scholarly journals Mechanism of the decrease in hexose transport by mouse mammary epithelial cells caused by fasting

1988 ◽  
Vol 249 (1) ◽  
pp. 149-154 ◽  
Author(s):  
C G Prosser
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Binding Sites ◽  
Cytochalasin B ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Transport Activity ◽  
Intact Cells ◽  
Equilibrium Exchange ◽  
New Protein

The basal carrier-mediated uptake of 0.5 mM-3-O-methylglucose by mammary epithelial cells from lactating mice was calculated to be 227 +/- 9 pmol/min per microgram of DNA (mean +/- S.E.M., n = 11). Fasting the mice for 16 h overnight resulted in a decrease in this rate to 65 +/- 4 pmol/min per microgram of DNA (n = 10). Refeeding the fasted mouse for 3 h before isolation of the cells restored the transport activity to 230 +/- 12 pmol/min per microgram of DNA (n = 12). The Vmax. for equilibrium exchange entry of 3-O-methylglucose by intact cells was decreased from 6.6 +/- 0.4 to 0.9 +/- 0.2 nmol/min per microgram of DNA (mean +/- S.E.M., n = 3) by fasting. The number of D-glucose-inhibitable cytochalasin-B-binding sites in a plasma-membrane-enriched fraction of the cells was also decreased from 5.7 +/- 1.5 to 1.7 +/- 0.1 pmol/mg of membrane protein (mean +/- S.E.M., n = 3). Again, refeeding the fasted mouse for 3 h reversed both these effects. These results are consistent with a decrease in the number of functional glucose carriers in the plasma membrane of the mammary epithelial cells. Since the restoration of transporter activity after refeeding does not appear to require the synthesis of new protein, the effect of fasting probably involves not a loss of transporters, but a change in their orientation within the plasma membrane or a redistribution within the cell.

Investigation of the role of microtubules in protein secretion from lactating mouse mammary epithelial cells

1992 ◽  
Vol 102 (2) ◽  
pp. 239-247 ◽  
Author(s):  
M.E. Rennison ◽  
S.E. Handel ◽  
C.J. Wilde ◽  
R.D. Burgoyne
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Early Stage ◽  
Major Effect ◽  
Vesicle Transport ◽  
Mammary Epithelial ◽  
Mammary Cells

Disruption of microtubules has been shown to reduce protein secretion from lactating mammary epithelial cells. To investigate the involvement of microtubules in the secretory pathway in these cells we have examined the effect of nocodazole on protein secretion from mammary epithelial cells derived from the lactating mouse. Mouse mammary cells have extensive microtubule networks and 85% of their tubulin was in a polymeric form. Treatment with 1 micrograms/ml nocodazole converted most of the tubulin into a soluble form. In a continuous labelling protocol it was found that nocodazole did not interfere with protein synthesis but over a 5 h period secretion was markedly inhibited. To determine whether the inhibition was at the level of early or late stages of the secretory pathway mammary cells were pulse-labelled for 1 h to label protein throughout the secretory pathway before nocodazole treatment. When secretion was subsequently assayed it was found to be slower and only partially inhibited. These findings suggest that the major effect of nocodazole is on an early stage of the secretory pathway and that microtubules normally facilitate vesicle transport to the plasma membrane. An involvement of microtubules in vesicle transport to the plasma membrane is consistent with an observed accumulation of casein vesicles in nocodazole-treated cells. Exocytosis stimulated by the calcium ionophore ionomycin was unaffected by nocodazole treatment. We conclude from these results that the major effect of nocodazole is at an early stage of the secretory pathway, one possible target being casein vesicle biogenesis in the trans-Golgi network.

scholarly journals Analysis of Cd44-Containing Lipid Rafts

1999 ◽  
Vol 146 (4) ◽  
pp. 843-854 ◽  
Author(s):  
Snezhana Oliferenko ◽  
Karin Paiha ◽  
Thomas Harder ◽  
Volker Gerke ◽  
Christoph Schwärzler ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Lipid Rafts ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Annexin Ii ◽  
Membrane Cholesterol ◽  
Plasma Membrane Cholesterol

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II–p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II–lipid raft complexes were stabilized by addition of GTPγS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.

17-β-estradiol treatment maintains differentiative potential of virgin mouse mammary gland in culture and its responsiveness to insulin

1988 ◽  
Vol 119 (4) ◽  
pp. 543-548
Author(s):  
Hiroko Komura ◽  
Hideto Fukui ◽  
Hiroshi Wakimoto ◽  
Tetsuo Miyake ◽  
Naoki Terakawa ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Binding Sites ◽  
Thymidine Incorporation ◽  
Mammary Epithelial Cells ◽  
Milk Proteins ◽  
Insulin Binding ◽  
Mouse Mammary Gland ◽  
Mammary Epithelial ◽  
Estradiol Treatment ◽  
Lactogenic Hormones

Abstract. Synthesis of the milk proteins, casein and α-lactalbumin was not induced in cultured mammary explants from C3H/HeN castrated virgin mice in the presence of lactogenic hormones such as insulin, cortisol and PRL. Replacement therapy with 17-β-estradiol to the castrated mice completely restored the differentiative potentials of mammary explants, inducing synthesis of the two milk proteins. [3H] thymidine incorporation into DNA synthesized in cultured mammary explants was also decreased by castration to less than 50% (P< 0.001) of that obtained in intact mice, and 17-β-estradiol treatment to castrated animals restored DNA synthesis to 90% of the intact level. The addition of insulin to culture medium significantly (P< 0.001) enhanced [3H] thymidine incorporation into DNA in cultured mammary explants from both intact and 17-β-estradiol-treated castrated mice but not from castrated animals. Insulin binding sites (1710 ± 260 sites/cell) to mammary epithelial cells from castrated mice were significantly (P<0.05) lower than those from both intact (2870 ± 300 sites/cell) and 17-β-estradiol-treated castrated animals (2860 ± 190 sites/cell). The present findings suggest that 17-β-estradiol maintains growth and differentiative responses of mammary epithelial cells to insulin, which may be through preserving the number of insulin binding sites in the cells.

Glucose transporter in bovine mammary epithelial cells is an asymmetric carrier that exhibits cooperativity and trans-stimulation

2003 ◽  
Vol 285 (5) ◽  
pp. C1226-C1234 ◽  
Author(s):  
Changting Xiao ◽  
John P. Cant
Keyword(s):  
Epithelial Cells ◽  
Glucose Transporter ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Isolated Cells ◽  
Experimental Conditions ◽  
Bovine Mammary Epithelial Cells ◽  
Equilibrium Exchange ◽  
Uptake Activity

Glucose transport kinetics were quantified in isolated bovine mammary epithelial cells using 3- O-methyl-d-glucose. Isolated cells retained satisfactory viability and glucose uptake activity, which was inhibited by cytochalasin B, phloretin, HgCl2, and low temperature. Initial rates of entry were measured over a 15-s interval at 37°C under zero- trans, equilibrium-exchange, high- cis, and high- trans concentrations of 3- O-methyl-d-glucose between 0 and 20 mM. The combined set of rate measurements from all experimental conditions was fit to the fixed-site carrier model by nonlinear regression to estimate parameters of transport. For the regression between predicted and observed initial rates, r2 was 0.97. Forward Vmax was estimated at 18.2 nmol·min-1·mg protein-1, and the Michaelis constant was 8.29 mM. The cooperativity parameter was 1.63, trans-stimulation was 2.13-fold, and asymmetry was 2.06-fold. On the basis of the kinetic parameters, variations in intracellular glucose concentrations are not responsible for the range of glucose uptakes by bovine mammary glands observed in vivo.

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