27 research outputs found
The facilitated glucose transporter GLUT12: What do we know and what would we like to know?
Human GLUT12 was isolated from the breast cancer cell line MCF-7 by its homology with GLUT4. Glucose has been described as its main substrate, but it also can transport other sugars. In humans, GLUT12 protein is expressed mainly in insulin sensitive tissues. Functional analysis has showed that GLUT12 transports sugars down its concentration gradient, but it can also work as a proton-coupled symporter. Studies from our laboratory, performed in Xenopus laevis oocytes expressing GLUT12, show that glucose uptake increases in the presence of Na+ and induces inward current. These findings suggest a transport mechanism never described for other GLUTs, which would indicate a distinct functional role for GLUT12. In relation with its physiological and pathophysiological function, GLUT12 has been mainly studied due to its role as a secondary insulin-sensitive glucose transporter and its possible implication in impaired insulin signalling pathologies. Its expression in some tumour tissues has been described and recently, it has been proposed as one of the key proteins in the glucose supply to malignant cells. Overall, even though a lot of information about GLUT12 has been released during the last years, its functional characteristics, physiological role or implication in the development of some diseases is still unclear. Therefore, this review of the literature can help to address further investigations needed to elucidate these issues that, in our view, are of great interest mainly due to the direct GLUT12 relation with cancer and probably with diabetes development
Electrophysiological characterization of the human Na(+)/nucleoside cotransporter 1 (hCNT1) and role of adenosine on hCNT1 function.
We previously reported that the human Na(+)/nucleoside transporter pyrimidine-preferring 1 (hCNT1) is electrogenic and transports gemcitabine and 5'-deoxy-5-fluorouridine, a precursor of the active drug 5-fluorouracil. Nevertheless, a complete electrophysiological characterization of the basic properties of hCNT1-mediated translocation has not been performed yet, and the exact role of adenosine in hCNT1 function has not been addressed either. In the present work we have used the two-electrode voltage clamp technique to investigate hCNT1 transport mechanism and study the kinetic properties of adenosine as an inhibitor of hCNT1. We show that hCNT1 exhibits presteady-state currents that disappear upon the addition of adenosine or uridine. Adenosine, a purine nucleoside described as a substrate of the pyrimidine-preferring transporters, is not a substrate of hCNT1 but a high affinity blocker able to inhibit uridine-induced inward currents, the Na(+)-leak currents, and the presteady-state currents, with a K(i) of 6.5 microM. The kinetic parameters for uridine, gemcitabine, and 5'-deoxy-5-fluorouridine were studied as a function of membrane potential; at -50 mV, K(0.5) was 37, 18, and 245 microM, respectively, and remained voltage-independent. I(max) for gemcitabine was voltage-independent and accounts for approximately 40% that for uridine at -50 mV. Maximal current for 5'-DFUR was voltage-dependent and was approximately 150% that for uridine at all membrane potentials. K(0.5)(Na(+)) for Na(+) was voltage-independent at hyperpolarized membrane potentials (1.2 mM at -50 mV), whereas I(max)(Na(+)) was voltage-dependent, increasing 2-fold from -50 to -150 mV. Direct measurements of (3)H-nucleoside or (22)Na fluxes with the charge-associated revealed a ratio of two positive inward charges per nucleoside and one Na(+) per positive inward charge, suggesting a stoichiometry of two Na(+)/nucleoside
Luminal leptin inhibits L-glutamine transport in rat small intestine: involvement of ASCT2 and B0AT1.
L-glutamine is the primary metabolic fuel for enterocytes. Glutamine from the diet is transported into the absorptive cells by two sodium-dependent neutral amino acid transporters present at the apical membrane: ASCT2/SLC1A5 and B(0)AT1/SLC6A19. We have demonstrated that leptin is secreted into the stomach lumen after a meal and modulates the transport of sugars after binding to its receptors located at the brush border of the enterocytes. The present study was designed to address the effect of luminal leptin on Na(+)-dependent glutamine (Gln) transport in rat intestine and identify the transporters involved. We found that 0.2 nM leptin inhibited uptake of Gln and phenylalanine (Phe) (substrate of B(0)AT1) using everted intestinal rings. In Ussing chambers, 10 mM Gln absorption followed as Na(+)-induced short-circuit current was inhibited by leptin in a dose-dependent manner (maximum inhibition at 10 nM; I(C50) = approximately 0.1 nM). Phe absorption was also decreased by leptin. Western blot analysis after 3-min incubation of the intestinal loops with 10 mM Gln, showed marked increase of ASCT2 and B(0)AT1 protein in the brush-border membrane that was reduced by rapid preincubation of the intestinal lumen with 1 nM leptin. Similarly, the increase in ASCT2 and B(0)AT1 gene expression induced by 60-min incubation of the intestine with 10 mM Gln was strongly reduced after a short preincubation period with leptin. Altogether these data demonstrate that, in rat, leptin controls the active Gln entry through reduction of both B(0)AT1 and ASCT2 proteins traffic to the apical plasma membrane and modulation of their gene expression
Characterization of the rat Na+/nucleoside cotransporter 2 and transport of nucleoside-derived drugs using electrophysiological methods.
The Na(+)-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. We used the two-electrode voltage-clamp technique to investigate rat CNT2 (rCNT2) transport mechanism and study the interaction of nucleoside-derived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides, and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates, apparent affinity (K(0.5)) was in the low micromolar range (12-34) and was voltage independent for hyperpolarizing membrane potentials, whereas maximal current (I(max)) was voltage dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. Lack of the 2'-hydroxyl group decreased affinity but increased I(max). Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and I(max). Fludarabine and formycin B were also transported with higher I(max) than uridine and moderate affinity (102 +/- 10 and 66 +/- 6 microM, respectively). Analysis of the pre-steady-state currents revealed a half-maximal activation voltage of about -39 mV and a valence of about -0.8. K(0.5) for Na(+) was 2.3 mM at -50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge associated showed a ratio of two positive inward charges per nucleoside, suggesting a stoichiometry of two Na(+) per nucleoside. This discrepancy in the number of Na(+) molecules that bind rCNT2 may indicate a low degree of cooperativity between the Na(+) binding sites
Leptin regulates sugar and amino acids transport in the human intestinal cell line Caco-2
Aim: Studies in rodents have shown that leptin controls sugars and glutamine entry in the enterocytes by regulating membrane transporters. Here, we have examined the effect of leptin on sugar and amino acids absorption in the human model of intestinal cells Caco-2 and investigated the transporters involved.
Methods: Substrate uptake experiments were performed in Caco-2 cells, grown on plates, in the presence and the absence of leptin and the expression of the different transporters in brush border membrane vesicles was analysed by Western blot.
Results: Leptin inhibited 0.1 mM α-methyl-D-glucoside uptake after 5 or 30 min treatment, and decreased SGLT1 protein abundance in the apical membrane. Uptake of 20 µM glutamine and 0.1 mM phenylalanine was also inhibited by leptin, indicating sensitivity to the hormone of the Na+-dependent neutral amino acid transporters ASCT2 and B0AT1. This inhibition was accompanied by a reduction of the transporters expression at the brush-border membrane. Leptin also inhibited 1 mM proline and β-alanine uptake in Na+ medium at pH 6, conditions for optimal activity of the H+-dependent neutral amino acid transporter PAT1. In this case, abundance of PAT1 in the brush-border membrane after leptin treatment was not modified. Interestingly, leptin inhibitory effect on β-alanine uptake was reversed by the PKA inhibitor H-89 suggesting involvement of PKA pathway in leptin´s regulation of PAT1 activity.
Conclusion: These data show in human intestinal cells that leptin can rapidly control the activity of physiologically relevant transporters for rich-energy molecules, i.e D-glucose (SGLT1) and amino acids (ASCT2, B0AT1 and PAT1)
The molecular basis of glucose galactose malabsorption in a large Swedish pedigree
Glucose-galactose malabsorption (GGM) is due to mutations in the gene coding for the intestinal sodium glucose cotransporter SGLT1 (SLC5A1). Here we identify the rare variant Gln457Arg (Q457R) in a large pedigree of patients in the Vasterbotten County in Northern Sweden with the clinical phenotype of GGM. The functional effect of the Q457R mutation was determined in protein expressed in Xenopus laevis oocytes using biophysical and biochemical methods. The mutant failed to transport the specific SGLT1 sugar analog alpha-methyl-D-glucopyranoside (alphaMDG). Q457R SGLT1 was synthesized in amounts comparable to the wild-type (WT) transporter. SGLT1 charge measurements and freeze-fracture electron microscopy demonstrated that the mutant protein was inserted into the plasma membrane. Electrophysiological experiments, both steady-state and presteady-state, demonstrated that the mutant bound sugar with an affinity lower than the WT transporter. Together with our previous studies on Q457C and Q457E mutants, we established that the positive charge on Q457R prevented the translocation of sugar from the outward-facing to inward-facing conformation. This is contrary to other GGM cases where missense mutations caused defects in trafficking SGLT1 to the plasma membrane. Thirteen GGM patients are now added to the pedigree traced back to the late 17th century. The frequency of the Q457R variant in Vasterbotten County genomes, 0.0067, is higher than in the general Swedish population, 0.0015..
Could GLUT12 be a potential therapeutic target in cancer treatment? A preliminary report
Background: Recent studies proposed GLUT12 to be a major glucose transporter involved in the
glycolytic metabolism of cancer cells.
Methods: GLUT12 expression was determined by immunohistochemistry in a selection of cancer
cell lines and a tumour spheroid model.
Results: GLUT12 expression was high in A549 and RH-36; low in HT29; and absent in NB-EB
cancer cell lines. GLUT12 expression was located in the necrotic centre of HT29 spheroids, which
is characterised by anaerobic metabolism.
Conclusion: The data supports the involvement of GLUT12 in the glycolytic metabolism of cancer
cells and therefore, its potential as a novel therapeutic target for cancer treatment
Further Characterization of the Electrogenicity and pH Sensitivity of the Human Organic Anion-Transporting Polypeptides OATP1B1 and OATP1B3
Organic anion-transporting polypeptides (OATPs) are involved in the liver uptake of many endogenous and xenobiotic compounds, such as bile acids and drugs, respectively. Using Xenopus laevis oocytes and Chinese hamster ovary (CHO) cells expressing rat Oatp1a1, human OATP1B1, or OATP1B3, the sensitivity of these transporters to extracellular/intracellular pH (pHo/pHi) and changes in plasma membrane potential (ΔΨ) was investigated. In X. laevis oocytes, nonspecific plasma membrane permeability increased only at pHo below 4.5. Above this value, both using oocytes and CHO cells, extracellular acidification affected differently the specific transport of taurocholic acid (TCA) and estradiol 17β-d-glucuronide (E217βG) by Oatp1a1 (stimulation), OATP1B1 (inhibition), and OATP1B3 (stimulation). Changes in substrate uptake in the presence of valinomycin (K+-ionophore), carbonyl cyanide 3-chlorophenylhydrazone and nigericin (protonophores), and amiloride (Na+/H+-inhibitor) and cation replacement in the medium were studied with fluorescent probes for measuring substrate uptake (cholylglycyl amidofluorescein) and changes in pHi (SNARF-4F) and ΔΨ [DilC1(5)]. The results suggest that activity of these three carriers is sodium/potassium-independent and affected differently by changes in pHo and ΔΨ: Oatp1a1 was confirmed to be an electroneutral anion exchanger, whereas the function of both OATP1B1 and OATP1B3 was markedly affected by the magnitude of ΔΨ. Moreover, electrophysiological measurements revealed the existence of a net anion influx associated to OATP1B1/OATP1B3-mediated transport of TCA, E217βG, and estrone-3-sulfate. Furthermore, a leakage of Na+ through OATP1B1 and OATP1B3, which is not coupled to substrate transport, was found. In conclusion, these results suggest that OATP1B1 and OATP1B3 are electrogenic transporters whose activity may be strongly affected under circumstances of displacement of local pH
Role of the human concentrative nucleoside transporter (hCNT1) in the cytotoxic action of 5[Prime]-deoxy-5-fluorouridine, an active intermediate metabolite of capecitabine, a novel oral anticancer drug.
We attempt to identify the plasma membrane transporter involved in the uptake of 5'-deoxy-5-fluorouridine (5'-DFUR), an intermediate metabolite of capecitabine. This novel oral fluoropyrimidine is used in cancer treatments and is a direct precursor of the cytostatic agent 5'-fluorouracil. We also examine the role of the transporter in 5'-DFUR cytotoxicity. The human concentrative nucleoside transporter (hCNT1) was cloned from human fetal liver and expressed in Xenopus laevis oocytes. The two-electrode voltage-clamp technique was used to demonstrate that 5'-DFUR, but not capecitabine or 5'-FU, is an hCNT1 substrate. Then, hCNT1 was heterologously expressed in the mammalian cell line Chinese hamster ovary-K1. Functional expression was demonstrated by monitoring transport of radiolabeled substrates and by using a monospecific polyclonal antibody generated against the transporter. hCNT1-expressing cells were more sensitive to 5'-DFUR than vector-transfected or wild-type cells. The sensitivity of the three cell types to other agents such as cisplatin or 5'-FU was identical. In conclusion, this study shows that 1) the pharmacological profile of a nucleoside transporter can be determined by an electrophysiological approach; 2) the hCNT1 transporter is involved in 5'-DFUR uptake; and 3) hCNT1 expression may increase cell sensitivity to 5'-DFUR treatment. This study also reports for the first time the generation of an antibody against hCNT1, which may be useful in the elucidation of the relationship between hCNT1 expression and tumor response to capecitabine treatmen
Interaction of nucleoside inhibitors of HIV-1 reverse transcriptase with the concentrative nucleoside transporter-1 (SLC28A1)
Human concentrative nucleoside transporter-1 (hCNT1) (SLC28A1) is a widely expressed, high-affinity, pyrimidine-preferring, nucleoside transporter implicated in the uptake of naturally occurring pyrimidine nucleosides as well as a variety of derivatives used in anticancer treatment. Its putative role in the uptake of other pyrimidine nucleoside analogues with antiviral properties has not been studied in detail to date. Here, using a hCNT1 stably transfected cell line and the two-electrode voltage-clamp technique, we have assessed the interaction of selected pyrimidine-based antiviral drugs, inhibitors of HIV-1 reverse transcriptase such as zidovudine (AZT), stavudine (d4T), lamivudine (3TC) and zalcitabine (ddC), with hCNT1. hCNT1 transports AZT and d4T with low affinity, whereas 3TC and ddC are not translocated, the latter being able to bind the transporter protein. Selectivity appears to rely mostly upon the presence of a hydroxyl group in the 3'-position of the ribose ring. Thus, hCNT1 cannot be considered a broad-selectivity pyrimidine nucleoside carrier; in fact, very slight changes in substrate structure provoke a dramatic shift in selectivity