scholarly journals AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain

2005 ◽  
Vol 391 (1) ◽  
pp. 87-93 ◽  
Author(s):  
Cristinel P. Mîinea ◽  
Hiroyuki Sano ◽  
Susan Kane ◽  
Eiko Sano ◽  
Mitsunori Fukuda ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Protein Domain ◽  
Rab Gtpase ◽  
Significant Activity ◽  
Glut4 Translocation ◽  
Gtpase Activating Protein ◽  
Akt Phosphorylation ◽  
Intracellular Vesicles ◽  
Glucose Transporter Glut4 ◽  
Protein Kinase Akt

Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.

scholarly journals Substrate specificity and effect on GLUT4 translocation of the Rab GTPase-activating protein Tbc1d1

2007 ◽  
Vol 403 (2) ◽  
pp. 353-358 ◽  
Author(s):  
William G. Roach ◽  
Jose A. Chavez ◽  
Cristinel P. Mîinea ◽  
Gustav E. Lienhard
Keyword(s):  
Plasma Membrane ◽  
Glucose Transporter ◽  
Insulin Treatment ◽  
Ectopic Expression ◽  
Rab Gtpase ◽  
Glut4 Translocation ◽  
Gtpase Activating Protein ◽  
Insulin Stimulation ◽  
Glucose Transporter Glut4 ◽  
Stimulation Of

Insulin stimulation of the trafficking of the glucose transporter GLUT4 to the plasma membrane is controlled in part by the phosphorylation of the Rab GAP (GTPase-activating protein) AS160 (also known as Tbc1d4). Considerable evidence indicates that the phosphorylation of this protein by Akt (protein kinase B) leads to suppression of its GAP activity and results in the elevation of the GTP form of a critical Rab. The present study examines a similar Rab GAP, Tbc1d1, about which very little is known. We found that the Rab specificity of the Tbc1d1 GAP domain is identical with that of AS160. Ectopic expression of Tbc1d1 in 3T3-L1 adipocytes blocked insulin-stimulated GLUT4 translocation to the plasma membrane, whereas a point mutant with an inactive GAP domain had no effect. Insulin treatment led to the phosphorylation of Tbc1d1 on an Akt site that is conserved between Tbc1d1 and AS160. These results show that Tbc1d1 regulates GLUT4 translocation through its GAP activity, and is a likely Akt substrate. An allele of Tbc1d1 in which Arg125 is replaced by tryptophan has very recently been implicated in susceptibility to obesity by genetic analysis. We found that this form of Tbc1d1 also inhibited GLUT4 translocation and that this effect also required a functional GAP domain.

scholarly journals Estradiol-induced regulation of GLUT4 in 3T3-L1 cells: involvement of ESR1 and AKT activation

2017 ◽  
Vol 59 (3) ◽  
pp. 257-268 ◽  
Author(s):  
Raquel S Campello ◽  
Luciana A Fátima ◽  
João Nilton Barreto-Andrade ◽  
Thais F Lucas ◽  
Rosana C Mori ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Subcellular Distribution ◽  
Glucose Transporter ◽  
Biological Effects ◽  
Glut4 Translocation ◽  
Akt Phosphorylation ◽  
Akt Activation ◽  
Glut4 Expression ◽  
Protein Kinase Akt

Impaired insulin-stimulated glucose uptake involves reduced expression of the GLUT4 (solute carrier family 2 facilitated glucose transporter member 4, SLC2A4 gene). 17β-estradiol (E2) modulates SLC2A4/GLUT4 expression, but the involved mechanisms are unclear. Although E2 exerts biological effects by binding to estrogen receptors 1/2 (ESR1/2), which are nuclear transcriptional factors; extranuclear effects have also been proposed. We hypothesize that E2 regulates GLUT4 through an extranuclear ESR1 mechanism. Thus, we investigated the effects of E2 upon (1) subcellular distribution of ESRs and the proto-oncogene tyrosine-protein kinases (SRC) involvement; (2) serine/threonine-protein kinase (AKT) activation; (3) Slc2a4/GLUT4 expression and (4) GLUT4 subcellular distribution and glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were cultivated or not with E2 for 24 h, and additionally treated or not with ESR1-selective agonist (PPT), ESR1-selective antagonist (MPP) or selective SRC inhibitor (PP2). Subcellular distribution of ESR1, ESR2 and GLUT4 was analyzed by immunocytochemistry; Slc2a4 mRNA and GLUT4 were quantified by qPCR and Western blotting, respectively; plasma membrane GLUT4 translocation and glucose uptake were analyzed under insulin stimulus for 20 min or not. E2 induced (1) translocation of ESR1, but not of ESR2, from nucleus to plasma membrane and AKT phosphorylation, effects mimicked by PPT and blocked by MPP and PP2; (2) increased Slc2a4/GLUT4 expression and (3) increased insulin-stimulated GLUT4 translocation and glucose uptake. In conclusion, E2 treatment promoted a SRC-mediated nucleus-plasma membrane shuttle of ESR1, and increased AKT phosphorylation, Slc2a4/GLUT4 expression and plasma membrane GLUT4 translocation; consequently, improving insulin-stimulated glucose uptake. These results unravel mechanisms through which estrogen improves insulin sensitivity.

scholarly journals Insulin stimulates the phosphorylation of the exocyst protein Sec8 in adipocytes

2009 ◽  
Vol 29 (4) ◽  
pp. 229-235 ◽  
Author(s):  
Patrick D. Lyons ◽  
Grantley R. Peck ◽  
Arminja N. Kettenbach ◽  
Scott A. Gerber ◽  
Liya Roudaia ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Transporter ◽  
Kinase Inhibitor ◽  
Signal Transduction Pathway ◽  
Current Evidence ◽  
Rab Gtpase ◽  
Gtpase Activating Protein ◽  
Phosphoinositide 3 Kinase ◽  
Glucose Transporter Glut4

The signal transduction pathway leading from the insulin receptor to stimulate the fusion of vesicles containing the glucose transporter GLUT4 with the plasma membrane in adipocytes and muscle cells is not completely understood. Current evidence suggests that in addition to the Rab GTPase-activating protein AS160, at least one other substrate of Akt (also called protein kinase B), which is as yet unidentified, is required. Sec8 is a component of the exocyst complex that has been previously implicated in GLUT4 trafficking. In the present study, we report that insulin stimulates the phosphorylation of Sec8 on Ser-32 in 3T3-L1 adipocytes. On the basis of the sequence around Ser-32 and the finding that phosphorylation is inhibited by the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, it is likely that Akt is the kinase for Ser-32. We examined the possible role of Ser-32 phosphorylation in the insulin-stimulated trafficking of GLUT4, as well as the TfR (transferrin receptor), to the plasma membrane by determining the effects of overexpression of the non-phosphorylatable S32A mutant of Sec8 and the phosphomimetic S32E mutant of Sec8. Substantial overexpression of both mutants had no effect on the amount of GLUT4 or TfR at the cell surface in either the untreated or insulin-treated states. These results indicate that insulin-stimulated phosphorylation of Sec8 is not part of the mechanism by which insulin enhances the fusion of vesicles with the plasma membrane.

Abstract 158: A Tether Containing a UBX Domain (TUG) Mediates Glucose Transporter Translocation in the Ischemic Heart

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Ji Li ◽  
Yina Ma ◽  
Jonathan Bogan
Keyword(s):  
Cell Surface ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Ex Vivo ◽  
Ischemia And Reperfusion ◽  
Ampk Activation ◽  
Glut4 Translocation ◽  
Heart Perfusion ◽  
Intracellular Vesicles

Introduction: The adaptive metabolic regulation of glucose and fatty acid in the heart plays a critical role in limiting cardiac damage caused by ischemia and reperfusion (I/R). TUG (tether containing a UBX domain, for GLUT4) can be cleaved to mobilize glucose transporter GLUT4 from intracellular vesicles to the cell surface in skeletal muscle and adipose in response to insulin stimulation. The energy sensor AMP-activated protein kinase (AMPK) plays an important cardioprotective role in response to ischemic insults by modulating GLUT4 translocation. Hypothesis: TUG is one of the downstream targets of AMPK in the heart. TUG could be phosphorylated by ischemic AMPK and cleaved to dissociate with GLUT4 and increase GLUT4 translocation in the ischemic heart. Methods: In vivo regional ischemia by ligation of left anterior coronary artery and ex vivo isolated mouse heart perfusion Langendorff system were used to test the hypothesis. Results: Antithrombin (AT) is an endogenous AMPK agonist in the heart and used to define the role of TUG in regulating GLUT4 trafficking during ischemia and reperfusion in the heart. AT showed its cardioprotective function through recovering cardiac pumping function and activating AMPK. The results showed that AMPK activation by AT treatment was through LKB1 and Sesn2 complex. Furthermore, the ex vivo heart perfusion data demonstrated that AT administration significantly increase GLUT4 translocation, glucose uptake, glycolysis and glucose oxidation during ischemia and reperfusion (p<0.05 vs . vehicle). Moreover, AT treatment increased abundance of a TUG cleavage product (42 KD) in response to I/R. The TUG protein was clearly phosphorylated by activated AMPK in HL-1 cardiomyocytes. The in vivo myocardial ischemia results demonstrated that ischemic AMPK activation triggers TUG cleavage and significantly increases GLUT4 translocation to the cell surface. Moreover, an augmented interaction between AMPK and TUG was observed during ischemia. Conclusions: Cardiac AMPK activation stimulates TUG cleavage and causes the dissociation between TUG and GLUT4 in the intracellular vesicles. TUG is a critical mediator that modulates cardiac GLUT4 translocation to cell surface and enhances glucose uptake by AMPK signaling pathway.

scholarly journals Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs

2013 ◽  
Vol 24 (16) ◽  
pp. 2544-2557 ◽  
Author(s):  
L. Amanda Sadacca ◽  
Joanne Bruno ◽  
Jennifer Wen ◽  
Wenyong Xiong ◽  
Timothy E. McGraw
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Receptor Activation ◽  
Glut4 Translocation ◽  
Insulin Stimulation ◽  
Transport Vesicles ◽  
Glucose Transporter Glut4

Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation.

Rab10 in insulin-stimulated GLUT4 translocation

2008 ◽  
Vol 411 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Hiroyuki Sano ◽  
William G. Roach ◽  
Grantley R. Peck ◽  
Mitsunori Fukuda ◽  
Gustav E. Lienhard
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Subcellular Distribution ◽  
Protein Kinase B ◽  
Muscle Cells ◽  
Low Density ◽  
Glut4 Translocation ◽  
Gtpase Activating Protein ◽  
Intracellular Vesicles

In fat and muscle cells, insulin stimulates the movement to and fusion of intracellular vesicles containing GLUT4 with the plasma membrane, a process referred to as GLUT4 translocation. Previous studies have indicated that Akt [also known as PKB (protein kinase B)] phosphorylation of AS160, a GAP (GTPase-activating protein) for Rabs, is required for GLUT4 translocation. The results suggest that this phosphorylation suppresses the GAP activity and leads to the elevation of the GTP form of one or more Rabs required for GLUT4 translocation. Based on their presence in GLUT4 vesicles and activity as AS160 GAP substrates, Rabs 8A, 8B, 10 and 14 are candidate Rabs. Here, we provide further evidence that Rab10 participates in GLUT4 translocation in 3T3-L1 adipocytes. Among Rabs 8A, 8B, 10 and 14, only the knockdown of Rab10 inhibited GLUT4 translocation. In addition, we describe the subcellular distribution of Rab10 and estimate the fraction of Rab10 in the active GTP form in vivo. Approx. 5% of the total Rab10 was present in GLUT4 vesicles isolated from the low-density microsomes. In both the basal and the insulin state, 90% of the total Rab10 was in the inactive GDP state. Thus, if insulin increases the GTP form of Rab10, the increase is limited to a small portion of the total Rab10. Finally, we report that the Rab10 mutant considered to be constitutively active (Rab10 Q68L) is a substrate for the AS160 GAP domain and, hence, cannot be used to deduce rigorously the function of Rab10 in its GTP form.

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

2005 ◽  
Vol 19 (4) ◽  
pp. 1067-1077 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document