Abstract
Insulin modulates glucose uptake into adipocytes by regulating the trafficking of the GLUT4 glucose transporter. GLUT4 is mostly excluded from the surface of unstimulated cells because it is much more slowly exocytosed than it is endocytosed. GLUT4 traffics through an adipocyte-specific, specialized endosomal recycling pathway that only partially overlaps with compartments of the general endosomal recycling pathway. Insulin stimulates GLUT4 exocytosis and partially inhibits its endocytosis, resulting in GLUT4 redistribution to the cell surface. Insulin does not stimulate glucose uptake into adipocytes lacking the CCAAT/enhancer-binding protein alpha (C/EBPalpha) transcription factor. Here we show that these adipocytes do not properly traffic GLUT4. In these adipocytes, GLUT4 was rapidly exocytosed in basal conditions, resulting in an accumulation of GLUT4 on the plasma membrane. Although the kinetics of GLUT4 trafficking were altered, GLUT4 was still targeted to specialized intracellular compartments in adipocytes lacking C/EBPalpha, demonstrating an uncoupling of the targeting of GLUT4 to a specialized, adipocyte-specific insulin-regulated pathway from the regulation of the movement of GLUT4 through this pathway. Re-expression of C/EBPalpha in adipocytes lacking C/EBPalpha restored normal GLUT4 trafficking. We propose that C/EBPalpha controls the expression of the proteins that determine the basal, slow exocytosis of GLUT4, but not the proteins required to make the adipocyte-specific compartments through which GLUT4 traffics. Furthermore, these data support a model in which insulin stimulates GLUT4 exocytosis by releasing an inhibitor of GLUT4 movement to the cell surface, and it is this clamp on basal exocytosis that is missing in adipocytes lacking C/EBPalpha.
Highlights
Inhibits internalization, and both of these changes promote a net redistribution of GLUT4 to the cell surface
To calculate the fraction of HA-GLUT4-GFP in the specialized compartment the following was used: fTR Ϫ fIRAP-TR/fTOTAL Ϫ fIRAP-TR, where fTR is the Cy3/GFP fluorescence remaining after ablation by Tf-HRP taken up via the TR, fIRAP-TR is the fluorescence remaining after ablation by Tf-HRP taken up via the IRAP-transferrin receptor (IRAP-TR), and fTOTAL is the fluorescence in cells that were not incubated with Tf-HRP
Regardless, our results show that re-expression of C/EBP␣ restores insulin-responsive GLUT4 trafficking to CCAAT/enhancer-binding protein ␣ (C/EBP␣)Ϫ/Ϫ adipocytes, thereby explaining the previous observations that re-expression of C/EBP␣ restores insulin-regulated glucose uptake [21, 30]
Summary
Inhibits internalization, and both of these changes promote a net redistribution of GLUT4 to the cell surface. IRAP, a transmembrane insulin-regulated aminopeptidase, is the only other protein known to traffic through the GLUT4 pathway [5, 6]. In addition to the insulin-regulated pathway being highly selective for cargo molecules (e.g. GLUT4 and IRAP), this specialized trafficking pathway is most highly developed in fat and muscle cells, the two cell types involved in disposal of dietary glucose. The complex intracellular itinerary of GLUT4 has been intensively studied, and the pathway has not been completely elucidated, it is clear that GLUT4 traffics through both general endosomes and specialized compartments as it cycles between the interior and cell surface [8, 9]. GLUT4 does not return directly from endosomes to the cell surface, but rather it is transported to an intracellular compartment that is not along the general endocytic-recycling pathway.
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