Hexose transporter mRNAs for GLUT4, GLUT5, and GLUT12 predominate in human muscle

In the past few years, 8 additional members of the facilitative hexose transporter family have been identified, giving a total of 14 members of the SLC2A family of membrane-bound hexose transporters. To determine which of the new hexose transporters were expressed in muscle, mRNA concentrations of 11 glucose transporters (GLUTs) were quantified and compared. RNA from muscle from 10 normal volunteers was subjected to RT-PCR. Primers were designed that amplified 78- to 241-base fragments, and cDNA standards were cloned for GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, and GAPDH. Seven of these eleven hexose transporters were detectable in normal human muscle. The rank order was GLUT4, GLUT5, GLUT12, GLUT8, GLUT11, GLUT3, and GLUT1, with corresponding concentrations of 404 +/- 49, 131 +/- 14, 33 +/- 4, 5.5 +/- 0.5, 4.1 +/- 0.4, 1.2 +/- .0.1, and 0.9 +/- 0.2 copies/ng RNA (means +/- SE), respectively, for the 10 subjects. Concentrations of mRNA for GLUT4, GLUT5, and GLUT12 were much higher than those for the remainder of the GLUTs and together accounted for 98% of the total GLUT isoform mRNA. Immunoblots of muscle homogenates verified that the respective proteins for GLUT4, GLUT5, and GLUT12 were present in normal human muscle. Immunofluorescent studies demonstrated that GLUT4 and GLUT12 were predominantly expressed in type I oxidative fibers; however, GLUT5 was expressed predominantly in type II (white) fibers.     

Structural analysis of the GLUT1 facilitative glucose transporter (review).

The structure of the human erythrocyte facilitative glucose transporter (GLUT1) has been intensively investigated using a wide array of chemical and biophysical approaches. Despite the lack of a crystal structure for any of the facilitative monosaccharide transport proteins, detailed information regarding primary and secondary structure, membrane topology, transport kinetics, and functionally important residues has allowed the construction of a sophisticated working model for GLUT1 tertiary structure. The existing data support the formation of a central aqueous channel formed by the juxtaposition of several amphipathic transmembrane-spanning alpha-helices. The results of extensive mutational analysis of GLUT1 have elucidated many of the structural determinants of the glucose permeation pathway. Continued application of currently available technologies will allow further refinement of this working model. In addition to providing insights into the molecular basis of both normal and disordered glucose homeostasis, this detailed understanding of structure/function relationships within GLUT1 can provide a basis for understanding transport carried out by other members of the major facilitator superfamily.     

Expression and regulation of the human GLUT4/muscle-fat facilitative glucose transporter gene in transgenic mice.

To study the molecular basis of tissue-specific expression of the GLUT4/muscle-fat facilitative glucose transporter gene, we generated lines of transgenic mice carrying 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene (hGLUT4[2.4]-CAT). This reporter gene construct was specifically expressed in tissues that normally express GLUT4 mRNA, which include both brown and white adipose tissues as well as cardiac, skeletal, and smooth muscle. In contrast, CAT reporter activity was not detected in brain or liver, two tissues that do not express the GLUT4 gene. In addition, the relative levels of CAT mRNA driven by the human GLUT4 promoter in various tissues of these transgenic animals mirrored those of the endogenous mouse GLUT4 mRNA. Since previous studies have observed alterations in GLUT4 mRNA levels induced by fasting and refeeding (Sivitz, W. I., DeSautel, S. L., Kayano, T., Bell, G. I., and Pessin, J. E. (1989) Nature 340, 72-74), the regulated expression the hGLUT4[2.4]-CAT transgene was also assessed in these animals. Fasting was observed to decrease CAT activity in white adipose tissue which was super-induced upon refeeding. These alterations in CAT expression occurred in parallel to the changes in endogenous mouse GLUT4 mRNA levels. Although CAT expression in skeletal muscle and brown adipose tissue was unaffected, the endogenous mouse GLUT4 mRNA was also refractory to the effects of fasting/refeeding in these tissues. These data demonstrate that 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene contain all the necessary sequence elements to confer tissue-specific expression and at least some of the sequence elements controlling the hormonal/metabolic regulation of this gene.     

Structural analysis of the GLUT1 facilitative glucose transporter

The structure of the human erythrocyte facilitative glucose transporter (GLUT1) has been intensively investigated using a wide array of chemical and biophysical approaches. Despite the lack of a crystal structure for any of the facilitative monosaccharide transport proteins, detailed information regarding primary and secondary structure, membrane topology, transport kinetics, and functionally important residues has allowed the construction of a sophisticated working model for GLUT1 tertiary structure. The existing data support the formation of a central aqueous channel formed by the juxtaposition of several amphipathic transmembrane-spanning α-helices. The results of extensive mutational analysis of GLUT1 have elucidated many of the structural determinants of the glucose permeation pathway. Continued application of currently available technologies will allow further refinement of this working model. In addition to providing insights into the molecular basis of both normal and disordered glucose homeostasis, this detailed understanding of structure/function relationships within GLUT1 can provide a basis for understanding transport carried out by othermembers of the major facilitator super family.     

Replacement of intracellular C-terminal domain of GLUT1 glucose transporter with that of GLUT2 increases Vmax and Km of transport activity.

The intracellular C-terminal domain is diverse in size and amino acid sequence among facilitative glucose transporter isoforms. The characteristics of glucose transport are also divergent, and GLUT2 has far higher Km and Vmax values compared with GLUT1. To investigate the role of the intracellular C-terminal domain in glucose transport, we expressed in Chinese hamster ovary cells the mutated GLUT1 protein whose intracellular C-terminal domain was replaced with that of GLUT2 by means of engineering the chimeric cDNA. Cytochalasin B, for which GLUT2 protein has much lower affinity, bound to this chimeric protein in a fashion similar to GLUT1. In contrast, greater transport activity was observed in this chimeric glucose transporter compared with the wild-type GLUT1 at 10 mM 2-deoxy-D-glucose concentration. The kinetic studies on 2-deoxy-D-glucose uptake revealed a 3.8-fold increase in Km and a 4.3-fold increase in Vmax in this chimeric glucose transporter compared with the wild-type GLUT1. Thus, replacement of the intracellular C-terminal domain confers the GLUT2-like property on the glucose transporter. These results strongly suggest that the diversity of intracellular C-terminal domain contributes to the diversity of glucose transport characteristics among isoforms.     

Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer

Malignant cells are known to have accelerated metabolism, high glucose requirements, and increased glucose uptake. Transport of glucose across the plasma membrane of mammalian cells is the first rate-limiting step for glucose metabolism and is mediated by facilitative glucose transporter (GLUT) proteins. Increased glucose transport in malignant cells has been associated with increased and deregulated expression of glucose transporter proteins, with overexpression of GLUT1 and/or GLUT3 a characteristic feature. Oncogenic transformation of cultured mammalian cells causes a rapid increase of glucose transport and GLUT1 expression via interaction with GLUT1 promoter enhancer elements. In human studies, high levels of GLUT1 expression in tumors have been associated with poor survival. Studies indicate that glucose transport in breast cancer is not fully explained by GLUT1 or GLUT3 expression, suggesting involvement of another glucose transporter. Recently, a novel glucose transporter protein, GLUT12, has been found in breast and prostate cancers. In human breast and prostate tumors and cultured cells, GLUT12 is located intracellularly and at the cell surface. Trafficking of GLUT12 to the plasma membrane could therefore contribute to glucose uptake. Several factors have been implicated in the regulation of glucose transporter expression in breast cancer. Hypoxia can increase GLUT1 levels and glucose uptake. Estradiol and epidermal growth factor, both of which can play a role in breast cancer cell growth, increase glucose consumption. Estradiol and epidermal growth factor also increase GLUT12 protein levels in cultured breast cancer cells. Targeting GLUT12 could provide novel methods for detection and treatment of breast and prostate cancer.     

Detection of the GLUT3 facilitative glucose transporter in rat L6 muscle cells: regulation by cellular differentiation, insulin and insulin-like growth factor-I.

The GLUT3 facilitative glucose transporter protein was found to be expressed in rat L6 muscle cells. It was detected at both the myoblast and myotube stage. GLUT3 protein content per mg of total membrane protein increased significantly during L6 cell differentiation. Subcellular fractionation demonstrated that the GLUT3 protein was predominantly localized in plasma membrane-enriched fractions of either myoblasts or myotubes. Short-term exposure of L6 myotubes to IGF-I or insulin caused a redistribution of GLUT3 protein from an intracellular membrane fraction to the plasma membrane, without affecting total membrane GLUT3 protein content. Long-term exposure of L6 myotubes to IGF-I produced an increase of GLUT3 protein in total membranes and all subcellular membrane fractions, especially the plasma membrane. We propose that the GLUT3 glucose transporter may play an important role in glucose metabolism in developing muscle.     

Phylogenesis and Biological Characterization of a New Glucose Transporter in the Chicken (Gallus gallus), GLUT12

In mammals, insulin-sensitive GLUTs, including GLUT4, are recruited to the plasma membrane of adipose and muscle tissues in response to insulin. The GLUT4 gene is absent from the chicken genome, and no functional insulin-sensitive GLUTs have been characterized in chicken tissues to date. A nucleotide sequence is predicted to encode a chicken GLUT12 ortholog and, interestingly, GLUT12 has been described to act as an insulin-sensitive GLUT in mammals. It encodes a 596 amino acid protein exhibiting 71% identity with human GLUT12. First, we present the results of a phylogenetic study showing the stability of this gene during evolution of vertebrates. Second, tissue distribution of chicken SLC2A12 mRNA was characterized by RT-PCR. It was predominantly expressed in skeletal muscle and heart. Protein distribution was analysed by Western blotting using an anti-human GLUT12 antibody directed against a highly conserved region (87% of identity). An immuno-reactive band of the expected size (75kDa) was detected in the same tissues. Third a physiological characterization was performed: SLC2A12 mRNA levels were significantly lowered in fed chickens subjected to insulin immuno-neutralization. Finally, recruitment of immuno-reactive GLUT12 to the muscle plasma membrane was increased following 1h of intraperitoneal insulin administration (compared to a control fasted state). Thus insulin administration elicited membrane GLUT12 recruitment. In conclusion, these results suggest that the facilitative glucose transporter protein GLUT12 could act in chicken muscle as an insulin-sensitive transporter that is qualitatively similar to GLUT4 in mammals.     

Functional expression and characterisation of a gut facilitative glucose transporter, NlHT1, from the phloem-feeding insect Nilaparvata lugens (rice brown planthopper)

Phloem-sap feeding Hemipteran insects have access to a sucrose-rich diet but are dependent on sucrose hydrolysis and hexose transport for carbon nutrition. A cDNA library from Nilaparvata lugens (rice brown planthopper) was screened for clones encoding potential transmembrane transporters. A selected cDNA, NlHT1, encodes a 53kDa polypeptide with sequence similarity to facilitative hexose transporters of eukaryotes and prokaryotes, including GLUT1, the human erythrocyte hexose transporter. NlHT1 was expressed as a recombinant protein in the methylotropic yeast Pichia pastoris, and was identified in a membrane fraction isolated from transformed yeast cells. Transport experiments using membrane vesicles containing NlHT1 showed that the protein is a saturable, sodium independent transporter, with a relatively low affinity for glucose (K(m) 3.0mM), which can be inhibited by cytochalasin B. Competition experiments with fructose demonstrate NlHT1 is glucose specific. In situ localisation studies revealed that NlHT1 mRNA is expressed in N. lugens gut tissue, mainly in midgut regions, and that expression is absent in hindgut and Malpighian tubules. NlHT1 is therefore likely to play an important role in glucose transport from the gut, and in carbon nutrition in vivo. This is the first report of a facilitative glucose transporter from a phloem-feeding insect pest.