Determination of in vivo kinetics of the starvation-induced Hxt5 glucose transporter of Saccharomyces cerevisiae

We have investigated the role and the kinetic properties of the Hxt5 glucose transporter of Saccharomyces cerevisiae. The HXT5 gene was not expressed during growth of the yeast cells in rich medium with glucose or raffinose. However, it became strongly induced during nitrogen or carbon starvation. We have constructed yeast strains constitutively expressing only Hxt5, Hxt1 (low affinity) or Hxt7 (high affinity), but no other glucose transporters. Aerobic fed-batch cultures at quasi steady-state conditions, and aerobic and anaerobic chemostat cultures at steady-state conditions of these strains were used for estimation of the kinetic properties of the individual transporters under in vivo conditions, by investigating the dynamic responses of the strains to changes in extracellular glucose concentration. The K(m) value and the growth properties of the HXT5 single expression strain indicate that Hxt5 is a transporter with intermediate affinity.     

Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters

The yeast glucose transporters Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 and Gal2, individually expressed in an hxt1-7 null mutant strain, demonstrate the phenomenon of countertransport. Thus, these transporters, which are the most important glucose transporters in Saccharomyces cerevisiae, are facilitated diffusion transporters. Apparent K(m)-values from high to low affinity, determined from countertransport and initial-uptake experiments, respectively, are: Hxt6 0.9+/-0.2 and 1.4+/-0.1 mM, Hxt7 1.3+/-0.3 and 1.9+/-0.1 mM, Gal2 1.5 and 1.6+/-0.1 mM, Hxt2 2.9+/-0.3 and 4.6+/-0.3 mM, Hxt4 6.2+/-0.5 and 6.2+/-0.3 mM, Hxt3 28.6+/-6.8 and 34.2+/-3.2 mM, and Hxt1 107+/-49 and 129+/-9 mM. From both independent methods, countertransport and initial uptake, the same range of apparent K(m)-values was obtained for each transporter. In contrast to that in human erythrocytes, the facilitated diffusion transport mechanism of glucose in yeast was symmetric. Besides facilitated diffusion there existed in all single glucose transport mutants, except for the HXT1 strain, significant first-order behaviour.     

The glucose signaling network in yeast

Background

Most cells possess a sophisticated mechanism for sensing glucose and responding to it appropriately. Glucose sensing and signaling in the budding yeast Saccharomyces cerevisiae represent an important paradigm for understanding how extracellular signals lead to changes in the gene expression program in eukaryotes.

Scope of review

This review focuses on the yeast glucose sensing and signaling pathways that operate in a highly regulated and cooperative manner to bring about glucose-induction of HXT gene expression.

Major conclusions

The yeast cells possess a family of glucose transporters (HXTs), with different kinetic properties. They employ three major glucose signaling pathways—Rgt2/Snf3, AMPK, and cAMP-PKA—to express only those transporters best suited for the amounts of glucose available. We discuss the current understanding of how these pathways are integrated into a regulatory network to ensure efficient uptake and utilization of glucose.

General significance

Elucidating the role of multiple glucose signals and pathways involved in glucose uptake and metabolism in yeast may reveal the molecular basis of glucose homeostasis in humans, especially under pathological conditions, such as hyperglycemia in diabetics and the elevated rate of glycolysis observed in many solid tumors.     

Overexpression of glucose transporter modulates insulin biosynthesis in insulin producing cell line

Glucose transporter (GT) has been suggested to be involved in the insulin biosynthesis. However, the functional relationship between GT and insulin biosynthesis is not well understood. In this report, we have generated rat pancreatic B cell lines (RINr) that stably overexpress a cDNA encoding the brain type GT. These cell lines showed 3- to 4-fold increase in insulin mRNA and protein. These results suggest that GT might have some relationship to the insulin biosynthesis in the pancreatic B cells.     

Oxime-based inhibitors of glucose transporter 1 displaying antiproliferative effects in cancer cells

An analysis of the main pharmacophoric features present in the still limited number of inhibitors of glucose transporter GLUT1 led to the identification of new oxime-based inhibitors, which proved to be able to efficiently hinder glucose uptake and cell growth in H1299 lung cancer cells. The most important interactions of a representative inhibitor were indicated by a novel computational model of GLUT1, which was purposely developed to explain these results and to provide useful indications for the design and the development of new and more efficient GLUT1 inhibitors.     

Localization of erythrocyte/HepG2-type glucose transporter (GLUT1) in human placental villi

Summary The syncytiotrophoblast covering the surface of the placental villi contains the machinery for the transfer of specific substances between maternal and fetal blood, and also serves as a barrier. Existence of a facilitated-diffusion transporter for glucose in the syncytiotrophoblast has been suggested. Using antibodies to erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of the facilitated-diffusion glucose transporters, we detected a 50 kD protein in human placenta at term. By use of immunohistochemistry, GLUT1 was found to be abundant in both the syncytiotrophoblast and cytotrophoblast. Endothelial cells of the fetal capillaries also showed positive staining for GLUT1. Electron-microscopic examination revealed that GLUT1 was concentrated at both the microvillous apical plasma membrane and the infolded basal plasma membrane of the syncytiotrophoblast. Plasma membrane of the cytotrophoblast was also positive for GLUT1. GLUT1 at the apical plasma membrane of the syncytiotrophoblast may function for the entry of glucose into its cytoplasm, while GLUT1 at the basal plasma membrane may be essential for the exit of glucose from the cytoplasm into the stroma of the placental villi. Thus, GLUT1 at the plasma membranes of syncytiotrophoblast and endothelial cells may play an important role in the transport of glucose across the placental barrier.     

Glucose transport in nongrowing yeast

The inducible, nonenergy-requiring glucose transport system of the yeast Kluyveromyces lactis is inactivated upon starving cells of glucose by (1) transferring logarithmic phase glucose-grown cells to synthetic medium containing a nonglycolytic carbon source, and (2) upon transition of logarithmic phase glucose-grown cells to stationary phase. The steady-state accumulation of nonmetabolizeable 6-deoxyglucose and the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of transport of 6-deoxyglucose is the same in stationary phase cells and in logarithmic phase cells. The rate of transport is lower in the nongrowing cells. Restoration of activity requires energy and protein synthesis as well as inducer.     

Molecular analysis of the mechanism of potassium uptake through the TRK1 transporter of Saccharomyces cerevisiae

The TRK-HKT family of K⁺ transporters mediates K⁺ and Na⁺ uptake in fungi and plants. In this study, we have investigated the molecular mechanism involved in the movement of alkali cations through the TRK1 transporter of Saccharomyces cerevisiae. The model that best explains the activity of ScTRK1 is a cotransport of two K⁺ or Rb⁺, both of which bind the two binding sites of ScTRK1 with very high affinities in K⁺-starved cells. Na⁺ can be transported in the same way but it exhibits a much lower affinity for the second binding site. Therefore, only at critical concentration ratios between K⁺ and Na⁺, or Rb⁺ and Na⁺, the transporter takes up Na⁺ together with K⁺ or Rb⁺. Mutation analyses suggest that the two binding sites are located in the P fragment of the first MPM motif of the transporter, and that Gln⁹⁰ is involved in these binding sites. ScTRK1 can be in two states, medium or high affinity, and we have found that Leu⁹⁴⁹ is involved in the oscillation of the transporter between these two states. ScTRK1 mediates active K⁺ uptake. This is not Na⁺-coupled and direct coupling of ScTRK1 to a source of chemical energy seems more probable than K⁺-H⁺ cotransport.     

Asymmetry of glucose transport in the yeast, Kluyveromyces lactis

Uptake and efflux of 6-deoxy-d-[³H]glucose and of 2-deoxy-d-[¹⁴C]glucose by the yeast Kluyveromyces lactis was studied. The tritiated, nonphosphorylatable hexose analogue leaves the cell in the absence and presence of intracellular 2-deoxy-d-glucose 6-phosphate. In energy-rich cells containing pools of hexose 6-phosphate, 2-deoxy-d-glucose is trapped in the cells, for it neither effluxes into glucose-free medium nor exchanges with external, free sugar. In starved, poisoned cells containing negligible amounts of 2-deoxy-d-glucose 6-phosphate, 2-deoxy-d-glucose does leave the cells upon transfer to glucose-free medium. An involvement of analogue structure and availability of metabolites of energy-rich cells in hexose retention is suggested. An internal pool of 6-deoxy-d-glucose does not affect the rate of uptake of 6-deoxy-d-[³H]glucose, nor does internal 2-deoxy-d-[¹⁴C]glucose 6-phosphate influence that rate. Hence, transport of glucose by this yeast is probably not regulated by internal pools of glucose 6-phosphate.     

Immunolocalization of glucose transporter GLUT1 in the rat placental barrier: possible role of GLUT1 and the gap junction in the transport of glucose across the placental barrier

GLUT1 is an isoform of facilitated-diffusion glucose transporters and has been shown to be abundant in cells of blood-tissue barriers. Using antibodies against GLUT1, we investigated the immunohistochemical localization of GLUT1 in the rat placenta. Rat placenta is of the hemotrichorial type. Three cell layers (from the maternal blood side inward) cytotrophoblast and syncytiotrophoblasts I and II, lie between the maternal and fetal bloodstreams. GLUT1 was abundant along the invaginating plasma membrane facing the cytotrophoblast and the syncytiotrophoblast I. Also, the infolded basal plasma membrane of the syncytiotrophoblast II was rich in GLUT1. Apposing plasma membranes of syncytiotrophoblasts I and II, however, had only a small amount of GLUT1. Numerous gap junctions were seen between syncytiotrophoblasts I and II. Taking into account the localization of GLUT1 and the gap junctions, we suggest a possible major transport route of glucose across the placental barrier, as follows: glucose in the maternal blood passes freely through pores of the cytotrophoblast. Glucose is then transported into the cytoplasm of the syncytiotrophoblast I via GLUT1. Glucose enters the syncytiotrophoblast II throught the gap junctions. Finally glucose leaves the syncytiotrophoblast II via GLUT1 and enters the fetal blood through pores of the endothelial cells.     

Expression of glucose transporter 1 is associated with loss of heterozygosity of chromosome 1p in oligodendroglial tumors WHO grade II

Objective Oligodendroglial tumors with a loss of heterozygosity of 1p (LOH1p) appear to have a better prognosis than oligodendrogliomas without LOH. Previously, we reported glucose uptake in low-grade oligodendroglial tumors to be related to LOH 1p status. Here, we performed an immunohistochemical study of the common glucose transporters (GLUT) in relation to LOH1p. Material and methods We examined 17 oligodendrogliomas (O II, 11 with LOH1p), 16 oligoastrocytomas (OA II, 5 with LOH1p) and 7 astrocytomas (A II, none with LOH1p). Confocal microscopy was performed for p53, GLUT-1, −3 and −12. Immunoreaction was rated semiquantitatively by percentage of positive cells and staining intensity on immunohistological stainings. Results Confocal microscopy depicted immunoreaction for GLUT-1, −3 and −12 in the cytoplasm of the tumor cells. Oligodendrogliomas revealed a lower immunoreactivity for GLUT-1 than oligoastrocytomas and astrocytomas (P = 0.0263). No differences in immunoreactivity were found for GLUT-3 and GLUT-12. GLUT-1 expression in tumors with LOH 1p was significantly lower than in tumors with wild type 1p status (P = 0.0017). GLUT-3 and GLUT-12 immunoreactivity was not correlated with LOH 1p. Conclusion Expression of GLUT-1 is significantly reduced in low-grade oligodendroglial tumors harboring LOH 1p. Further studies should address the functional role of GLUT-1 in regard to chemosensitivity of oligodendrogliomas.     

The amino‐terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid

Hxt2 is a glucose repressed, high affinity glucose transporter of the yeast Saccharomyces cerevisiae and is subjected to high glucose induced degradation. Hxt11 is a sugar transporter that is stably expressed at the membrane irrespective the sugar concentration. To transfer this property to Hxt2, the N‐terminal tail of Hxt2 was replaced by the corresponding region of Hxt11 yielding a chimeric Hxt11/2 transporter. This resulted in the stable expression of Hxt2 at the membrane and improved the growth on 8% d ‐glucose and 4% d ‐xylose. Mutation of N361 of Hxt11/2 into threonine reversed the specificity for d ‐xylose over d ‐glucose with high d ‐xylose transport rates. This mutant supported efficient sugar fermentation of both d ‐glucose and d ‐xylose at industrially relevant sugar concentrations even in the presence of the inhibitor acetic acid which is normally present in lignocellulosic hydrolysates. Biotechnol. Bioeng. 2017;114: 1937–1945. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Glucose deprivation and hypoxia increase the expression of the GLUT1 glucose transporter via a specific mRNA cis-acting regulatory element

The expression of the glucose transporter type-1 (GLUT1) gene is up-regulated in hypoxia and glucose deprivation. A 10 nucleotide (nt) cis-acting regulatory element (CAE), which is located within nt 2181-2190 of the GLUT1 3'-untranslated region (CAE2181-2190), increases the expression of a GLUT1-luciferase reporter gene and decreases its mRNA decay. The present study investigated the role of the GLUT1 CAE2181-2190 in glucose deprivation and hypoxia using stable transfectants. Glucose and O2 deprivation produced a marked increase in the expression of the GLUT1 reporter gene carrying the CAE2181-2190, and this effect was additive. Glucose deprivation and/or hypoxia induced no significant changes in the expression of the reporter gene wherein the GLUT1 CAE2181-2190 was site-directed deleted. Data presented here suggest that the GLUT1 CAE2181-2190 participates in the increase of GLUT1 gene expression in glucose deprivation and hypoxia.     

N-glycosylation is critical for the stability and intracellular trafficking of glucose transporter GLUT4

The facilitative glucose transporter GLUT4 plays a key role in regulating whole body glucose homeostasis. GLUT4 dramatically changes its distribution upon insulin stimulation, and insulin-resistant diabetes is often linked with compromised translocation of GLUT4 under insulin stimulation. To elucidate the functional significance of the sole N-glycan chain on GLUT4, wild-type GLUT4 and a GLUT4 glycosylation mutant conjugated with enhanced GFP were stably expressed in HeLa cells. The N-glycan contributed to the overall stability of newly synthesized GLUT4. Moreover, cell surface expression of wild-type GLUT4 in HeLa cells was elevated upon insulin treatment, whereas the glycosylation mutant lost the ability to respond to insulin. Subcellular distribution of the mutant was distinct from that of wild-type GLUT4, implying that the subcellular localization required for insulin-mediated translocation was impaired in the mutant protein. Interestingly, kifunensine-treated cells also lost sensitivity to insulin, suggesting the functional importance of the N-glycan structure for GLUT4 trafficking. The K(m) or turnover rates of wild-type and mutant GLUT4, however, were similar, suggesting that the N-glycan had little effect on transporter activity. These findings underscore the critical roles of the N-glycan chain in quality control as well as intracellular trafficking of GLUT4.     

Functional characterization of a LAHC sucrose transporter isolated from grape berries in yeast

Large amounts of sugar are imported into grape berries from source leaves during ripening, and sucrose transporters play a key role during this process. In this study, a putative grape sucrose transporter gene VvSUC27, primarily expressed in sink tissue, was transformed into a yeast strain to characterize its function as a sucrose transporter. Sucrose was taken up by yeast transformed with VvSUC27 at an optimum pH of 4.0–5.0 and a K _m of 8.0–10.5 mM, indicating VvSUC27 is a LAHC (low-affinity/high-capacity) sucrose transporter. The ability of sucrose uptake in transformed yeast was activated by monosaccharides and inhibited by maltose and DEPC. Ya Li Zhang and Qing Yong Meng contributed equally to the paper.     

Subcellular distribution of glucose transporter (GLUT-1) during development of the blood-brain barrier in rats

Electron microscopy was used to quantify the subcellular distribution of the GLUT-1 isoform of the glucose transporter in developing microvessels of the brain of embryonic rats from E (embryonic stage) 13 to E19 and in adult rats. Gold-conjugated secondary antibodies were used to localize, on ultrathin sections of brain, a rabbit polyclonal antiserum (anti-GLUT-1) raised against a synthetic peptide encoding 13 amino acids of the C-terminus of the human glucose transporter. Staining was weak at E13 but increased in density during development into adulthood. The increase represented an increase in the absolute amount of transporter per vessel profile, with a concomitant decrease in vessel size with the narrowing of the wall. At early stages, the percentages of total particles per profile of lumenal membrane, ablumenal membrane, and cytoplasm were approximately equivalent. The ratio of lumenal to ablumenal particle density then shifted from below 1 at E13 to above 2 at E19 and to 4 in the adult. In contrast, vessels of the choroid plexus were devoid of labeling, but the choroid plexus epithelium stained as early as E15. In the brain, no astrocytes, neurons, or pericytes were stained at any stage examined. Developmental upregulation of the GLUT-1 glucose transporter therefore seems to occur at the blood-brain barrier, and the modulation of the subcellular distribution of the transporter can be correlated with other observed changes in the microvessels as they develop the blood-brain barrier phenotype. Received: 18 November 1995 / Accepted: 12 January 1996     

Up-regulation of Na-coupled glucose transporter SGLT1 by caveolin-1

The Na⁺-coupled glucose transporter SGLT1 (SLC5A1) accomplishes concentrative cellular glucose uptake even at low extracellular glucose concentrations. The carrier is expressed in renal proximal tubules, small intestine and a variety of nonpolarized cells including several tumor cells. The present study explored whether SGLT1 activity is regulated by caveolin-1, which is known to regulate the insertion of several ion channels and carriers in the cell membrane. To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of caveolin-1 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes, but not in oocytes injected with water or caveolin-1 alone, the addition of glucose to the extracellular bath generated an inward current (I_g), which was increased following coexpression of caveolin-1. Kinetic analysis revealed that caveolin-1 increased maximal I_g without significantly modifying the glucose concentration required to trigger half maximal I_g (K_M). According to chemiluminescence and confocal microscopy, caveolin-1 increased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of I_g, which was similar in the absence and presence of caveolin-1. In conclusion, caveolin-1 up-regulates SGLT1 activity by increasing carrier protein abundance in the cell membrane, an effect presumably due to stimulation of carrier protein insertion into the cell membrane.     

Glucose transporter 10 and arterial tortuosity syndrome: The vitamin C connection

Arterial Tortuosity Syndrome (ATS) is a heritable disease characterized by twisting and lengthening of the major arteries, hypermobility of the joints, and laxity of skin. ATS is caused by mutations in SLC2A10, encoding Glucose Transporter 10 (GLUT10). The current model of ATS holds that loss of GLUT10 at the nuclear periphery induces a glucose-dependent increase in Transforming Growth Factor-β (TGFβ) that stimulates vessel wall cell proliferation. Instead, we propose that GLUT10 transports ascorbate, a cofactor for collagen and elastin hydroxylases, into the secretory pathway. In ATS, loss of GLUT10 results in defective collagen and/or elastin. TGFβ activation represents a secondary response to a defective extracellular matrix.