Regulated dynamic subcellular GLUT4 localization revealed by proximal proteome mapping in human muscle cells.

Regulation of glucose transport, which is central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter (also known as SLC2A4) in the plasma membrane (PM) of fat and muscle cells. Physiologic signals [such as activated insulin receptor or AMP-activated protein kinase (AMPK)] increase PM GLUT4. Here, we show that the distribution of GLUT4 between the PM and interior of human muscle cells is dynamically maintained, and that AMPK promotes PM redistribution of GLUT4 by regulating exocytosis and endocytosis. Stimulation of exocytosis by AMPK is mediated by Rab10 and the Rab GTPase-activating protein TBC1D4. APEX2 proximity mapping reveals that GLUT4 traverses both PM-proximal and PM-distal compartments in unstimulated muscle cells, further supporting retention of GLUT4 by a constitutive retrieval mechanism. AMPK-stimulated translocation involves GLUT4 redistribution among the same compartments traversed in unstimulated cells, with a significant recruitment of GLUT4 from the Golgi and trans-Golgi network compartments. Our comprehensive proximal protein mapping provides an integrated, high-density, whole-cell accounting of the localization of GLUT4 at a resolution of ∼20 nm that serves as a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in a physiologically relevant cell type.

GLUT4 dynamic subcellular localization is controlled by AMP kinase activation as revealed by proximal proteome mapping in human muscle cells.

Regulation of glucose transport into muscle and adipocytes, central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter in the plasma membrane ( PM ). Physiologic signals (activated insulin receptor or AMP kinase [ AMPK ]), acutely increase PM GLUT4 to enhance glucose uptake. Here we show in kinetic studies that intracellular GLUT4 is in equilibrium with the PM in unstimulated cultured human skeletal muscle cells, and that AMPK promotes GLUT4 redistribution to the PM by regulating both exocytosis and endocytosis. AMPK-stimulation of exocytosis requires Rab10 and Rab GTPase activating protein TBC1D4, requirements shared with insulin control of GLUT4 in adipocytes. Using APEX2 proximity mapping, we identify, at high-density and high-resolution, the GLUT4 proximal proteome, revealing GLUT4 traverses both PM proximal and distal compartments in unstimulated muscle cells. These data support intracellular retention of GLUT4 in unstimulated muscle cells by a dynamic mechanism dependent on the rates of internalization and recycling. AMPK promoted GLUT4 translocation to the PM involves redistribution of GLUT4 among the same compartments traversed in unstimulated cells, with a significant redistribution of GLUT4 from the PM distal Trans Golgi Network Golgi compartments. The comprehensive proximal protein mapping provides an integrated, whole cell accounting of GLUT4's localization at a resolution of ∼20 nm, a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in physiologically relevant cell type and as such, sheds new light on novel key pathways and molecular components as potential therapeutic approaches to modulate muscle glucose uptake.