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Inhibition of GLUT4 Translocation by Tbc1d1, a Rab GTPase-activating Protein Abundant in Skeletal Muscle, Is Partially Relieved by AMP-activated Protein Kinase Activation

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2008; 283(14):9187-95. DOI: 10.1074/jbc.M708934200
Source: PubMed

ABSTRACT 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.

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    • "Therefore it is conceivable that GLUT4 behavior in skeletal muscle cells is more complex than that in adipocytes . Previous observations suggested that actions of Tbc1d1 dominate over those of AS160 since ectopic expression of Tbc1d1 strongly inhibited insulin-responsive GLUT4 translocation in 3T3L1 adipocytes (Roach et al., 2007; Chavez et al., 2008). This inhibitory action partially diminished in the presence of AICAR, indicating that Tbc1d1 and AS160 have distinct modes of action in regulating GLUT4 translocation, which is consistent with the present observations . "
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    ABSTRACT: Tbc1d1 is key to skeletal muscle GLUT4 regulation. Herein, employing GLUT4 nanometry combined with a cell-based reconstitution model, we revealed a regulatory mode shift of Tbc1d1, by showing that Tbc1d1 temporally acquires insulin-responsiveness which triggers GLUT4 trafficking only after exercise-mimetic stimuli such as AICAR-pretreatment. The functional acquisition of insulin-responsiveness requires Ser237 phosphorylation and an intact PTB1 domain. Mutations in PTB1 including R125W (a natural mutant) thus result in complete loss of insulin-responsiveness acquisition, while AICAR-responsive GLUT4 liberation activity remains intact. Thus, our data provide novel insights into temporal acquisition of Tbc1d1 insulin-responsiveness, relying on the PTB1 domain, possibly a key factor in the beneficial effects of exercise on muscle insulin potency.
    Molecular biology of the cell 01/2013; 24(6). DOI:10.1091/mbc.E12-10-0725 · 5.98 Impact Factor
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    • "As previously reported (27), incubation of EDL ex vivo with AICAR promoted robust phosphorylation of the AMPKα catalytic subunit at Thr172 in the T-loop and activation of both AMPKα1 (∼twofold) and AMPKα2 (∼fourfold) compared with unstimulated muscle (Fig. 1A and B). Consistent with these observations, AICAR treatment increased phosphorylation of known AMPK substrates such as ACC2 at Ser212, raptor at Ser792 (30), and TBC1D1 at Ser231 (25,31) (Fig. 1B). "
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    ABSTRACT: During energy stress, AMP-activated protein kinase (AMPK) promotes glucose transport and glycolysis for ATP production, while it is thought to inhibit anabolic glycogen synthesis by suppressing the activity of glycogen synthase (GS) to maintain the energy balance in muscle. Paradoxically, chronic activation of AMPK causes an increase in glycogen accumulation in skeletal and cardiac muscles, which in some cases is associated with cardiac dysfunction. The aim of this study was to elucidate the molecular mechanism by which AMPK activation promotes muscle glycogen accumulation. We recently generated knock-in mice in which wild-type muscle GS was replaced by a mutant (Arg582Ala) that could not be activated by glucose-6-phosphate (G6P), but possessed full catalytic activity and could still be activated normally by dephosphorylation. Muscles from GS knock-in or transgenic mice overexpressing a kinase dead (KD) AMPK were incubated with glucose tracers and the AMPK-activating compound 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) ex vivo. GS activity and glucose uptake and utilization (glycolysis and glycogen synthesis) were assessed. Even though AICAR caused a modest inactivation of GS, it stimulated muscle glycogen synthesis that was accompanied by increases in glucose transport and intracellular [G6P]. These effects of AICAR required the catalytic activity of AMPK. Strikingly, AICAR-induced glycogen synthesis was completely abolished in G6P-insensitive GS knock-in mice, although AICAR-stimulated AMPK activation, glucose transport, and total glucose utilization were normal. We provide genetic evidence that AMPK activation promotes muscle glycogen accumulation by allosteric activation of GS through an increase in glucose uptake and subsequent rise in cellular [G6P].
    Diabetes 02/2011; 60(3):766-74. DOI:10.2337/db10-1148 · 8.47 Impact Factor
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    • "TBC1D1 is similar to AS160 in terms of size and structure [20]. However, the tissue distribution of the two proteins is considerably different, with TBC1D1 having little if any expression in adipocytes, and the highest level of expression in skeletal muscle [21,22]. We found that contraction, insulin and the AMPK (AMP-activated protein kinase) activator AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) increase TBC1D1 phosphorylation on PAS (phospho-Akt substrate) motifs in mouse skeletal muscle, demonstrating that TBC1D1 is an additional point of convergence for insulin and contraction signalling [21]. "
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    ABSTRACT: TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab-GAP (GTPase-activating protein) that is highly expressed in skeletal muscle, but little is known about TBC1D1 regulation and function. We studied TBC1D1 phosphorylation on three predicted AMPK (AMP-activated protein kinase) phosphorylation sites (Ser231, Ser660 and Ser700) and one predicted Akt phosphorylation site (Thr590) in control mice, AMPKα2 inactive transgenic mice (AMPKα2i TG) and Akt2-knockout mice (Akt2 KO). Muscle contraction significantly increased TBC1D1 phosphorylation on Ser231 and Ser660, tended to increase Ser700 phosphorylation, but had no effect on Thr590. AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) also increased phosphorylation on Ser231, Ser660 and Ser700, but not Thr590, whereas insulin only increased Thr590 phosphorylation. Basal and contraction-stimulated TBC1D1 Ser231, Ser660 and Ser700 phosphorylation were greatly reduced in AMPKα2i TG mice, although contraction still elicited a small increase in phosphorylation. Akt2 KO mice had blunted insulin-stimulated TBC1D1 Thr590 phosphorylation. Contraction-stimulated TBC1D1 Ser231 and Ser660 phosphorylation were normal in high-fat-fed mice. Glucose uptake in vivo was significantly decreased in tibialis anterior muscles overexpressing TBC1D1 mutated on four predicted AMPK phosphorylation sites. In conclusion, contraction causes site-specific phosphorylation of TBC1D1 in skeletal muscle, and TBC1D1 phosphorylation on AMPK sites regulates contraction-stimulated glucose uptake. AMPK and Akt regulate TBC1D1 phosphorylation, but there must be additional upstream kinases that mediate TBC1D1 phosphorylation in skeletal muscle.
    Biochemical Journal 10/2010; 431(2):311-20. DOI:10.1042/BJ20101100 · 4.78 Impact Factor
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