Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Submitted 8 May 2003 ; accepted in final form 4 July 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
model; cow; mammary epithelial cell
GLUT-1 has been identified as the major glucose transporter in lactating mammary tissue of the cow (42), rat (5, 37), mouse (2, 26), and human (4). GLUT-1-mediated transport of sugars has been extensively studied in human erythrocytes (7, 8, 10), where the kinetics display a few striking features. The transport is not symmetrical, with Vmax and apparent Km [Km(app)] much higher for zero-trans efflux than for zero-trans entry (1). Export and import sites cooperatively interact with each other (9, 15, 36), so that binding on one side influences binding on the other. Unidirectional glucose entry is accelerated by the presence of glucose on the opposite, trans, side (21, 39). It is possible that the lack of correlation between extracellular glucose concentration and lactose synthesis rate in mammary tissue is due to the effect of a variable intracellular glucose concentration on net glucose uptake. In ruminants with arterial glucose concentrations of 2-4 mM and mammary venous concentrations 20% lower, concentrations inside mammary secretory cells have been estimated at 0.1-0.5 mM (13, 33).
Several models have been proposed for GLUT-1-mediated glucose transport in human erythrocytes. The simple carrier model assumes that GLUT-1 presents one sugar-translocating site at a time, alternating between efflux and influx of sugar (40). A fixed-site carrier model that assumes simultaneous presentation of intracellular and extracellular sites was proposed to account for transport kinetics in red blood cells (18, 25). Recent ligand-binding studies suggest that the erythrocyte glucose transporter functions as a homotetramer that presents two export sites and two import sites at all times (9, 15), which causes initial entry rate measurements to behave according to the fixed-site carrier model (7, 18). The objective of this study was to characterize the kinetics of steady-state glucose transport across the plasma membrane of bovine mammary epithelial cells in response to extracellular and intracellular glucose concentrations. The fixed-site carrier model was used to quantify the degree of apparent asymmetry, cooperativity, and trans-stimulation. From the parameter values obtained, it was concluded that the variation in mammary glucose uptake in vivo is too large to be simply a consequence of the kinetics of transport.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Procedures involving animals were approved by the Animal Care Committee of the University of Guelph. The isolation of mammary epithelial cells was a modification of the procedure described by Prosser and Topper (31). Mammary tissue (30 g) was collected at slaughter from lactating Holstein cows. Visible connective and adipose tissues were trimmed off. The remaining tissue was rinsed in ice-cold phosphate-buffered saline (PBS, pH 7.4) and transferred on ice to the laboratory within 5 min. The tissue was then chopped with a surgical scalpel and minced with scissors in a dissection dish. The mince was washed three times with ice-cold PBS and transferred to a 500-ml flask with 200 ml of digestion medium containing HBSS supplemented with 0.1% (wt/vol) collagenase (crude, type IA), 4% (wt/vol) bovine serum albumin, 5 mM CaCl2, and 10 µM MgSO4. The flask was stoppered and reciprocally shaken at 120 strokes/min in a water bath at 37°C for 75 min. The choices of the collagenase concentration and digestion time were based on a series of preliminary experiments to obtain cells with satisfactory yield and viability.
The digested tissue was filtered, and the filtrate was centrifuged at 100 g for 5 min for collection of epithelial cells. Adipocytes and fibroblasts suspended in the supernatant were discarded. Cell pellets were resuspended in HBSS and filtered through a 149-µm polypropylene macroporous filter (Fisher Scientific, Whitby, ON, Canada). The cells were collected by centrifugation and washed twice in HBSS. After the final wash, cell pellets were resuspended in ice-cold glucose-deficient DMEM base supplemented with 5 µg/ml insulin, 1 µg/ml prolactin, 1 µg/ml hydrocortisone, and 2 mM acetate, to give a final protein concentration of 2 mg/ml. Cells were divided into aliquots and stored on ice until use.
Determination of protein content. An aliquot of the cells was homogenized and sonicated. The protein content of the homogenate was measured by the Bio-Rad assay (Bio-Rad Laboratories, Mississauga, ON, Canada), with bovine serum albumin used as a standard. Neither centrifugation nor incubation of the cells with various buffers resulted in significant loss of protein.
Viability of isolated cells. Cell viability was assayed over time by measuring the ability of the cells to exclude trypan blue according to Phillips (29). The last viability check was carried out after 6 h of cell storage on ice.
Time course of 3-OMG uptake. Glucose transport activity in isolated mammary epithelial cells was determined using the labeled analog 3-OMG. Aliquots of 100 µl of cell suspension in microcentrifuge tubes were prewarmed for 30 min at room temperature and then for 15 min at 37°C. Uptake was initiated by addition of 100 µl of labeled 3-OMG in PBS to give a final concentration of 5 mM 3-OMG and 1 µCi/tube. Incubation intervals were controlled by a GrabLab timer (Fisher Scientific) that was connected to a foot switch and had an accuracy of 0.1 s. The uptake was terminated at appropriate time intervals by addition of 500 µl of ice-cold stopper solution containing 20 µM cytochalasin B, 100 µM phloretin, and 100 µM HgCl2. The cells were immediately loaded onto a Whatman GF/C glass fiber filter (Fisher Scientific, Whitby, ON, Canada) that was premoistened with ice-cold PBS and mounted on a vacuum filtration system (Millipore, Bedford, MA). Filters were washed within 20 s with 30 ml of ice-cold PBS and then transferred to 20-ml scintillation vials, soaked for 30 min with 0.5 ml of water, and filled with liquid scintillation fluid (ICN, Costa Mesa, CA). After they sat overnight, vials were counted on a liquid scintillation counter (model 6000, Beckman Instruments, Fullerton, CA). Blanks were prepared by addition of stopper solution to cell suspension before addition of incubation medium; then they were immediately filtered and washed. Rates of uptake were calculated by subtracting blank counts from the total sample counts and were expressed on a cell protein basis. Triplicates were measured for each time point.
Inhibition study. Aliquots of 100 µl of cell suspension were incubated at 37°C with 100 µl of incubation buffer supplemented with different inhibitors to give a final concentration of 5 mM 3-OMG and 20 µM cytochalasin B, 100 µM phloretin, 100 µM HgCl2, or 1 mM HgCl2. Uptakes of 3-OMG were also measured without inhibitor at 37°C, 22°C, and 0°C. Incubation time was 2 min for each inhibitor and temperature point.
Kinetic parameters of 3-OMG transport. All cells were prewarmed for 30 min at room temperature and then for 15 min at 37°C. Transport of 3-OMG into cells was measured under four different experimental conditions. 1) Zero-trans assays were initiated by mixing 100 µl of cell suspension and 100 µl of incubation buffer in microfuge tubes to give final extracellular 3-OMG concentrations of 0.5, 1.0, 2.5, 5.0, 10, and 20 mM, all with 1 µCi/tube. 2) For equilibrium exchange assays, 100 µl of cells were incubated with 100 µl of incubation buffer of 1.0, 2.5, 5.0, 10, 20, or 40 mM unlabeled 3-OMG during the 15-min prewarming period. Final extracellular and intracellular concentrations of 3-OMG in the 200-µl mixture were assumed to be one-half the original concentrations in the incubation buffer. Assays were initiated by addition of 10 µl of tracer to the preloaded cells to give 1 µCi/tube. 3) For high-cis entry, aliquots of 100 µl of cells were incubated with 100 µl of preloading buffer containing 0, 2.0, 5.0, 10, 20, or 40 mM 3-OMG during the prewarming period and then centrifuged at 100 g for 3 min to remove the extracellular 3-OMG. Assays were initiated by resuspending the preloaded cell pellets in 200 µl of incubation buffer containing 20 mM 3-OMG with 1 µCi/tube. 4) For high-trans conditions, 100 µl of cells were incubated with 100 µl of 40 mM 3-OMG during the prewarming period and centrifuged to remove the extracellular 3-OMG. Assays were initiated by resuspending the preloaded cell pellets with 200 µl of incubation buffer to give final extracellular 3-OMG concentrations of 0.5, 1.0, 2.5, 5.0, 10, and 20 mM, all with 1 µCi/tube. In all cases, transport was terminated after 15 s by addition of 500 µl of ice-cold stopper solution. The cells were collected, washed, and counted as described above. Triplicates were performed for each combination of extracellular and intracellular concentrations.
The kinetic parameters Vmax and Km(app) for transport of 3-OMG under each experimental condition were computed by nonlinear least-squares regression analysis (35) of unidirectional initial rates of 3-OMG entry vs. extracellular 3-OMG concentrations according to the Michaelis-Menten equation
![]() |
Carrier model parameters of 3-OMG transport. The experimental procedure is briefly illustrated in Fig. 1. All cells were prewarmed for 5 min at room temperature and then for 5 min at 37°C. Cells were then incubated with appropriate 3-OMG concentrations for the next 10 min to give an intracellular 3-OMG concentration of 0-20 mM. All cells were centrifuged at 100 g for 3 min to remove extracellular 3-OMG. Several tubes were randomly chosen and put aside on ice after centrifugation for later protein analysis. Transport was initiated by resuspending cell pellets in 200 µl of incubation buffer with appropriate concentrations of labeled 3-OMG prewarmed to 37°C. Initial rates of 3-OMG entry were measured for extracellular 3-OMG concentrations of 1.0, 2.5, 5.0, 10, and 20 mM, each with intracellular 3-OMG equal to 0 mM, 20 mM, and the extracellular 3-OMG concentration. Incubations with 20 mM extracellular 3-OMG were also carried out with 1, 2.5, 5.0, 7.5, 10, 12.5, and 15 mM intracellular 3-OMG. Transport was terminated after 15 s by addition of 3 ml of ice-cold stopper solution. Filtration, washing, and counting were carried out as described above. Triplicate measurements were obtained for every combination of extracellular and intracellular 3-OMG concentrations.
|
Parameters of the fixed-site carrier model were estimated from the set of all 66 measured initial rates of 3-OMG uptake from each experiment, using a nonlinear, least-squares method (35). The fixed-site carrier model was adopted from Helgerson and Carruthers (18) with the assumption of rapid equilibrium kinetics
|
where P is the intracellular substrate concentration, A is the extracellular substrate concentration, E is the carrier, Vmax f is forward Vmax, K is the Michaelis-Menten constant, represents the degree of cooperativity between sugar export and import sites,
is the trans-stimulation of sugar transport, and
is the degree of asymmetry in transport. Unidirectional sugar entry in this model follows Eq. A8 (derived in the APPENDIX)
![]() |
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the cell viability assay, >90% of the cells excluded trypan blue dye, and this exclusion was maintained for 6 h after cell preparation when the last viability evaluation was carried out. The following transport measurements were finished within 4 h and were therefore conducted on cells of satisfactory viability.
Uptake of 3-OMG. After 10 min at 5 mM 3-OMG, labeled 3-OMG accumulation in cells approached a plateau (Fig. 2), indicating equilibrium across the cell membrane. In our study, equilibrium was reached later than the 2 min observed in rat mammary cells (38). This might be due to species differences or the different approaches adopted for cell preparation. Metabolic rate per unit of mammary tissue is expected to be slower in the cow than in the rat, but we also used different media and a longer digestion procedure to dissociate cells from the tough bovine mammary matrix. Lactogenic hormones were included in the medium for cell storage. Sodium acetate (2 mM) was also included to provide the preferred energy substrate for bovine mammary epithelial cells.
|
In conjunction with the viability assay, the 3-OMG uptake activity demonstrated that the cell preparation was suitable for measurement of glucose transport. In the following experiments, uptake was measured over 2 min (inhibition study) or over the first 15 s of incubation (kinetic experiments) with labeled 3-OMG.
Inhibition study. Two-minute uptakes of 3-OMG were substantially inhibited by 20 µM cytochalasin B, 100 µM phloretin, 100 µM HgCl2, and 1 mM HgCl2 (Fig. 3). These results are in agreement with previous observations in murine mammary epithelial cell preparations (2, 30, 38) and human erythrocytes (10). Low temperature functioned more potently than the chemical inhibitors to decrease the uptake. Although Threadgold et al. (38) found that cold saline could effectively terminate the glucose transport process, a combination of the above inhibitors (20 µM cytochalasin B and 100 µM phloretin) was included in the cold stopper solution in our study. Eilam and Stein (10) used 100 µM phloretin, 1 mM HgCl2, and 1.25 mM KI to stop glucose transport in human erythrocytes. HgCl2 denatures cell membrane protein in general. Because high concentrations of HgCl2 might cause leakage of 3-OMG across the cell membrane (Fig. 3), this substance was omitted in the stopper solution for the following kinetic measurements.
|
Kinetic parameters of 3-OMG transport. Zero-trans entry rates were measured when intracellular 3-OMG was absent (Fig. 4A). Calculated Km(app) and Vmax from three separate experiments were 6.95 ± 1.01 mM and 24.92 ± 3.41 nmol·min-1·mg protein-1, respectively (Table 1). Threadgold et al. (38) obtained an average Km of 16 mM and Vmax of 56 nmol·min-1·mg protein-1 for zero-trans entry of 2-deoxy-D-glucose into lactating rat mammary acini. If it is assumed that the affinities for 3-OMG and 2-deoxy-D-glucose are not significantly different, which has been noted in human erythrocytes (8), affinity was nearly twofold greater in mammary cells of the cow than of the rat for extracellular sugar when intracellular sugar was absent (38). Plasma glucose concentration in the lactating cow is maintained at 2.5-3.5 mM, about one-half the normal concentration in the rat. Erythrocyte transporters have much greater affinity under zero-trans conditions, with Km of 0.2-1.6 mM (8, 18, 39). It was reported that purified GLUT-1 exhibits a Km of 5-7 mM for glucose (24). Intracellular volume was calculated from the extracellular 3-OMG concentration and the 3-OMG uptake at 30 min, with the assumption that 3-OMG uptake at 30 min in Fig. 2 represents full equilibrium. The calculated intracellular volume of 2.45 µl/mg protein was in agreement with 2.76 µl/mg protein reported for rat mammary cells (41). With the use of 2.45 µl/mg protein, Vmax for zero-trans entry in bovine mammary epithelial cells at 37°C (Table 1) was 10.16 mM/min, which is three- to fivefold slower than 31-52 mM/min measured in human erythrocytes at 20-25°C (39).
|
|
Glucose transport rates were not significantly different among cell preparations from cows with daily milk yields of 15-36 kg (P > 0.46). The regression of zero-trans Vmax vs. daily milk yield was as follows: Vmax (nmol·min-1·mg protein-1) = 21.74 + 0.13 x milk yield (kg/day; r2 = 0.09, n = 8). The lack of a significant regression is consistent with the observation that differences in milk production between cows are primarily due to differences in secretory cell number, not activity per cell (19).
Km(app) (17.78 ± 1.74 mM) and Vmax (39.77 ± 0.91 nmol·min-1·mg protein-1) for 3-OMG entry under equilibrium exchange conditions (Table 1) were 2.5 and 1.7 times higher, respectively, than those obtained in zero-trans assays. In human erythrocytes, equilibrium exchange of sugar at ice temperature typically exhibits 50- to 100-fold higher Km and Vmax over zero-trans entry (8, 39). The difference was only fourfold in rat erythrocytes, which express 200- to 400-fold fewer GLUT-1 proteins on the cell surface than human erythrocytes (18). A Km of 14 mM and a Vmax of 6.6 nmol·min-1·µg DNA-1 were reported for equilibrium exchange of 3-OMG by isolated mammary cells of the mouse (30, 31). Given a DNA-to-protein ratio of 1:15.06 ± 0.27 (n = 4) in our cell preparation, the Vmax for equilibrium exchange in the cow was 2.64 nmol·min-1·µg DNA-1, which is approximately one-third of that obtained in mouse cells.
High-trans entry rates were measured in cells preloaded with 20 mM 3-OMG (Fig. 4D). The mean Km of 12.36 ± 0.84 mM from three separate experiments was 1.8 times higher than the Km for zero-trans entry, which falls in the range of 1.5- to 4.0-fold increases reported for the red blood cell (8, 39). This indicates a similar level of cooperativity or degree to which binding of intracellular sugar to the transporter reduces its affinity for extracellular sugar. However, the Vmax increased by a factor of only 1.3 with saturating intracellular 3-OMG compared with 30- to 60-fold increases in erythrocytes (8, 39). The potential for trans-stimulation is therefore much lower in bovine mammary epithelial cells.
The equilibrium exchange and high-trans experiments suggested less than twofold increases in entry rates when cells were preloaded with 3-OMG, which was directly demonstrated by the high-cis entry study (Fig. 4C). When intracellular 3-OMG concentration increased from 0 to 20 mM, the uptake rate increased 30%. There was a more dramatic 6- to 10-fold increase in trans-stimulation of saturating extracellular 3-OMG transport into erythrocytes when intracellular sugar concentration was increased from 0 to 20 mM (8, 18).
Carrier model parameters of 3-OMG transport. Simultaneous estimation of the cooperativity (), trans-stimulation (
), and asymmetry (
) factors was accomplished by fitting the fixed-site carrier model to the set of data from all incubations for each cow (Table 2). Estimates of
,
, and
were associated with errors ranging from 0.43 to 2.23 of the estimate, which suggests that these parameters are not highly identifiable from the four sets of incubation conditions commonly used. Nevertheless, among cows, estimates of all parameters agreed well, indicating repeatability of the estimation and consistency of carrier properties. Vmax f and K were estimated with relatively low errors, despite the high correlation between them (Table 3). The positive correlation is due to the fact that Vmax f and K appear on opposite sides of the quotient, so that increasing the value of one in an iterative optimization routine necessitates an increase in the other for a given point estimate of transport rate. The plateau of transport rate at higher [A] forces a solution to Vmax f and, hence, K. Parameters
and
were also highly correlated (Table 3), which, in this case, may have contributed to the difficulty in their identification. Mean estimates for
,
, and
were significantly different from 1.0 (P < 0.05), so the fixed-site carrier model did not overdefine mammary glucose transport and successfully simulated the four kinetic curves (Fig. 5). Furthermore, for the regression between predicted and observed initial rates (Fig. 6), r2 was 0.97, slope was not different from 1.0 (P = 0.19), and intercept was not different from 0 (P = 0.21). The model was therefore adequate to describe, in an accurate and unbiased manner, 15-s 3-OMG accumulation in mammary cells over a range of intracellular and extracellular sugar concentrations between 0 and 20 mM. On the basis of estimated parameter values, sugar transport by bovine mammary epithelial cells is twofold asymmetric, with moderate cooperativity between binding sites on the external and internal sides of the membrane and with a potential for twofold trans-stimulation.
|
|
|
|
The asymmetry of GLUT-1-mediated sugar transport is well defined in human erythrocytes, where it has been shown that Vmax and Km(app) are much greater for efflux into a glucose-free medium than for influx into glucose-depleted cells (1, 8). However, symmetry of sugar transport kinetics has been reported for rat and rabbit erythrocytes (18, 32); and
in rat erythrocytes were estimated to be 1. It has been suggested that the transport process is intrinsically symmetrical, but apparent asymmetry arises from the presence of more than one intracellular compartment. Carruthers and coworkers (15-17, 36) proposed a model in which the erythrocyte sugar carrier is a homotetramer of GLUT-1 proteins. Each copy of the GLUT-1 protein functions in isolation as a simple carrier undergoing reversible conformational change between exporting and importing states. Interactions between GLUT-1 proteins within the tetramer cause the transporter complex to present two export and two import sites at any instant, thereby acting as a fixed-site carrier. Allosteric binding of intracellular ATP is proposed to cause cytoplasmic domains of the GLUT-1 proteins to form a cage immediately under the cell membrane (16, 20). Sugar transported into the cage might be recycled for export, bound to high-affinity sugar-binding sites in the cage, or slowly released into the cytosol. Presence of the cage would result in underestimation of intracellular glucose concentration and inaccuracy in initial rate measurements because of significant backflux of radiolabel within the 5-15 s of incubation (1, 7). The consequence of the cage is an apparent asymmetry of transport. It has been demonstrated in human erythrocytes that cage formation is prompted by intracellular ATP binding to GLUT-1 protein (16, 20). Under circumstances that deplete cellular ATP or prevent ATP binding to GLUT-1, apparent asymmetry of the transporter would be lost (16). We have used the carrier model, which describes 15-s accumulation kinetics well, to identify a twofold asymmetry of glucose transport by bovine mammary epithelial cells. Although this asymmetry was not to the same degree as in human erythrocytes, GLUT-1 may be arranged in a similar homotetrameric manner in bovine mammary epithelial cells. ATP for cage formation would have been plentiful, given that the medium contained 2.0 mM acetate, the preferred energy substrate in the cow (14).
Implications of glucose transport parameters. Having estimated all parameters of the carrier model for sugar transport, the potential impact of intracellular glucose in the mammary glands in vivo can be considered. The assumption in these considerations is that the binding affinities estimated for 3-OMG can be applied directly to its analog, glucose. The kinetics of 3-OMG uptake by mammary secretory cells have not been compared with those of D-glucose uptake, but in the human erythrocyte at ice temperature, Km for zero-trans entry was 0.38 ± 0.13 and 0.46 ± 0.09 mM for the two sugars, respectively (18). Km values for infinite-trans entry of 3-OMG and glucose into erythrocytes were 1.57 ± 0.10 and 2.19 ± 0.36 mM, respectively (18). The similarity in binding affinities suggests that the 3-OMG kinetics measured here can be used as approximations of glucose kinetics.
Intracellular glucose concentrations are notoriously difficult to measure, but milk glucose concentration is often used as a surrogate. Faulkner et al. (13) observed a milk concentration of 0.14 mM, and Rigout et al. (33) found 0.5 mM. Km(app) values for net sugar entry, calculated as
![]() |
![]() |
|
![]() |
APPENDIX |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
![]() |
![]() | (A1) |
![]() | (A2) |
![]() | (A3) |
![]() | (A4) |
![]() | (A5) |
![]() | (A6) |
![]() | (A7) |
![]() | (A8) |
![]() |
![]() | (A9) |
![]() | (A10) |
![]() | (A11) |
![]() | (A12) |
![]() | (A13) |
![]() | (A14) |
![]() |
![]() | (A15) |
![]() |
DISCLOSURES |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bennett BL, Grigor MR, and Prosser CG. Glucose transport in a murine mammary epithelial cell line. Biochem Mol Biol Int 42: 315-323, 1997.[ISI][Medline]
3. Bickerstaffe R and Annison EF. The metabolism of glucose, acetate, lipids and amino acids in lactating dairy cows. J Agric Sci 82: 71-85, 1974.[ISI]
4. Brown RS and Wahl RL. Overexpression of Glut-1 glucose transporter in human breast cancer. An immunohistochemical study. Cancer 72: 2979-2985, 1993.[ISI][Medline]
5. Burnol AF, Leturque A, Loizeau M, Postic C, and Girard J. Glucose transporter expression in rat mammary gland. Biochem J 270: 277-279, 1990.[ISI][Medline]
6. Cant JP, DePeters EJ, and Baldwin RL. Mammary uptake of energy metabolites in dairy cows fed fat and its relationship to milk protein depression. J Dairy Sci 76: 2254-2265, 1993.
7. Carruthers A. Mechanisms for the facilitated diffusion of substrates across cell membranes. Biochemistry 30: 3898-3906, 1991.[ISI][Medline]
8. Cloherty EK, Heard KS, and Carruthers A. Human erythrocyte sugar transport is incompatible with available carrier models. Biochemistry 35: 10411-10421, 1996.[ISI][Medline]
9. Cloherty EK, Levine KB, and Carruthers A. The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry 40: 15549-15561, 2001.[ISI][Medline]
10. Eilam Y and Stein WD. Kinetic studies of transport across red blood cell membranes. In: Methods in Membrane Biology, edited by Edward DK. New York: Plenum, 1974, p. 283-354.
11. Emerman TJ, Bartley JC, and Bissell MJ. Interrelationship of glycogen metabolism and lactose synthesis in mammary epithelial cells of mice. Biochem J 192: 695-702, 1980.[ISI][Medline]
12. Faulkner A. Glucose availability and lactose synthesis in the goat. Biochem Soc Trans 13: 496-497, 1985.[ISI]
13. Faulkner A, Chaiyabutr N, Peaker M, Carrick DT, and Kuhn NJ. Metabolic significance of milk glucose. J Dairy Res 48: 51-56, 1981.[ISI][Medline]
14. Forsberg NE, Baldwin RL, and Smith NE. Roles of glucose and its interactions with acetate in maintenance and biosynthesis in bovine mammary tissue. J Dairy Sci 68: 2544-2549, 1985.[ISI][Medline]
15. Hamill S, Cloherty EK, and Carruthers A. The human erythrocyte sugar transporter presents two sugar import sites. Biochemistry 38: 16974-16983, 1999.[ISI][Medline]
16. Heard KS, Fidyk N, and Carruthers A. ATP-dependent substrate occlusion by the human erythrocyte sugar transporter. Biochemistry 39: 3005-3014, 2000.[ISI][Medline]
17. Hebert DN and Carruthers A. Glucose transporter oligomeric structure determines transporter function. Reversible redox-dependent interconversions of tetrameric and dimeric GLUT1. J Biol Chem 267: 23829-23838, 1992.
18. Helgerson AL and Carruthers A. Analysis of protein-mediated 3-O-methylglucose transport in rat erythrocytes: rejection of the alternating conformation carrier model for sugar transport. Biochemistry 28: 4580-4594, 1989.[ISI][Medline]
19. Knight CH, Peaker M, and Wilde CJ. Local control of mammary development and function. Rev Reprod 3: 104-112, 1998.
20. Levine KB, Cloherty EK, Fidyk NJ, and Carruthers A. Structural and physiologic determinants of human erythrocyte sugar transport regulation by adenosine triphosphate. Biochemistry 37: 12221-12232, 1998.[ISI][Medline]
21. Lowe AG and Walmsley AR. The kinetics of glucose transport in human red blood cells. Biochim Biophys Acta 857: 146-154, 1986.[ISI][Medline]
22. Mepham TB. Physiology of Lactation. Philadelphia, PA: Open University Press, 1987.
23. Miller PS, Reis BL, Calvert CC, DePeters EJ, and Baldwin RL. Relationship of early lactation and bovine somatotropin on nutrient uptake by cow mammary glands. J Dairy Sci 74: 3800-3806, 1991.
24. Mueckler M, Hresko RC, and Sato M. Structure, function, and biosynthesis of GLUT1. Biochem Soc Trans 22: 951-954, 1997.
25. Naftalin RJ and Rist RJ. Re-examination of hexose exchanges using rat erythrocytes: evidence inconsistent with a one-site sequential exchange model, but consistent with a two-site simultaneous exchange model. Biochim Biophys Acta 1191: 65-78, 1994.[ISI][Medline]
26. Nemeth BA, Tsang SW, Geske RS, and Haney PM. Golgi targeting of the GLUT1 glucose transporter in lactating mouse mammary gland. Pediatr Res 47: 444-450, 2000.
27. Page T and Kuhn N. Intracellular glucose in the mammary gland of the lactating rat. Biochem Soc Trans 15: 1095-1096, 1987.[ISI]
28. Park CS, Smith JJ, Sasaki M, Eigel WN, and Keenan TW. Isolation of functionally active acini from bovine mammary gland. J Dairy Sci 62: 537-545, 1979.[ISI][Medline]
29. Phillips HJ. Dye exclusion test for cell viability. In: Tissue Culture Methods and Applications, edited by Kruse PF and Patterson MK. New York: Academic, 1973, p. 406-408.
30. Prosser CG. Mechanism of the decrease in hexose transport by mouse mammary epithelial cells caused by fasting. Biochem J 249: 149-154, 1988.[ISI][Medline]
31. Prosser CG and Topper YJ. Changes in the rate of carrier-mediated glucose transport by mouse mammary epithelial cells during ontogeny: hormone dependence delineated in vitro. Endocrinology 119: 91-96, 1986.[Abstract]
32. Regen DM and Morgan HE. Studies of the glucose-transport system in the rabbit erythrocyte. Biochim Biophys Acta 79: 151-166, 1964.[ISI]
33. Rigout S, Lemosquet S, van EJ, Blum JW, and Rulquin H. Duodenal glucose increases glucose fluxes and lactose synthesis in grass silage-fed dairy cows. J Dairy Sci 85: 595-606, 2002.
34. Rook JAF and Hopwood JB. The effects of intravenous infusion of insulin and of sodium succinate on milk secretion in the goat. J Dairy Res 37: 193-198, 1970.[ISI]
35. SAS Institute. User's Guide: Statistics, version 8.01. Cary, NC: SAS Institute, 1999.
36. Sultzman LA and Carruthers A. Stop-flow analysis of cooperative interactions between GLUT1 sugar import and export sites. Biochemistry 38: 6640-6650, 1999.[ISI][Medline]
37. Takata K, Fujikura K, Suzuki M, Suzuki T, and Hirano H. GLUT1 glucose transporter in the lactating mammary gland in the rat. Acta Histochem Cytochem 30: 623-628, 1997.[ISI]
38. Threadgold LC, Coore HG, and Kuhn NJ. Monosaccharide transport into lactating-rat mammary acini. Biochem J 204: 493-501, 1982.[ISI][Medline]
39. Wheeler TJ and Whelan JD. Infinite-cis kinetics support the carrier model for erythrocyte glucose transport. Biochemistry 27: 1441-1450, 1988.[ISI][Medline]
40. Widdas WF. Inability of diffusion to account for placental glucose transfer in the sheep and consideration of the kinetics of a possible carrier transfer. J Physiol 118: 23-39, 1952.[ISI][Medline]
41. Wilde CJ and Kuhn NJ. Lactose synthesis and the utilisation of glucose by rat mammary acini. Int J Biochem 13: 311-316, 1981.[ISI][Medline]
42. Zhao FQ, Dixon WT, and Kennelly JJ. Localization and gene expression of glucose transporters in bovine mammary gland. Comp Biochem Physiol B Biochem Mol Biol 115: 127-134, 1996.[ISI][Medline]