffold protein MICAL-L2 in this process. RNA interference–mediated knockdown of MICAL-L2 or truncated MICAL-L2 (MICAL-L2-CT) impaired insulin-stimulated GLUT4 translocation. Insulin increased Rab13 binding to MICAL-L2, assessed by pull down and colocalization under confocal fluorescence and structured illumination microscopies. Association was also visualized at the cell periphery using TIRF microscopy. Insulin further increased binding of MICAL-L2 to α-actinin-4 (ACTN4), a protein involved in GLUT4 translocation. Rab13, MICAL-L2, and ACTN4 formed an insulin-dependent complex assessed by pull down and confocal fluorescence imaging. Of note, GLUT4 associated with the complex in response to insulin, requiring the ACTN4-binding domain in MICAL-L2. This was demonstrated by pull down with distinct fragments of MICAL-L2 and confocal and structured illumination microscopies. Finally, expression of MICAL-L2-CT abrogated the insulin-dependent colocalization of Rab13 with ACTN4 or Rab13 with GLUT4. Our findings suggest that MICAL-L2 is an effector of insulin-activated Rab13, which links to GLUT4 through ACTN4, localizing GLUT4 vesicles at the muscle cell periphery to enable their fusion with the membrane.

Download Full-text

Differentiation with elaidate tends to impair insulin-dependent glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes

International Journal of Food Sciences and Nutrition ◽

10.3109/09637486.2016.1144721 ◽

2016 ◽

Vol 67 (2) ◽

pp. 99-110 ◽

Cited By ~ 5

Author(s):

Kenichi Ishibashi ◽

Kana Nehashi ◽

Toshiyuki Oshima ◽

Naoki Ohkura ◽

Gen-Ichi Atsumi

Keyword(s):

Glucose Uptake ◽

Glut4 Translocation ◽

Insulin Dependent

Download Full-text

Regulation of glucose transporters by insulin and extracellular glucose in C2C12 myotubes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00194.2006 ◽

2006 ◽

Vol 291 (4) ◽

pp. E817-E828 ◽

Cited By ~ 69

Author(s):

Taku Nedachi ◽

Makoto Kanzaki

Keyword(s):

Skeletal Muscle ◽

Glucose Uptake ◽

Glucose Transport ◽

Glucose Transporter ◽

C2c12 Myotubes ◽

Glut4 Translocation ◽

Insulin Stimulation ◽

Extracellular Glucose ◽

Insulin Dependent ◽

Stimulation Of

It is well established that insulin stimulation of glucose uptake in skeletal muscle cells is mediated through translocation of GLUT4 from intracellular storage sites to the cell surface. However, the established skeletal muscle cell lines, with the exception of L6 myocytes, reportedly show minimal insulin-dependent glucose uptake and GLUT4 translocation. Using C2C12 myocytes expressing exofacial-Myc-GLUT4-enhanced cyan fluorescent protein, we herein show that differentiated C2C12 myotubes are equipped with basic GLUT4 translocation machinery that can be activated by insulin stimulation (∼3-fold increase as assessed by anti-Myc antibody uptake and immunostaining assay). However, this insulin stimulation of GLUT4 translocation was difficult to demonstrate with a conventional 2-deoxyglucose uptake assay because of markedly elevated basal glucose uptake via other glucose transporter(s). Intriguingly, the basal glucose transport activity in C2C12 myotubes appeared to be acutely suppressed within 5 min by preincubation with a pathophysiologically high level of extracellular glucose (25 mM). In contrast, this activity was augmented by acute glucose deprivation via an unidentified mechanism that is independent of GLUT4 translocation but is dependent on phosphatidylinositol 3-kinase activity. Taken together, these findings indicate that regulation of the facilitative glucose transport system in differentiated C2C12 myotubes can be achieved through surprisingly acute glucose-dependent modulation of the activity of glucose transporter(s), which apparently contributes to obscuring the insulin augmentation of glucose uptake elicited by GLUT4 translocation. We herein also describe several methods of monitoring insulin-dependent glucose uptake in C2C12 myotubes and propose this cell line to be a useful model for analyzing GLUT4 translocation in skeletal muscle.

Download Full-text