Identification and characterization of glucose transport proteins in plasma membrane- and Golgi vesicle-enriched fractions prepared from lactating rat mammary gland

Abstract

Plasma membrane-and Golgi vesicle-enriched membrane fractions were prepared from day-10 lactating rat mammary glands. Each fraction was found to contain a single set of D-glucose-inhibitable cytochalasin B-binding sites: plasma membranes and Golgi vesicles bound 20+ 2 and 53 +4 pmol of cytochalasin/mg of membrane protein (means + S.E.M.), with dissociation constants of 259 + 47 and 520 + 47 nm respectively. Anti-peptide antibodies against the C-terminal region (residues 477-492) of the rat brain/human erythrocyte glucose transporter labelled a sharp band of apparent Mr 50000 on Western blots of both fractions. Treatment with endoglycosidase F before blotting decreased the apparent Mr of this band to 38000, indicating that it corresponded to a glycoprotein. Confirmation that this immunologically cross-reactive band was a glucose transporter was provided by the demonstration that it could be photoaffinity-labelled, in a D-glucosesensitive fashion, with cytochalasin B. Quantitative Western blotting studies yielded values of 28 + 5 and 23 + 3 pmol of immunologically cross-reactive glucose transporters/mg of membrane protein in the plasma membrane and Golgi vesicle fractions respectively. From comparison with the concentration of cytochalasin B-binding sites, it is concluded that a protein homologous to the rat brain glucose transporter cons'titutes the major glucose transport species in the plasma membranes of mammary gland epithelial cells. Glucose transporters are also found in the Golgi membranes of these cells, at least half of them being similar, if not identical, to the transporters of the plasma membrane. However, their function in this location remains unclear.

GLUT1 Glucose Transporter in the Lactating Mammary Gland in the Rat.

“…In the rat mammary gland, a cell fractionation study suggested that GLUT1 or a related transporter might be present in the Golgi apparatus in addition to the plasma membrane [13]. It was suggested that the glucose transporter in the Golgi membrane may play an important role in supplying glucose for lactose synthesis within the Golgi lumen [13]. However, we were unable to demonstrate GLUT1 in the Golgi apparatus by immunofluorescence or immunoelectron microscopy using anti-GLUT1 antibody.…”

“…GLUT1 localized at the basolateral domain in these metabolically active epithelial cells may serve for the uptake of glucose from the intercellular space of connective tissues adjacent to blood vessels. In the rat mammary gland, a cell fractionation study suggested that GLUT1 or a related transporter might be present in the Golgi apparatus in addition to the plasma membrane [13]. It was suggested that the glucose transporter in the Golgi membrane may play an important role in supplying glucose for lactose synthesis within the Golgi lumen [13].…”

“…Glucose is utilized as a source of ATP production and a substrate of lactose and proteins secreted into the milk. Immunohistochemical and biochemical analyses have demonstrated the presence of GLUT1 in the lactating mammary gland [5,6,13], although the precise localization has yet to be determined. In our present study, we examined the localization of GLUT1 in the rat mammary gland by immunofluorescence microscopy and immunoperoxidase electron microscopy.…”

Expression of constitutively activated Akt in the mammary gland leads to excess lipid synthesis during pregnancy and lactation

“…There is good evidence that glucose uptake by mammary epithelial cells for use during de novo fatty acid synthesis, as well as synthesis of lactose, is mediated by the glucose transporter, glucose transporter-1 (GLUT1). Expression of GLUT1 increases during lactation, and a change in its subcellular localization has been observed in mammary epithelial cells during lactation (14)(15)(16).…”

Mammary gland secretion: hormonal coordination of endocytosis and exocytosis

“…Therefore the uptake of glucose and its transport across the basolateral membrane are important processes during milk synthesis. The glucose transporter (GLUT1) is present at the basolateral membrane of rodent MEC (Madon et al, 1990;Nemeth et al, 2000) and is the predominant glucose transporter in the lactating bovine mammary gland (Zhao and Keating, 2007). The expression of genes encoding the glucose transporters is under the control of lactogenic hormones in the mammary gland during pregnancy and lactation (Shennan and Peaker, 2000 and references therein; Zhao and Keating, 2007).…”