Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada, N1G 2W1
Submitted 22 August 2003 ; accepted in final form 21 November 2003
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ABSTRACT |
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compartmentalization; milk synthesis
The mechanism of sugar binding and transport by GLUT-1 that results in the features of asymmetry, trans-stimulation, and cooperativity is under investigation. The fixed-site carrier model (12) can accommodate these features. However, because the Vmax and Km for zero-trans glucose efflux from human erythrocytes predicted from effects of intracellular sugar on glucose entry were different from those observed, it was suggested that some sort of unstirred glucose layer on the intracellular aspect of the plasma membrane confounded initial rate measurements, causing an artifactual apparent asymmetry (16). It has been recently proposed that the glucose transporter in human erythrocytes is a homotetramer of GLUT-1 molecules. Although each GLUT-1 monomer functions as a simple carrier, cooperative interactions between GLUT-1 dimers cause the carrier to present two export and import sites simultaneously at any instant (6, 9, 11, 21). On allosteric regulation by intracellular ATP binding, the GLUT-1 cytosolic domains form a cage. Sugar is translocated across the cell membrane and into the cage, where the sugar molecules can be recycled back to the extracellular space, bound to binding sites within the cage, or released into the cytosol (10).
Heard et al. (10), using a mathematical model of glucose translocation, occlusion, binding, and release, were able to simulate the 60-fold asymmetry of glucose transport into human erythrocytes. Our objective in this study was to examine possible compartmentalization of intracellular sugar and to determine whether this compartmentalization results in the observed kinetic characteristics of glucose transport by isolated bovine mammary epithelial cells. Typically, kinetics of transport are described by a Michaelis-Menten equation, such as that for the fixed-site carrier, which is parametized from transmembrane fluxes measured at various initial sugar concentrations. Here, we have combined time course and concentration dependence data to fit a three-compartment model that accounts for the apparent asymmetry, trans-stimulation, and cooperativity of mammary glucose transport.
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MATERIALS AND METHODS |
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Cell isolation. Procedures involving animals were approved by the Animal Care Committee of the University of Guelph. Cells were prepared as a modification of the procedure described by Prosser and Topper (18). In brief, fresh tissue was obtained from lactating Holstein dairy cow (milk yield 15 kg/day) mammary gland and digested with 0.1% (wt/vol) collagenase in HBSS. Cells were collected by centrifugation and washed in phosphate-buffered saline (PBS, pH 7.4). Washed cells were resuspended in glucose-deficient DMEM base at a protein content of 24 mg/ml and stored on ice for further experiments. Cell protein was analyzed by the Bio-Rad method with bovine serum albumin as the standard.
Net uptake of 3-OMG by isolated bovine mammary epithelial cells. Time course of glucose transport was measured at 37°C by incubating 100 µl of cell suspension with 100 µl of DMEM base medium containing 10 mM 3-OMG and 1 µCi of tracer. Uptake was terminated after a range of incubation times between 15 s and 20 min by addition of 3 ml of ice-cold PBS (pH 7.4) containing 20 µM cytochalasin B. Cells were then loaded onto Whatman GF/C filters (Fisher Scientific, Whitby, ON, Canada) premoistened in PBS and mounted on a Millipore vacuum filtration unit. Filters were immediately washed with 30 ml of ice-cold PBS. Filters were transferred into 20-ml scintillation vials and soaked with 0.5 ml of water for 30 min, and then 10 ml of scintillation fluid (ICN, Costa Mesa, CA) was added to each vial. Vials were counted in a Beckman 6000 scintillation counter. Blanks (time 0 uptake) were prepared by addition of stopping solution before addition of incubation medium containing 3-OMG and radiolabel followed by an immediate wash. Nonspecific binding of radiolabel to filters was obtained by filtration and washing of incubation medium alone. Uptakes for each time point were measured in triplicate.
Calculation of rate constants. The time course of intracellular radioactivity accumulation in disintegrations per min (DPMt) was fit to the following single-, double-, or triple-exponential equations, respectively
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Derivation of three-compartment model. On the basis of curve-fitting results and the model of Heard et al. (10) for glucose transport in human erythrocytes, the model structure of Fig. 1 was adopted for interpretation of time course and kinetic data.
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According to this model, extracellular (E) glucose (GlE) is first translocated across the cell membrane and into an occlusion compartment by a symmetric carrier exhibiting a simple Vmax and Km. Exchange between the occlusion (O) and intracellular (I) compartments is a first-order process governed by rate constants kOI and kIO.
The differential equations describing sugar fluxes (U; nmol/min) in the system are
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For radiolabeled sugar (RA; dpm/min)
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Calculation of model parameters. Values for rate constants kOI and kIO and volumes evol, ivol, and ovol were derived from compartmental analysis of the time course of 3-OMG net uptake assuming a closed, sequential three-pool system and first-order interchanges between pools (20). Attempts to estimate Km and Vmax from the time course curve failed to yield unique parameter solutions because of the high correlation between Km and Vmax and the narrow range of extracellular 3-OMG concentration ([3-OMG]) manifest during 20 min of incubation. Hence, least-squares estimates of Km and Vmax were obtained by iteratively simulating, with Eqs. 49 and kOI, kIO, evol, ivol, and ovol from the compartmental analysis, the 15-s incubations of mammary epithelial cells with varied initial extracellular and intracellular concentrations of 3-OMG reported previously (24). In these simulations, initial [GlE] was set to the concentration in the incubation medium. In cases in which cells were preloaded with different concentrations of intracellular sugar, initial [GlO] and [GlI] were both set to the same loaded concentrations. Rates of 3-OMG entry for comparison with observed were obtained from the 15-s values of RAO and RAI as (RAO + RAI)/(iSAE x 0.25 min), where iSAE is the initial specific activity in incubation medium before initiation of uptake.
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RESULTS |
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Compartmental analysis of time course of 3-OMG net uptake. First-order compartmental analysis of the time course curves yielded the parameter values summarized in Table 1. The rate constant for backflux of sugar from the occluded space, kOE, was an order of magnitude greater than kOI for entry into the inner compartment, as observed in erythrocytes (8). All estimated volumes were expressed per milligram of protein for purposes of simulation. Although evol merely represents the volume of incubation medium used, it was estimated that the occluded space accounted for 32.6 ± 5.7% of the total 3-OMG distribution volume, whereas the true intracellular space represented 67.4 ± 5.7%. These results are in close agreement with the 29% and 71% obtained in human erythrocytes for the relative distribution volume of these two compartments, respectively (8). In human erythrocytes, it was suggested that the small compartment is formed within the cytoplasmic domain of the glucose transporter protein, immediately under the plasma membrane, and that the multistep transport is sequential rather than parallel (8). Because zero-time binding was subtracted from the net uptake and cells were extensively washed in this study, the two compartments are not located on the exofacial aspect of the cell membrane, i.e., they may also be located on the cytoplasmic side.
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Parameters for three-compartment model. Sugar exchange rates between the two posttranslocation compartments were estimated from compartmental analysis to be kIO = 0.29 ± 0.03 min1 and kOI = 0.16 ± 0.03 min1 (Table 1). When these parameters were used in the model shown in Fig. 1, least-squares estimates of Km and Vmax from fits to an additional set of kinetic data were 7.88 mM and 29.65 nmol·min1·mg protein1, respectively. Parameter estimates for the three-compartment model were associated with low standard errors among cows (Table 2). The fully parameterized three-compartment model mimicked the time course of 3-OMG uptake (Fig. 2A) with an r2 of 0.98 and the kinetics of 15-s intracellular accumulation with an r2 of 0.99 (Figs. 3 and 4).
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DISCUSSION |
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Three-compartment model. On the basis of similarities between erythrocytes and mammary cells in sugar distribution spaces and the ratio of exponents in the biexponential time course equation, we modeled glucose transport in bovine mammary epithelial cells according to the above cage hypothesis. In this model, two serial steps are considered: saturable, symmetric, carrier-mediated sugar translocation followed by slow, diffusive exchange with intracellular glucose.
The model was used to explore the origin of the asymmetry, trans-stimulation, and cooperativity exhibited in kinetics of sugar accumulation in mammary epithelial cells (Fig. 3). Intracellular radiolabeled sugar accumulation in 15-s incubations was simulated under zero-trans conditions where initial intracellular glucose concentration was 0 and initial extracellular glucose concentration ranged from 0 to 20 mM. The apparent Vmax of the simulated zero-trans curve (20.13 nmol·min1·mg protein1; Table 3) was 68% of the Vmax of the translocation step (29.65 nmol·min1·mg protein1; Table 2) because of rapid backflux of label from the occlusion compartment within 15 s (Fig. 5). The apparent Km of zero-trans-accumulation kinetics (10.18 mM) was elevated 30% above the Km of the translocation process (7.88 mM) because the proportion of incoming radiolabel that exits cells within 15 s declined as initial glucose concentration increased. Under high-trans conditions, where initial intracellular glucose concentration was maintained at 20 mM for all initial extracellular concentrations, the backflux of label was lessened by dilution in the occlusion compartment so that the apparent Vmax of sugar entry simulated over 15 s (24.50 nmol·min1·mg protein1; Table 3) was elevated to 83% of the translocation Vmax. The apparent Vmax of equilibrium exchange kinetics (30.80 nmol·min1·mg protein1), where initial extracellular and intracellular glucose concentrations are equal, was essentially equivalent to the Vmax of translocation because the apparent Vmax is an extrapolation to infinitely high intracellular glucose where backflux of radiolabel approaches zero. As glucose concentrations increased in the equilibrium-exchange experiment, dilution in the occlusion compartment also increased so that backflux as a proportion of influx declined, causing the apparent Km to be highest of all the three saturation curves.
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Parameters of the fixed-site carrier equation
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Kinetics of glucose transport by mammary epithelial cells are better represented by a three-compartment model with four parameters than a carrier model with five parameters. To study the effect of experimental treatments on mammary glucose transport, the parameters describing first-order exchange between the two intracellular compartments can be obtained from the time course of sugar accumulation. The Km and Vmax of transmembrane translocation are equivalent to those of short-term infinite-trans accumulation. Unfortunately, osmotic effects of high intracellular sugar concentrations make the infinite-trans experiment a practical impossibility. Instead, parameters of the translocation step of mammary glucose transport can be obtained from a combined set of time course and concentration dependence data.
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FOOTNOTES |
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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.
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