GLUT4 is internalized by a cholesterol-dependent nystatin-sensitive mechanism inhibited by insulin

Abstract

Insulin slows GLUT4 internalization by an unknown mechanism. Here we show that in unstimulated adipocytes, GLUT4 is internalized by two mechanisms. Approximately 80% of GLUT4 is internalized by a mechanism that is sensitive to the cholesterol‐aggregating drug nystatin, and is independent of AP‐2 clathrin adaptor and two putative GLUT4 endocytic motifs. The remaining GLUT4 is internalized by an AP‐2‐dependent, nystatin‐resistant pathway that requires the FQQI GLUT4 motif. Insulin inhibits GLUT4 uptake by the nystatin‐sensitive pathway and, consequently, GLUT4 is internalized by the AP‐2‐dependent pathway in stimulated adipocytes. The phenylalanine‐based FQQI GLUT4 motif promotes AP‐2‐dependent internalization less rapidly than a tyrosine‐based motif, the classic form of aromatic‐based motifs. Thus, both a change in the predominant endocytosis pathway and the specific use of a suboptimal internalization motif contribute to the slowing of GLUT4 internalization in insulin‐stimulated adipocytes. Insulin also inhibits the uptake of cholera‐toxin B, indicating that insulin broadly regulates cholesterol‐dependent uptake mechanisms rather than specially targeting GLUT4. Our work thus identifies cholesterol‐dependent uptake as a novel target of insulin action in adipocytes.