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Methods Mol Biol. 2010;637:231-44. doi: 10.1007/978-1-60761-700-6_12.

Imaging of protein translocation in situ in skeletal muscle of living mice.

Author information

1
Research Division, Joslin Diabetes Center and Harvard Medical School, Boston, MA, USA.

Abstract

Skeletal muscle plays a key role in regulating whole body glucose homeostasis and severe dysfunction in insulin-mediated glucose uptake is the hallmark of insulin-resistant states and type II diabetes. Therefore it is highly pathophysiologically relevant to perform detailed studies of insulin signaling inside skeletal muscle cells in order to elucidate the specific molecular events during both normal and insulin-resistant conditions. So far, cell biology imaging techniques have been limited to in vitro cultured muscle originating from primary or cell line-based myoblasts. However, these types of cultured muscle lack many characteristics of fully differentiated muscle cells. By performing intravital protein translocation analysis directly in situ in living animals, we have been able to give a high-resolution account of the spatial and temporal details during insulin signaling in vivo in muscle that does not have the limitations of in vitro cultures. We have shown that after i.v. insulin injection, PI3-kinase activation and, in turn, GLUT4 translocation are initiated at the plasma membrane proper, the sarcolemma. Then insulin signaling progresses into the t-tubules with a velocity corresponding to the diffusion of sulforhodamine B-conjugated insulin molecules. By using intravital confocal time-lapse analysis we have revealed that the t-tubules are the membrane surface where the majority of the insulin signaling is located.

PMID:
20419438
DOI:
10.1007/978-1-60761-700-6_12
[Indexed for MEDLINE]

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