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. Author manuscript; available in PMC: 2015 Feb 23.
Published in final edited form as: J Dairy Sci. 2012 Mar;95(3):1188–1197. doi: 10.3168/jds.2011-4430

Characterization of Bovine Glucose Transporter 1 Kinetics and Substrate Specificities in Xenopus Oocytes

P A Bentley *,1, Y Shao *, Y Misra *, A D Morielli †, F-Q Zhao *,2
PMCID: PMC4337999  NIHMSID: NIHMS664308  PMID: 22365203

Abstract

Glucose is an essential substrate for lactose synthesis and an important energy source in milk production. Glucose uptake in the mammary gland therefore plays a critical role in milk synthesis. Facilitative glucose transporters (GLUTs) mediate glucose uptake in the mammary gland. GLUT1 is the major facilitative glucose transporter expressed in bovine mammary gland and has been shown to localize to the basolateral membrane of mammary epithelial cells. GLUT1 is therefore thought to play a major role in glucose uptake during lactation. The objective of this study was to determine the transport kinetic properties and substrate specificity of bovine GLUT1 using the Xenopus oocyte model. Bovine GLUT1 was expressed in Xenopus oocytes by microinjection of in vitro transcribed cRNA and was found to be localized to the plasma membrane, which resulted in increased glucose uptake. This bGLUT1-mediated glucose uptake was dramatically inhibited by specific facilitative glucose transport inhibitors, cytochalasin B and phloretin. Kinetic analysis of bovine and human GLUT1 was conducted under zero-trans conditions using radio-labeled 2-deoxy-D-glucose and the principles of Michaelis-Menten kinetics. Bovine GLUT1 exhibited a KM of 9.8 ± 3.0 mM for 2-deoxy-D-glucose, similar to 11.7 ± 3.7 mM for human GLUT1. Transport by bovine GLUT1 was inhibited by mannose and galactose, but not fructose, indicating that bovine GLUT1 may also be able to transport mannose and galactose. Our data provides functional insight into the transport properties of bovine GLUT1 in taking up glucose across mammary epithelial cells for milk synthesis.

Keywords: bovine glucose transporter, glucose transport, kinetics, substrate specificity

INTRODUCTION

Glucose is the primary precursor of lactose which is a principle component of milk and functions as the primary osmotic regulator of milk volume (Peaker, 1977). The mammary gland uses approximately 3 kg of glucose for every 40 kg of milk produced. Mammary epithelial cells lack the ability to synthesize glucose, therefore glucose must be obtained from the blood. Glucose transport into the mammary epithelial cells is primarily mediated by the energy independent facilitative glucose transporters (GLUTs, gene name: SLC2A) (Zhao and Keating, 2007a, b). The GLUT family currently includes 13 members and its structure is characterized by 12-transmebrane domains and a single N-glycosyation site (Mueckler et al., 1985). GLUT1 has been shown to be the major GLUT isoform expressed in bovine mammary gland and its expression is developmentally regulated (Zhao et al., 1993, Zhao and Keating, 2007a). From 40 days before parturition to 7 days post calving, GLUT1 expression in bovine mammary gland increases more than 100-fold (Finucane et al., 2008, Zhao and Keating, 2007a). This dramatic increase in GLUT1 expression may be responsible for supplying the mammary gland with increased glucose needed during lactation.

Individual glucose transporters vary in tissue distribution, kinetic characteristics (Km and Vmax) and substrate specificity, implying that each transporter plays a distinct role in tissue glucose utilization and maintenance of body glucose homeostasis. For instance, while the human and rodent GLUT1 has a Km of 6.9-17 mM for D-glucose (Burant and Bell, 1992, Gould et al., 1991, Nishimura et al., 1993), hepatocytes glucose transporter (GLUT2) has a 2-10-fold higher Km and a higher Vmax to allow glucose efflux following gluconeogenesis (Burant and Bell, 1992, Colville et al., 1993, Gould et al., 1991). GLUT3 and GLUT4 with lower Km values mediate the uptake of glucose by the brain and the insulin regulated glucose uptake by skeletal muscle, respectively (Burant and Bell, 1992, Colville et al., 1993, Nishimura et al., 1993). GLUT5 has a high-affinity for fructose, with a poor ability to transport glucose (Corpe et al., 2002). In addition, the transport kinetics and substrate specificity of some transporters have been shown to differ between species. For instance, while pig SGLT3 shows a tightly coupled sodium and glucose transport activity, its human homolog, hSGLT3, does not have this characteristics (Wright and Turk, 2004).

Because most mammalian cells express a number of different glucose transporters with high basal glucose transport activity, it is difficult to determine the transport kinetics and the substrate specificity of individual glucose transporters in situ. Characterization of the transport kinetics of individual transporters has been widely carried out in Xenopus oocytes by over-expressing the transporter (Colville et al., 1993, Keller and Mueckler, 1990, Keller et al., 1989, Nishimura et al., 1993). Xenopus oocytes exhibit extremely low levels of basal glucose transport activity and thus offer an ideal system for expression and functional characterization of heterologous glucose transporters using radiolabeled nonmetabolizable glucose analogues such as 2-deoxy-D-glucose (2-DG) and 3-O-methylglucose (3-O-MG). The general transport properties of GLUT derived from expression studies in Xenopus oocytes do relate well to the known characteristics of glucose transport in various mammalian tissues.

Considering that the normal blood glucose levels in bovine (2.5-3.5 mM), being a ruminant, are generally lower than that of human (3.6-5.8 mM) and other non-ruminant animals, we hypothesize that the bovine GLUT1, the isoform ubiquitously distributed and responsible for basal glucose uptake of most tissues, has a lower Km than the human GLUT1. The objectives of this study were to characterize the transport kinetics of bovine GLUT1 and to compare the kinetics properties of GLUT1 between bovine and human. We further aim to determine bGLUT1 substrate specificities. Knowing the characteristics of bovine GLUT1, the predominant isoform in lactating bovine mammary gland, will provide insights into the physiological function of GLUT1 in bovine mammary cells and its roles in supporting milk synthesis.

MATERIALS AND METHODS

Plasmid Constructs

The human GLUT1 plasmid construct, pSP64T-hGLUT1, was kindly donated by Dr. Gwyn Gould (Gould et al., 1991). The bovine GLUT1 (bGLUT1) cDNA (Zhao et al., 2004) was cloned into the Xenopus expression vector SP64T as following: the SP64T-hGLUT1 plasmid was firstly digested with Bgl II (New England Biolabs, Ipswich, MA) overnight at 37°C to remove the hGLUT1 insert and the vector product was then purified by phenol extraction and ethanol precipitation, resolved on a 1% agarose gel, and isolated by Qiaquick Gel Extraction Kit (Qiagen, Valencia, CA). bGLUT1 cDNA was excised from the pCDNA3.1(-)-bGLUT1 plasmid by digestion with Xba I and Hind III (New England Biolabs) and purified by gel extraction. bGLUT1 was then digested with Bam HI and ligated into the pSP64T vector at the Bgl II site between the 89 bp 5′- and 141 bp 3′- Xenopus β-globin flanking sequences. The resulting SP64T-bGLUT1 construct was verified by restriction digestion and sequencing.

In vitro Transcription of Human and Bovine GLUT1-SP64T Constructs

The hGLUT1 and bGLUT1-SP64T constructs were in vitro transcribed and 5′ capped using a SP6 RNA polymerase and the mMessage mMachine Kit (Ambion, Austin, TX). The plasmid DNA was first linearized by restriction digestion with Sal I (New England BioLabs). Linearized plasmid DNA (1 μg) was transcribed as per manufacturer’s instruction , which included DNase treatment for 15 min. The resulting cRNA was recovered using MEGAclear Kit (Ambion) and was eluted in 50 μL of Elution Solution. cRNA concentration was measured by optical density (260/280 nm) using the Nanodrop ND-1000 (Thermo Fisher Scientific, Wilmington, DE) and diluted to 300 ng/uL. cRNA quality was assessed by the Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA) under reducing conditions.

Xenopus Oocyte Harvest

The use of Xenopus and the experimental procedures were approved by the University of Vermont Institutional Animal Care and Use Committee. Female Xenopus laevis were maintained at the Small Animal Care Facility of the University of Vermont on a 12-hour light/dark cycle and fed a standard diet of bovine liver pellets. Animals were anesthetized for oocyte removal by submersion in 1 g/L of tricane methanesulfonate (Sigma, St. Louise, MO) (pH 7.0) in aged and de-chlorinated water for 15 minutes or until motor reflex was undetectable. Incisions were made parasagittally on the abdomen using a #11-scalpel blade. Oocytes were removed, severed by scissors and placed in calcium-free OR2 media [82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.5].

Excised oocytes were washed three times in 40 mL of calcium-free OR2 then incubated in 1 mg/mL of Collagenase Type II (Sigma) in calcium-free OR2 for 1 hour at room temperature with agitation. Oocytes were again washed three times in 40 mL of calcium-free OR2 and then transferred to a Petri dish and maintained at 18 °C in Barth’s media [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.41 mM CaCl2, 0.33 mM Ca(NO3)2, 5 mM HEPES, pH 7.6] with 10 μg/mL penicillin and 10 IU/mL streptomycin (Invitrogen, Carlsbad, CA). Oocytes were incubated for 30 minutes prior to injection of cRNA.

Injection of cRNA into Oocytes

Micropipettes were made using 3.5” glass tubes (Drummond Scientific, Broomall, PA) on a vertical puller (KOPF Needle and Pipette Puller). Pipettes were trimmed and filled with sterile mineral oil using a modified syringe. Pipettes were fitted to the Drummond Nanoject Automatic micro-injector and were back-filled with cRNA (300 ng/μL, unless otherwise noted) or sterile water. Oocytes were transferred into a 60 mm Petri dish containing ND-96 media [96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, pH 7.5] for injection. Under a light microscope (10X) individual mature oocytes (stage V and VI) were injected in the vegetal pole or midline with ~ 46 nL of cRNA or water. Oocytes were returned to Barth’s media and incubated at 18 °C for 72 hours unless otherwise specified in figure legends. Media was changed every twelve hours during the incubation period and dead oocytes were removed when observed.

2-Deoxy-D-Glucose Uptake by Oocytes

Injected oocytes were washed once in antibiotic free Barth’s media and were randomly selected for treatment groups and transferred into a 24-well cell culture dish (Corning, Corning, NY). 2-deoxy-D-[1-3H]-glucose ([3H]-2-DG) (Amersham, Piscataway, NJ) was used as a radio-tracer for 2-DG (Sigma) uptake. Concentrations of non-radioactive 2-DG ranging from 0 mM to 100 mM were prepared in Barth’s media containing no antibiotic. [3H]-2-DG was added to each 2-DG solution at a concentration of 3 μCi/mL (for kinetic assays) or 1 μCi/mL (for all other assays) of total volume. For assays conducted at one concentration, 5 mM 2-DG was utilized. Hexose mixtures were stored at 4°C but were equilibrated to room temperature for 12 hours prior to use. 300 μL of each glucose analogue solutions (2-DG + 3H-2-DG) was applied to each group of oocytes (n = 5-25) for specified times as indicated in figure legends. Times ranged from 0 to 90 minutes, 0 minute time points were conducted by application and immediate removal of 2-DG. When time was not a variable within an assay, oocytes were exposed to 2-DG + [3H]-2-DG for 15 minutes. After application of 2-DG, the 24-well plate was gently agitated to ensure oocytes were suspended. Glucose uptake was halted by aspiration of the 2-DG solution and immediate application of ~1 mL of ice-cold phosphate buffered saline (PBS) (Invitrogen) containing 0.1 mM phloretin (Sigma), an inhibitor of facilitative glucose transport (Krupka, 1985). The oocytes were washed three times within one minute with PBS containing phloretin before suspension in 2 mL of the same solution, individually transferred to 7 mL borosilicate glass scintillation vials (Fisher Scientific, Pittsburg, PA) containing 0.5 mL of 1% (wt/vol) sodium dodecyl sulfate (SDS) and incubated for at least 1 hour. 5 mL of Econo Safe Liquid Scintillation Fluid (Atlantic Nuclear, Rockland, MA) was added to the scintillation vial, which was then shaken until homogenous. Sample radioactivity was quantified using the Wallac 1400 DSA scintillation counter. Each sample was counted for 180 seconds. Where indicated, the GLUT1-injected oocyte 2-DG uptake was corrected for the 2-DG uptake in water-injected oocytes exposed to the same 2-DG concentration and time as the GLUT1-injected oocytes.

Inhibition of 2-DG Uptake by Cytochalasin B and Phloretin

Oocytes injected with bGLUT1 cRNA and water were exposed to 5 mM 2-DG + [3H]-2-DG in the presence of various concentrations of cytochalasin B (CCB) (Sigma) and phloretin (Sigma), two potent inhibitors of facilitative glucose transport (Krupka, 1985, Sogin and Hinkle, 1980). 2-DG + [3H]-2-DG with various concentrations of cytochalasin B and phloretin were applied to groups of 10 oocytes for 15 minutes. Oocytes were then washed in ice-cold PBS, transferred into scintillation vials containing SDS and counted as described above.

Substrate Specificity: Hexose Sugar Inhibition Assays

Inhibition of 2-DG uptake was measured in the presence of 30 mM of the following hexose sugars: L-glucose, D-glucose, D-mannose, D-fructose, 3-OMG, and D-galactose (Sigma). 2-DG was diluted to a final concentration of 5 mM in Barth’s media containing 30 mM individual inhibitor sugar. [3H]-2-DG was added at a concentration of 1 μCi/mL of the final volume. 30 mM L-glucose was employed as a negative control for inhibition of 2-DG uptake (Gould et al., 1991). 300 μL of 2-DG with [3H]-2-DG and inhibitor hexose were applied to groups (n =20) of bGLUT1 cRNA- or water- injected oocytes. 2-DG uptake from water-injected oocytes was subtracted from the cRNA injected oocytes.

Immunohistochemistry

Oocytes were fixed for immunohistochemistry in 4% (wt/vol) paraformaldehyde for 3 hours on ice. Oocytes were washed three times in PBS and snap frozen in Optimal Cutting Temperature (OCT) compound (Sukura Finetek, Torrance, CA). Oocyte blocks were sectioned and thaw-mounted on the surface of gelatin-coated slides.

Tissue sections were equilibrated to room temperature prior to incubation in PBS containing 10% (vol/vol) goat serum for 1 hour at room temperature and then washed two times for five minutes with PBS. Sections were then exposed to 10 μg/mL of rabbit polyclonal anti-GLUT1 antibody (Millipore, Billerica, MA) for 1 hour at room temperature and washed two times for five minutes with PBS. Sections were incubated with Alexa Fluor® 555-conjugated goat anti-rabbit IgG at a concentration of 2.5 μg/mL for 1 hour in the dark at room temperature. Sections were washed two times with PBS and once with ultrapure water before mounting under a cover slip with Aqua Poly/Mount (PolySciences, Warrington, PA). Tissue sections were examined using the Zeiss LSM 510 META Confocal Laser Scanning Imaging System at the Microscopy Imaging Center, University of Vermont and visualized using the Zeiss LSM 5 image browser.

Western Blot Analysis

Total protein was isolated from cRNA- and water- injected oocytes. Oocytes were washed in PBS and suspended in 10 μL per oocyte of homogenization buffer [0.25 mM sucrose, 0.010 mM HEPES, 1 mM EGTA, 2 mM MgCl2] containing protease inhibitors (100 mM PMSF, 1 mg/mL pepstatin A, 1 mg/mL leupeptin). Oocytes were disrupted by 10 strokes of a 200 μL pipette tip. The homogenate was stored at −20°C before use. Prior to use, samples were thawed on ice and centrifuged for 30 seconds to pellet yolk proteins. Bradford assay (Bio-Rad, Hercules, CA) was used to determine the protein concentration of the supernatant.

Oocyte cell homogenates were denatured at 100 °C for four minutes in the presence of 10 μL 6X sample buffer [62.5 mM Tris (pH 6.8), 2% (wt/vol) SDS, 10% (vol/vol) glycerol, 5% (vol/vol) 2-β-mercaptoethanol]. Proteins were electrophoresed on a 12% SDS-polyacrylamide gel and then electrophoretically transferred to Hybond-P PVDF membrane (Amersham) on ice. Membranes were then blocked in Tris-buffered saline (TBS) [20 mM Tris (pH 7.4), 137 mM NaCl] containing 5% (wt/vol) non-fat powdered milk (Bio-Rad) and 0.01% (vol/vol) Tween-20 (Fisher). Membranes were washed two times in TBS containing 0.01% tween for five minutes and then incubated for 1 hour at room temperature with 1 μg/mL rabbit anti-GLUT1 polyclonal antibody (Millipore) diluted in TBS-tween containing 5% (wt/vol) non-fat milk. Membranes were washed two times in TBS-tween and were then exposed to a horseradish peroxidaseconjugated anti-rabbit IgG secondary antibody (Amersham) at room temperature for 1 hour. Membranes were washed three times in TBS-tween and once in TBS. The immune complex was detected using the West Pico Chemiluminescent Kit (Pierce, Rockford, IL) and detected on X-ray film (Kodak). Bands were quantified by densitometry (Molecular Imager, Versa Doc 4100 MP).

Statistical Analysis

Data points in glucose uptake assays outside 1.5 times the inter-quartile range (IQR) were designated as outliers and were disregarded for both water and cRNA injected oocytes. 2-DG uptake in GLUT1 cRNA injected oocytes was corrected by subtraction of the mean 2-DG uptake for water-injected oocytes at the corresponding concentration and incubation time; any resulting negative uptake values were disregarded. Statistical significance was determined by Tukey-Kramer HSD test, Welch’s ANOVA test and Dunnett’s one way ANOVA as indicated in individual figure legends and the analyses were carried out using JMP 8.0 software (SAS, Cary, NC). Points indicate sample mean and error bars represent the standard error of the mean (SEM). Plots and curve-fitting analysis for Michaelis-Menten equation were carried out using GraphPad Prism 5.02 (GraphPad Software, La Jolla, CA).

RESULTS

Expression and Localization of Exogenous GLUT1 in Xenopus Oocytes

To examine the transport kinetics of bGLUT1 in Xenopus oocytes, bGLUT1 cDNA was sub-cloned into the Xenopus specific expression vector, pSP64T (Gould et al., 1991). The open reading frame of bGLUT1 cDNA was inserted between the 5′ and 3′ untranslated flanking sequences of the Xenopus β-globin gene to form pSP64T-bGLUT1 and was in vitro transcribed. The hGLUT1 or bGLUT1 cRNAs (15 ng/injection) or water were injected into oocytes, uninjected oocytes were also used to determine a base-line estimate of endogenous GLUT1 expression in Xenopus oocytes. The oocytes were harvested after 72 hours of incubation and expression of GLUT1 protein was detected by western blot analysis of whole oocyte lysates (Figure 1A), and quantified by densitometry (Figure 1B). Two distinct bands were detected in both the hGLUT1 and bGLUT1 injected oocytes at a molecular weight between 42-50 kDa, consistent with endogenous GLUT1 detected in the cells of a mouse mammary cell line HC11. Low levels of endogenous GLUT1 protein expression were detected in both the water-injected and uninjected oocytes while injection of bGLUT1 or hGLUT1 cRNA injection increased the GLUT1 protein levels by 5 to 10-fold relative to the controls. The presence of two bands may be due to glycolsylation of GLUT1 protein, a post-translational modification which has been shown to be vital for transport activity by increasing the affinity of GLUT1 for glucose (Ahmed and Berridge, 1999, Asano et al., 1991).

Figure 1.

Figure 1

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Expression of GLUT1 protein in Xenopus oocytes. Western blot analysis (A) was performed on homogenate of oocytes injected with either human GLUT1 (hGLUT1) or bovine GLUT1 (bGLUT1) cRNA. Water injected and uninjected oocytes were used for detection of endogenous GLUT1 expression. 75 μg of total protein were loaded in each lane. Relative GLUT1 band intensities were determined by densitometry (B).

Subcellular localization of hGLUT1, bGLUT1, and endogenous GLUT1 was assessed by immunofluorescence on cRNA or water-injected oocytes (Figure 2). bGLUT1 was primarily detected on the plasma membrane of the bGLUT1 cRNA-injected oocytes (Figure 2A). hGLUT1 was localized to both the plasma membrane and the intracellular pool (Figure 2B). Water injected oocytes had no detectable fluorescence staining (Data not shown). The specificity of the secondary antibody was tested by exposure of bGLUT1 cRNA injected oocytes to the secondary antibody alone or with normal rabbit serum (data not shown). These negative controls showed no detectable signal.

Figure 2.

Figure 2

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Immunofluorescence staining of exogenous GLUT1 in Xenopus oocytes injected with either bovine GLUT1 cRNA (A) or human GLUT1 cRNA (B). Scale bar = 50 μm.

Functional Characterization of Exogenous GLUT1 in Xenopus laevis Oocytes

The functional transport activity of exogenous bGLUT1 in Xenopus oocytes was evaluated by exposing bGLUT cRNA-injected oocytes to 5 mM 2-DG containing [3H]-2-DG for 15 minutes. The water-injected and uninjected oocytes were used to determine the endogenous levels of glucose transport. 2-DG uptake was significantly higher in the hGLUT1 and bGLUT1 cRNA injected oocytes than in the water-injected and uninjected controls (n ≥ 88, P < 0.0001) (Figure 3A). Oocytes injected with hGLUT1 and bGLUT1 cRNA had mean 2-DG uptake of 580.0 and 568.7 pmols/oocyte/15 min, respectively while the water-injected oocytes had mean 2-DG uptake of 44.6 nmols/oocytes/15 min. There was no significant difference in 2-DG uptake between water injected, uninjected oocytes and uninjected oocytes treated with cytochalasin B.

Figure 3.

Figure 3

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Transport activity of exogenous GLUT1 in Xenopus oocytes. A: Xenopus oocytes were injected with human GLUT1 (hGLUT1) or bovine GLUT1 (bGLUT1) cRNA (15 ng), water or were not injected (Uninj.). 100μM cytochalasin B (CCB) was added to the uptake reaction of a group of uninjected oocytes (Uninj. + CCB). Oocytes were exposed to 5 mM 2-DG containing 1 μCi/mL [3H]-2-DG for 15 minutes. Means of 2-DG uptake in the cRNA-injected oocytes versus water-injected or uninjected controls were compared using Tukey-Kraner HSD test, error bars represent SEM, p < 0.001. B: Oocytes were injected with 0.0015-45 ng of bovine GLUT1 cRNA and subjected to the transport analysis as in A. Data represent the uptake in the cRNA-injected oocytes after subtracting the uptake in water-injected oocytes. Statistical significance was determined by Welch ANOVA, p < 0.001 (***), comparing groups as indicated in the Figure.

To determine the quantity of cRNA needed to achieve maximal expression and transport activity of bGLUT1, we conducted 2-DG uptake assays in oocytes that were injected with various quantities of cRNA. No statistical difference in 2-DG uptake was observed in oocytes injected with 1.5 to 45 ng of cRNA (n = 10, P = 0.96), in addition, there was no significant difference in 2-DG uptake between the 0.015 and 0.0015 ng cRNA-injected oocytes (n ≥ 6, P = 0.16) (Figure 3B). There was, however, significant difference between the two cRNA groups of 1.5 to 45 ng and 0.015 to 0.0015 ng (P < 0.001). The concentration of 15 ng/injection of cRNA was selected for use in further uptake assays to ensure maximal expression of bGLUT1 in the oocytes.

Facilitative glucose transporters are known to be inhibited by several pharmacological agents, including CCB and phloretin. To test whether, in our system, CCB and phoretin inhibited bGLUT1-mediated 2-DG transport, Xenopus oocytes injected with bGLUT1 cRNA were exposed to increasing concentrations of these two inhibitors during the 2-DG transport assay. The inhibitory affect of CCB was tested by increasing concentrations from 0 μM to 100 μM (Figure 4A). Concentrations of CCB more than 25 μM significantly reduced bGLUT1-mediated 2-DG uptake in oocytes (n ≥ 6, P < 0.05). 2-DG uptake was highly reduced at 100 μM CCB, from 1165 to 47 pmol/oocyte/15 minutes (P < 0.01). Non-linear one phase decay analysis estimated an inhibitory plateau at 207.2 pmol/oocyte/15 minutes of 2-DG. The inhibitory affect of phloretin was tested by increasing concentrations from 0 μM to 500 μM (Figure 4B). Phloretin, at concentrations ≥ 50 μM, significantly decreased bGLUT1-mediated 2-DG uptake (n ≥ 5, P < 0.001). Maximal inhibition of uptake was observed at concentrations ≥ 200 μM. Nonlinear one phase decay analysis estimated an inhibitory plateau at 718 pmols/oocytes/15minutes.

Figure 4.

Figure 4

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Inhibition of bovine GLUT1-mediated 2-deoxy-D-glucose uptake by cytochalsin B and phloretin in Xenopus oocytes. Oocytes were injected with bovine GLUT1 cRNA or water. Oocyte 2-DG uptake was measured after exposure of oocytes to 5 mM 2-DG and 1 μCi/mL [3H]-2-DG for 15 minutes and various concentrations of cytochalasin B (A) or phloretin (B). 2-DG uptake from water-injected oocytes was subtracted from cRNA injected oocytes. One phase non-linear decay model was used to identify the inhibition trend. Statistical analysis was conducted using Dunnett’s ANOVA, with 0 μM cytochalasin B or phloretin as the control group. Statistical significance was observed at p < 0.05 (*) and p < 0.01(**), p < 0.001 (***).

The quaternary structure of GLUTs is known to be regulated by intracellular ATP concentration (Heard et al., 2000). We, therefore, sought to determine if the lack of energy substrate in Barth’s media affected 2-DG uptake by GLUT1. Oocytes injected with cRNA or water were cultured for 72 hours in the presence or absence of 2.5 mM sodium pyruvate in Barth’s media (Figure 5). The presence of sodium pyruvate had no effect on glucose uptake in oocytes injected with hGLUT1 (n ≥ 8, P = 0.65) or bGLUT1 (n ≥ 8, P = 0.33) cRNA.

Figure 5.

Figure 5

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Effect of sodium pyruvate as an energy source on 2-deoxy-D-glucose uptake in Xenopus oocytes. Oocytes were injected with bovine or human GLUT1 cRNA or water. All oocytes were incubated for 72 hours in the presence (black bars) or absence (dashed bars) of 2.5 mM sodium pyruvate in Barth’s media. Oocytes were exposed to 5 mM 2-DG and 1 μCi/mL [3H]-2-DG for 15 minutes. 2-DG uptake in water-injected oocytes was subtracted from cRNA injected oocytes. Statistical analysis was conducted using the Tukey-Kramer HSD test; error bars represent SEM.

Kinetic Analysis of Bovine and Human GLUT1 in Xenopus Oocytes

Determination of transport KM and VMAX values using Michaelis-Menten enzyme kinetics were carried out using 15 minute incubations, which were found to be within the linear range of uptake for bovine GLUT1 (data not shown), consistent with previous studies of human GLUT1 (Gould et al., 1991). Kinetic analysis of bovine and human GLUT1 was conducted using various concentrations of 2-DG ranging from 0 to 50 mM. The analysis was repeated on oocytes from multiple frogs (9 assays for bGLUT1 and 5 for human GLUT1). A representative assay for each bGLUT1 and hGLUT1 are shown in Figure 6. Michaelis-Menten curve fit analysis of 2-DG uptake in GLUT1-injected oocytes after correcting for the uptake into the water-injected oocytes revealed KM values ranging from 5.1 mM to 14.0 mM for bGLUT1 and from 7.9 mM to 17.8 mM for hGLUT1. The mean KM for bovine and human GLUT1 are 9.8 ± 3.0 mM and 11.7 ± 3.6 mM, respectively. There is no statistical difference between the Kmof bovine and human GLUT1 (P = 0.24). VMAX values ranged from 2582 to 4914 pmol/oocyte/15 minutes for bGLUT1 and 2773 to 4914 pmol/oocyte/15 minutes for hGLUT1. The mean VMAX is 3567 ± 721 pmol/oocyte/15 minutes for bGLUT1 and 3947 ± 790 pmol/oocyte/15 minutes for hGLUT1.

Figure 6.

Figure 6

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Kinetic analysis of 2-deoxy-D-glucose uptake by bovine (A) and human GLUT1 (B) in Xenopus oocytes. Oocytes were injected with bovine or human GLUT1 cRNA or water and exposed to various 2-DG concentrations containing 3 μCi/mL [3H]-2-DG for 15 minutes. Points represent 2-DG uptake in GLUT1-injected oocytes after correction for the uptake into water-injected oocytes. One representative assay is shown for each bGLUT1 and hGLUT1. Michaelis-Menten non-linear analysis was conducted in GraphPad Prism 5. Error bars represent SEM.

Determination of Bovine GLUT1 Substrate Specificities

The substrate specificity of bGLUT1 was determined by the competitive inhibition of hexose sugars (30 mM) on 2-DG uptake in oocytes expressing bGLUT1 (Figure 7). L-Glucose was used as a negative control for the inhibition as glucose transporters have stereoselectivity for the D-enantiomer (Keller et al., 1989). The uptake in the presence of L-glucose was consistent with 2-DG uptake in non-inhibitory conditions. The positive controls, 2-DG and D-glucose, both significantly inhibited the uptake to 480 ± 20 pmol/oocyte/15 minutes (n ≥ 18, P < 0.0001). 3-OMG, a non-metabolizable glucose analogue, significantly inhibited 2-DG uptake (n = 17, P < 0.01). 2-DG uptake was also inhibited by D-mannose and D-galactose (n ≥ 14, P < 0.01), but not by D-fructose (n = 19, P = 0.18).

Figure 7.

Figure 7

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Inhibition of bovine GLUT1-mediated uptake of 2-deoxy-D-glucose by hexose substrates in Xenopus oocytes. Oocytes were injected with bovine GLUT1 cRNA or water and were exposed to 30 mM of each inhibitor sugar and 5 mM 2-DG and 1 μCi/mL [3H]-2-DG for 15 minutes. 2-DG uptake from water-injected oocytes was subtracted from cRNA-injected oocytes. Statistical analysis was conducted using Dunnett’s ANOVA, with L-glucose as the control group. Statistical significance was observed at p < 0.01(**), p < 0.001 (***), error bars represent SEM.

DISCUSSION

Characterization of Bovine GLUT1 Expression and 2-DG Transport in Xenopus Oocytes

The objective of our study was to establish the Xenopus oocyte as a model for the expression of bovine GLUT1 and determine the functional kinetic properties of the transporter in this model. The Xenopus oocyte is an effective model for the study of glucose transporters as oocytes have low levels of endogenous glucose transport activity and allow for the expression of exogenous GLUTs (Keller and Mueckler, 1990). In the Xenopus oocytes, glucose transport is not limited by phosphorylation of the sugar by hexokinase, therefore, the model can accurately simulate the steep concentration gradient across the basolateral membrane within the mammary gland (Gould et al., 1991). Our results demonstrated that bGLUT1 can be successfully expressed in Xenopus oocytes and that the protein localizes to the plasma membrane. We have shown that bGLUT1 is functionally active in oocytes and that bGLUT1-mediated transport can be inhibited by cytochalasin B and phloretin, two widely used inhibitors of facilitative glucose transporters.

In our study, water-injected and uninjected oocytes displayed only very low levels of endogenous GLUT1 expression and very low, but detectable levels of 2-DG uptake, a finding consistent with previous reports (Keller and Mueckler, 1990). This uptake may be mediated by low endogenous GLUT1 present in frog embryos (Suzawa et al., 2007). We observed, however, no inhibitory effect of cytochalasin B on 2-DG uptake in uninjected oocytes. Thus, it appears that the 2-DG uptake in water-injected oocytes may be attributable to radioactivity retained on the oocytes after washing, and is not solely a function of endogenous GLUT-mediated transport.

The 2-DG uptake in oocytes injected with bGLUT1 cRNA was not affected by adding sodium pyruvate, an energy substrate for the oocytes (Liu, 2006). This indicates that, in our culture conditions, a lack of energy in the growth media is not affecting the function or expression of exogenous bGLUT1 protein in oocytes. Another potential rate-limiting step to bGLUT1 production in oocytes was the amount of bGLUT1 cRNA injected. Our results indicated that 1.5 ng of cRNA is enough to saturate the translational or trafficking machinery of the oocytes, as we observed no further increase in 2-DG uptake in oocytes injected with higher concentrations of bGLUT1 cRNA.

Kinetic Analysis of Bovine GLUT1 in Xenopus Oocytes

The kinetic parameters of bovine GLUT1 were determined in Xenopus oocytes under zero-trans conditions. The linear range of uptake for bovine GLUT1 was found to be within 45 minutes of incubation with the substrate, which is consistent with previous studies of hGLUT1 (Gould et al., 1991). In our kinetic assay with 15 minutes of exposure to substrate, bGLUT1 exhibited Michaelis-Menten type transport kinetics and was saturated at 2-DG concentrations greater than 25 mM. bGLUT1 has a mean KM of 9.8 ± 3.0 mM, which is not statistically different from the Km of human GLUT1 (11.7 ± 3.6 mM). These Km values are between the previously reported Km for rat GLUT1 (6.9 mM) (Burant and Bell, 1992) and human GLUT1 (17 mM) (Gould et al., 1991). These differences may be due to different experimental conditions. Nevertheless, our study does not support our hypothesis that bGLUT1 transports glucose with a higher affinity than the human GLUT1. Sequence alignment of bGLUT1 and hGLUT1 shows only 13 amino acid differences (Zhao and Keating, 2007b). These amino acids may not play a critical role in glucose transport activity of the GLUT1.

The KM of a plasma membrane transporter higher than the concentration of substrate in the blood would generally indicate that the transporter is functioning within its linear range of transport. This allows the transporter to decrease or increase its rate of uptake based on fluctuations in its substrate concentration in the blood. If the transport property of bGLUT1 in bovine cells in vivo is similar to that in Xenopus oocytes, our data indicate that in vivo, bGLUT1 on the plasma membrane is ~ 30 % saturated at the blood glucose level of 3-3.5 mM (Faulkner and Peaker, 1987) and glucose uptake by the cells would increase when blood glucose concentration rises without increase in GLUT1 protein abundance. Supporting this notion is that the Km value of glucose uptake has been shown to be 8.29 mM in the mammary epithelial cells in which GLUT1 is the predominant glucose transporter (Xiao and Cant, 2003). Increasing blood glucose levels by intravenous or intestinal glucose infusion enhances glucose uptake by the mammary gland and milk production in dairy cows (Huhtanen et al., 2002, Hurtaud et al., 2000, Rigout et al., 2002).

Substrate Specificity of Bovine GLUT1

Substrate specificity of bovine GLUT1 was assessed by inhibition of 2-DG uptake by different hexose sugars, including galactose, mannose, and fructose. The inhibitory effects of hexose sugars were compared to that of L-glucose, which has been previously shown to not be transported by GLUT1 (Gould et al., 1991). We observed that D-mannose and D-galactose inhibit 2-DG transport, while D-fructose does not. The inhibitory effect of D-glucose was similar to that of 2-DG, indicating that in Xenopus oocytes bGLUT1 transports the glucose analogue 2-DG as efficiently as D-glucose. The blood levels of hexose sugars other than glucose are generally low, thus, glucose is the major substrate for GLUT1 on the plasma membrane. In mammary epithelial cells, however, both glucose and galactose are transported into the Golgi apparatus for lactose synthesis. GLUT1 has been shown to be present in Golgi membrane of the mammary epithelial cells (Nemeth et al., 2000) and therefore bGLUT1 may be involved in the transport of both glucose and galactose into the Golgi.

CONCLUSIONS

GLUT1 plays a major role in the uptake of glucose necessary for milk production in the mammary gland. Here, we have characterized bovine GLUT1 transport kinetics and substrate specificities in Xenopus oocytes. Our study provides insight into the function of GLUT1 in glucose uptake in the mammary gland. However, it is important to point out that GLUT1 may function differently in Xenopus oocyte than in vivo. It may be necessary to study the transport kinetics of GLUT1 in mammary epithelial cells in future studies to obtain more accurate transport properties of GLUT1 in situ. The bovine mammary gland also expresses other isoforms of glucose transporters, including GLUT8 and 12 (Miller et al., 2005, Zhao et al., 2004). The transport kinetics and properties of these transporters should also be studied in order to fully understand the glucose uptake process in the mammary gland.

ACKNOWLEDGEMENTS

We acknowledge Dr. Gwyn Gould for providing us the human GLUT1 construct and pSP64T vector as well as extensive technical advice. Thanks to Dr. Mike Mueckler, Dr. Thomas McFadden, Dr. Chris Cheeseman and Katarzyna Witkowska for their technical advice. We also want to thank Dr. Sheryl White for making the bGLUT1 construct, Marilyn Wadsworth for assistance with confocal microscopy, and Alan Howard for statistical support. This project was supported by National Research Initiative Competitive Grant no. 2007-35206-18037 from the USDA National Institute of Food and Agriculture (to FQZ).

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