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Thr(649)Ala-AS160 knock-in mutation does not impair contraction/AICAR-induced glucose transport in mouse muscle

Thr(649)Ala-AS160 knock-in mutation does not impair contraction/AICAR-induced glucose transport in mouse muscle

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Original languageEnglish
PagesE1036-E1043
Number of pages8
JournalAmerican Journal of Physiology, Endocrinology and Metabolism
Journal publication dateMay 2012
Journal number9
Volume302
DOIs
StatePublished

Abstract

Ducommun S, Wang HY, Sakamoto K, MacKintosh C, Chen S. Thr(649)Ala-AS160 knock-in mutation does not impair contraction/AICAR-induced glucose transport in mouse muscle. Am J Physiol Endocrinol Metab 302: E1036-E1043, 2012. First published February 7, 2012; doi:10.1152/ajpendo.00379.2011.-AS160 and its closely related protein TBC1D1 have emerged as key mediators for both insulin-and contraction-stimulated muscle glucose uptake through regulating GLUT4 trafficking. Insulin increases AS160 phosphorylation at multiple Akt/PKB consensus sites, including Thr(649), and promotes its binding to 14-3-3 proteins through phospho-Thr(649). We recently provided genetic evidence that AS160-Thr(649) phosphorylation/14-3-3 binding plays a key role in mediating insulin-stimulated glucose uptake in muscle. Contraction has also been proposed to increase phosphorylation of AS160 and TBC1D1 via AMPK, which could be detected by a generic phospho-Akt substrate (PAS) antibody. Here, analysis of AS160 immunoprecipitates from muscle extracts with site-specific phospho-antibodies revealed that contraction and AICAR caused no increase but rather a slight decrease in phosphorylation of the major PAS recognition site AS160-Thr(649). In line with this, contraction failed to enhance 14-3-3 binding to AS160. Consistent with previous reports, we also observed that in situ contraction stimulated the signal intensity of PAS antibody immunoreactive protein of similar to 150-160 kDa in muscle extracts. Using a TBC1D1 deletion mutant mouse, we showed that TBC1D1 protein accounted for the majority of the PAS antibody immunoreactive signals of similar to 150-160 kDa in extracts of contracted muscles. Consistent with the proposed role of AS160-Thr(649) phosphorylation/14-3-3 binding in mediating glucose uptake, AS160-Thr(649)Ala knock-in mice displayed normal glucose uptake upon contraction and AICAR in isolated muscles. We conclude that the previously reported PAS antibody immunoreactive band similar to 150-160 kDa, which were increased upon contraction, does not represent AS160 but TBC1D1, and that AS160-Thr(649)Ala substitution impairs insulin-but neither contraction- nor AICAR-stimulated glucose uptake in mouse skeletal muscle.

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