Abstract 519 Neonatal Nutrition and Metabolism I Poster Symposium, Saturday, 5/1

The synthesis of lactose, the major carbohydrate and osmotic constituent of human milk, takes place in the Golgi. To provide substrate for lactose synthesis in mammary epithelial cells, free glucose must be transported into Golgi. Evidence from 2-deoxyglucose uptake studies, subcellular fractionation, and immunocytochemistry demonstrates that the GLUT1 glucose transporter is induced and specifically targeted to the Golgi apparatus during lactation. The current experiments test two hypotheses: 1) prolactin and hydrocortisone stimulate glycosylation of GLUT1, altering its targeting from plasma membrane to Golgi, and 2) GLUT1 is targeted specifically to cis- or medial-Golgi and not trans-Golgi. CIT3 mouse mammary epithelial cells, a subline of Comma-1-D cells (P. Neville, Univ. of Colorado), were maintained in growth medium (insulin, EGF) or treated for four days in secretion medium (insulin, prolactin, hydrocortisone) to stimulate differentiation. Methods included 1) subcellular fractionation and treatment with PNGase F, followed by SDS-PAGE and Western blotting, and 2) treatment with Brefeldin A followed by confocal immunofluorescent microscopy. Secretion medium enhanced GLUT1 glycosylation, causing an increase in apparent molecular weight from 50 kD to 53 kD. However, the higher apparent molecular weight was observed in both a Golgi-enriched and a plasma membrane enriched fraction as well as in homogenate. Enzymatic deglycosylation with PNGase F reduced the apparent molecular weight of all fractions from both growth and secretion medium to 37 kD. This indicates that differential glycosylation is responsible for differences in apparent molecular weight of GLUT1 between growth medium and secretion medium, but not for determining whether GLUT1 is targeted to plasma membrane or to Golgi. At the dose used, 500 nM, Brefeldin A disrupts trans-Golgi but not cis- or medial-Golgi of lactating mammary epithelial cells ( Pauloin et al., Eur. J. Cell Biol. (1997) 72, 324-336). After treatment of CIT3 cells with secretion medium, GLUT1 demonstrates a perinuclear distribution with a punctate pattern scattered through the cytoplasm characteristic of Golgi. However, it colocalizes neither with BODIPY-TR ceramide, a trans-Golgi marker, nor with a-mannosidase or b-COP, markers of medial- and cis-Golgi respectively. Brefeldin A causes disruption of the trans-Golgi but does not grossly affect targeting of GLUT1, a-mannosidase, or b-COP. However, Brefeldin A does cause colocalization of GLUT1 with a-mannosidase and b-COP, suggesting a subtle effect on membrane trafficking in cis- and medial-Golgi. Therefore, in mammary epithelial cells, prolactin and hydrocortisone enhance GLUT1 glycosylation but cause glycosylation-independent targeting of GLUT1 to a subcompartment of cis- and/or medial-Golgi, but not to trans-Golgi.