Acute effectors of GLUT1 glucose transporter subcellular targeting in CIT3 mouse mammary epithelial cells

Lactogenic hormones cause intracellular targeting of glucose transporter 1 (GLUT1) for transport of glucose to the site of lactose synthesis in mammary glands. Our aim was to study the intracellular trafficking mechanisms involved in GLUT1 targeting and recycling in CIT3 mouse mammary epithelial cells. Fusion proteins of GLUT1 and enhanced green fluorescent protein (EGFP) were expressed in CIT3 cells maintained in growth medium (GM), or exposed to secretion medium (SM), containing prolactin. Agents acting on Golgi and related subcellular compartments and on GLUT1 and GLUT4 targeting in muscle and fat cells were studied. Wortmannin and staurosporine effects on internalization of GLUT1 were not specific, supporting a basal constitutive GLUT1 membrane-recycling pathway between an intracellular pool and the cell surface in CIT3 cells, which targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly to a brefeldin A-sensitive compartment. Arrest of endosomal acidification by bafilomycin A1 disrupted this prolactin-induced GLUT1 intracellular trafficking with central coalescence of GLUT1-EGFP signal, suggesting that it is via endosomal pathways. This machinery offers another level of regulation of lactose synthesis by altering GLUT1 targeting within minutes to hours.