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

Jose A Chavez,William G Roach,Susanna R Keller,William S Lane,Gustav E Lienhard

doi:10.1074/jbc.m708934200

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.

Highlights

In the absence of insulin AS160 keeps Rab10 and possibly other Rabs in their inactive GDP form; insulin treatment causes Akt phosphorylation of AS160; phosphorylation suppresses its GTPase-activating protein (GAP) activity and thereby results in an elevation of the active GTP form of Rab10 and possibly other Rabs; the GTP form of the Rab(s) triggers the machinery for the docking of GLUT4 vesicles to the plasma membrane, which leads to the fusion with the membrane
Contraction and activation of AMPK, as well as insulin, stimulate GLUT4 translocation [1, 8, 9]. This discovery raised the possibility that Tbc1d1 participates in the regulation of GLUT4 translocation in response to contraction and/or AMPK activation. In support of this possibility we found that the inhibitory effect of overexpressed Tbc1d1 on insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes is partially relieved by AICAR, an agent that activates AMPK
We previously showed that insulin treatment of 3T3-L1 adipocytes causes phosphorylation of Tbc1d1 on Thr596 in the Akt phosphorylation motif RRRANTL [7], which can be detected with the PAS antibody

Summary

Introduction

In support of this possibility we found that the inhibitory effect of overexpressed Tbc1d1 on insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes is partially relieved by AICAR, an agent that activates AMPK. Effect of Akt Phosphorylation of Tbc1d1—Overexpression of Tbc1d1 in 3T3-L1 adipocytes markedly inhibits insulin-stimulated GLUT4 translocation, whereas overexpression of AS160 does not inhibit [2, 7].

Results

Conclusion