Characterization of bovine glucose transporter 1 kinetics and substrate specificities in Xenopus oocytes

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.

Structural comparison of GLUT1 to GLUT3 reveal transport regulation mechanism in Sugar Porter family

“…Xenopus oocytes expressing GLUT1 or GLUT3 show a similar increase in 2-DG accumulation, while water-injected oocytes show that 2-DG uptake from endogenous transporters in the oocyte membrane is negligible ( Fig 1B). Michaelis-Menten analysis of substrate saturation kinetics with GLUT1 resulted in a Km for 2-DG uptake of 9.5 mM ( Fig 1C), comparable with previous reports in the literature (Burant and Bell, 1992;Bentley et al, 2012). The uptake was inhibited by the GLUT inhibitor cytochalasin B. GLUT3 displays a similar cytochalasin B-dependent behavior with a Km of 2.6 mM ( Fig 1D).…”

FGT-1 Is a Mammalian GLUT2-Like Facilitative Glucose Transporter in Caenorhabditis elegans Whose Malfunction Induces Fat Accumulation in Intestinal Cells

“…Kinetic assays revealed that FGT-1 has a K m of 2.8 mM for 2-DG, a non-metabolized glucose analog. This K m value is much lower than the K m reported for hGLUT1 (11.7 mM) or hGLUT4 (4.6 mM) [17,18], which is not surprising because C. elegans lives in a low glucose soil environment and may require cells to have transporters with a high affinity for glucose for its acquisition.…”

“…Oocytes were harvested from Xenopus laevis . The synthesized cRNA (2 µg/µL) or sterile water was injected into isolated mature oocytes (stage V and VI) following previously established procedures [17]. …”

“…2-DG uptake and the kinetic and inhibition analyses of FGT-1 and R09B5.11 were carried out in Xenopus oocytes as described previously [17]. cRNA- or water-injected oocytes were incubated in Barth’s media [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO 3 , 0.82 mM MgSO 4 , 0.41 mM CaCl 2 , 0.33 mM Ca(NO 3 ) 2 , 5 mM HEPES, pH 7.6] with 10 µg/mL penicillin and 10 IU/mL streptomycin (Gibco) for three days and then subjected to the uptake assay using 10 mM 2-DG containing 1 µCi 3 H-2-DG.…”

“…Protein levels were analyzed using Western blot procedures as described previously [17]. Ten worms were collected in sample buffer [62.5 mM Tris (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, and 5% (v/v) 2-β-mercaptoethanol].…”