Nuestros investigadores

María del Pilar Lostao Crespo

Vicerrectorado de Relaciones Internacionales
Rectorado. Universidad de Navarra
Ciencias de la Alimentación y Fisiología
Facultad de Farmacia y Nutrición. Universidad de Navarra
Líneas de investigación
Estudio de transportadores de membrana mediante técnicas electrofisiológicas, Estudio del transportador de glucosa GLUT-12 y de su papel fisiológico., Efecto de los ácidos grasos omega-3 y sus derivados lipídicos sobre el efecto inhibitorio del TNF-alfa en la absorción de nutrientes, Sexenios CNEAI: 4 (1992-1997, 1998-2003, 2004-2009, 2010-2015)
Índice H
22, (Scopus, 18/10/2018)

Publicaciones científicas más recientes (desde 2010)

Autores: Castilla Madrigal, Rosa María; Gil Iturbe, Eva; de Calle, M. L. ; et al.
ISSN 0955-2863  Vol. 76  2020 
Tumor necrosis factor-alfa (TNF-alpha) is a pro-inflammatory cytokine highly-involved in intestinal inflammation. Omega-3 polyunsaturated fatty acids (n3-PUFAs) show anti-inflammatory actions. We previously demonstrated that the n3-PUFA EPA prevents TNF-alpha inhibition of sugar uptake in Caco-2 cells. Here, we investigated whether the n3-PUFA DHA and its derived specialized pro-resolving lipid mediators (SPMs) MaR1, RvD1 and RvD2, could block TNF-alpha inhibition of intestinal sugar and glutamine uptake. DHA blocked TNF-alpha-induced inhibition of alpha-methyl-D-glucose (alpha MG) uptake and SGLT1 expression in the apical membrane of Caco-2 cells, through a pathway independent of GPR120. SPMs showed the same preventive effect but acting at concentrations 1000 times lower. In diet-induced obese (DIO) mice, oral gavage of MaR1 reversed the up-regulation of pro-inflammatory cytokines found in intestinal mucosa of these mice. However, MaR1 treatment was not able to counteract the reduced intestinal transport of alpha MG and SGLT1 expression in the DIO mice. In Caco-2 cells, TNF-alpha also inhibited glutamine uptake being this inhibition prevented by EPA, DHA and the DHA-derived SPMs. Interestingly, TNF-alpha increased the expression in the apical membrane of the glutamine transporter B(0)AT1. This increase was partially blocked by the n-3 PUFAs. These data reveal DHA and its SPMs as promising biomolecules to restore intestinal nutrients transport during intestinal inflammation. (C) 2019 Elsevier Inc. All rights reserved.
Autores: Gil Iturbe, Eva; Félix Soriano, Elisa; Sáinz Amillo, Neira; et al.
ISSN 1715-5312  Vol. 45  Nº 9  2020  págs. 957 - 967
Obesity is characterized by excessive fat accumulation and inflammation. Aging has also been characterized as an inflammatory condition, frequently accompanied by accumulation of visceral fat. Beneficial effects of exercise and 11-3 long-chain polyunsaturated fatty acids in metabolic disorders have been described. Glucose transporter 12 (GLUT12) is one of the less investigated members of the GLUT family. Glucose, insulin, and tumor necrosis factor alpha (TNF-alpha) induce GLUT12 translocation to the membrane in muscle, adipose tissue, and intestine. We aimed to investigate GLUT12 expression in obesity and aging, and under diet supplementation with docosahexaenoic acid (DHA) alone or in combination with physical exercise in mice. Aging increased GLUT12 expression in intestine, kidney, and adipose tissue, whereas obesity reduced it. No changes on the transporter occurred in skeletal muscle. In obese 18-month-old mice, DHA further decreased GLUT12 in the 4 organs. Aerobic exercise alone did not modify GLUT12, but the changes triggered by exercise were able to prevent the DHA-diminishing effect, and almost restored GLUT12 basal levels. In conclusion, the downregulation of metabolism in aging would be a stimulus to upregulate GLUT12 expression. Contrary, obesity, an excessive energy condition, would induce GLUT12 downregulation. The combination of exercise and DHA would contribute to restore basal function of GLUT12.
Autores: Gil Iturbe, Eva; Solas Zubiaurre, Maite; Cuadrado Tejedor, María del Mar; et al.
ISSN 0893-7648  Vol. 57  Nº 2  2020  págs. 798 - 805
The brain depends on glucose as a source of energy. This implies the presence of glucose transporters, being GLUT1 and GLUT3 the most relevant. Expression of GLUT12 is found in mouse and human brain at low levels. We previously demonstrated GLUT12 upregulation in the frontal cortex of aged subjects that was even higher in aged Alzheimer's disease (AD) patients. However, the cause and the mechanism through which this increase occurs are still unknown. Here, we aimed to investigate whether the upregulation of GLUT12 in AD is related with aging or A beta deposition in comparison with GLUT1, GLUT3, and GLUT4. In the frontal cortex of two amyloidogenic mouse models (Tg2576 and APP/PS1) GLUT12 levels were increased. Contrary, expression of GLUT1 and GLUT3 were decreased, while GLUT4 did not change. In aged mice and the senescence-accelerated model SAMP8, GLUT12 and GLUT4 were upregulated in comparison with young animals. GLUT1 and GLUT3 did not show significant changes with age. The effect of beta-amyloid (A beta) deposition was also evaluated in A beta peptide i.c.v. injected mice. In the hippocampus, GLUT12 expression increased whereas GLUT4 was not modified. Consistent with the results in the amyloidogenic models, GLUT3 and GLUT1 were downregulated. In summary, A beta increases GLUT12 protein expression in the brain pointing out a central role of the transporter in AD pathology and opening new perspectives for the treatment of this neurodegenerative disease.
Autores: Gil Iturbe, Eva; Castilla Madrigal, Rosa María; Barreneche Huici, Jayone; et al.
ISSN 0021-9541  Vol. 234  Nº 4  2019  págs. 4396-4408
Autores: Castilla Madrigal, Rosa María; Gil Iturbe, Eva; Sáinz Amillo, Neira; et al.
ISSN 0021-9541  Vol. 234  Nº 4  2019  págs. 4352 - 4361
We have previously demonstrated in Caco-2 cells that tumor necrosis factor-alpha (TNF-alpha) inhibits sugar uptake, acting from the apical membrane, by decreasing the expression of the Na+-glucose cotransporter SGLT1 in the brush border membrane. The goal was to investigate the hypothesis that TNF-alpha from abdominal adipose tissue (adipocytes and macrophages) would decrease sugar and amino acid transport acting from the basolateral membrane of the enterocytes. TNF-alpha placed in the basal compartment of Caco-2 cells decreased alpha-methyl- d-glucose (alphaMG) and glutamine uptake. The apical medium derived from these Caco-2 cells apically placed in another set of cells, also reduced sugar and glutamine transport. Reverse-transcription polymerase chain reaction analysis demonstrated upregulation of TNF-alpha, IL-1beta, and MCP1 expression in Caco-2 cells exposed to basal TNF-alpha. Similarly, MG uptake was inhibited after Caco-2 cells were incubated, in the basal compartment, with medium from visceral human mesenchymal stem cells-derived adipocytes of overweight individuals. The apical medium collected from those Caco-2 cells, and placed in the upper side of other set of cells, also decreased sugar uptake. Basal presence of medium derived from lipopolysaccharide-activated macrophages and nonactivated macrophages decreased MG uptake as well. Diet-induced obese mice showed an increase in the visceral adipose tissue surrounding the intestine.
Autores: Gil Iturbe, Eva; Arbones-Mainar, J. M. ; Moreno Aliaga, María Jesús; et al.
ISSN 1748-1708  Vol. 226  Nº 4  2019 
AimThe facilitative glucose transporter GLUT12 was isolated from the breast cancer cell line MCF-7 by its homology with GLUT4. GLUT12 is expressed in insulin-sensitive tissues such as adipose tissue. The aim of this work was to investigate GLUT12 expression and hormonal regulation in 3T3-L1 adipocytes and in adipose tissue of lean and diet-induced obese mice. MethodsUptake studies were performed using radio-labelled sugars; alpha-methyl-d-glucose (alpha MG) was used as specific substrate of GLUT12. Expression and localization of GLUT12 in adipocytes were investigated by western blot and immunohistochemical methods. ResultsGLUT12 is expressed in the peri-nuclear region of mouse adipocytes. Insulin, by AKT activation, and TNF-alpha, by AMPK activation, increase alpha MG uptake by inducing GLUT12 translocation to the membrane. In contrast, leptin and adiponectin decrease GLUT12 activity through its internalization. Under hypoxia conditions GLUT12 expression is upregulated. The response of GLUT12 to TNF-alpha, leptin, adiponectin and hypoxia is the opposite to that of GLUT4. In diet-induced obese mice and obese subjects, GLUT12 protein is decreased. Intraperitoneal injection of insulin increases AKT phosphorylation and GLUT12 expression, but this effect is lost in obese animals. ConclusionWe hypothesize that GLUT12 would contribute to modulate sugar absorption in physiological and pathophysiological situations such as obesity.
Autores: Gil Iturbe, Eva; Solas Zubiaurre, Maite; Cuadrado Tejedor, María del Mar; et al.
ISSN 1748-1708  Vol. 227  Nº Supl. 718  2019  págs. 83 - 84
Autores: Castilla Madrigal, Rosa María; Barreneche Huici, Jayone; Moreno Aliaga, María Jesús; et al.
ISSN 0021-9541  Vol. 233  Nº 3  2018  págs. 2426 - 2433
The aim of the present work was to investigate in Caco-2 cells whether eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid, could block the inhibitory effect of tumor necrosis factor-alpha (TNF-alpha) on sugar transport, and identify the intracellular signaling pathways involved. After pre-incubation of the Caco-2 cells with TNF-alpha and EPA for 1 hr, EPA prevented the inhibitory effect of the cytokine on alpha-methyl-D-glucose (alpha MG) uptake (15 min) and on SGLT1 expression at the brush border membrane, measured by Western blot. The ERK1/2 inhibitor PD98059 and the AMPK activator AICAR also prevented the inhibitory effect of TNF-alpha on both alpha MG uptake and SGLT1 expression. Interestingly, the AMPK inhibitor, Compound C, abolished the ability of EPA to prevent TNF-alpha-induced reduction of sugar uptake and transporter expression. The GPR120 antagonist, AH7614, also blocked the preventive effect of EPA on TNF-alpha-induced decrease of alpha MG uptake and AMPK phosphorylation. In summary, TNF-alpha inhibits alpha MG uptake by decreasing SGLT1 expression in the brush border membrane through the activation of ERK1/2 pathway. EPA prevents the inhibitory effect of TNF-alpha through the involvement of GPR120 and AMPK activation.
Autores: Gil Iturbe, Eva; Castilla Madrigal, Rosa María; Barrenetxe, J.; et al.
ISSN 1742-464X  Vol. 284  Nº Supl. 1  2017  págs. 157 - 158
Autores: López Yoldi, Miguel; Castilla Madrigal, Rosa María; Lostao Crespo, María del Pilar; et al.
ISSN 1748-1708  Vol. 217  Nº 3  2016  págs. 217 - 226
rCT-1 effects on ¿-Methyl-D-glucoside uptake were assessed in everted intestinal rings from wild-type and CT-1(-/-) mice and in Caco-2 cells. rCT-1 actions on SGLT-1 expression in brush border membrane vesicles and the identification of the potential signalling pathways involved were determined by Western blot. RESULTS: In vivo administration (0.2 mg kg(-1) ) of rCT-1 caused a significant decrease on ¿-Methyl-D-glucoside uptake in everted intestinal rings from wild-type and CT-1(-/-) mice after short-term and long-term treatments. Similarly, in vitro treatment (1-50 ng mL(-1) ) with rCT-1 reduced ¿-Methyl-D-glucoside uptake in everted intestinal rings. In Caco-2 cells, rCT-1 treatment (20 ng mL(-1) , 1 and 24 h) lowered apical uptake of ¿-Methyl-D-glucoside in parallel with a decrease on SGLT-1 protein expression. rCT-1 promoted the phosphorylation of STAT-3 after 5 and 15 min treatment, but inhibited the activation by phosphorylation of AMPK after 30 and 60 min. Interestingly, pre-treatment with the JAK/STAT inhibitor (AG490) and with the AMPK activator (AICAR) reversed the inhibitory effects of rCT-1 on ¿-Methyl-D-glucoside uptake. AICAR also prevented the inhibition of SGLT-1 observed in rCT-1-treated cells. CONCLUSIONS: CT-1 inhibits intestinal sugar absorption by the reduction of SGLT-1 levels through the AMPK pathway, which could also contribute to explain the hypoglycaemic and anti-obesity properties of CT-1.
Autores: Etxeberría Aramburu, Usune; Castilla Madrigal, Rosa María; Lostao Crespo, María del Pilar; et al.
ISSN 0145-5680  Vol. 61  Nº 8  2015  págs. 9 - 16
A DNA microarray analysis was conducted in Caco-2 cells to analyse the protective effects of trans-resveratrol on enterocyte physiology and metabolism in pro-inflammatory conditions. Cells were pre-treated with 50 ¿¿ of trans-resveratrol and, subsequently, lipopolysaccharide (LPS) was added for 48 h. The microarray analysis revealed 121 genes differentially expressed between resveratrol-treated and non-treated cells (B> 0, is the odd thatthe gene is differentially expressed). Inhibitor of DNA binding 1 (ID1), histidine-rich glycoprotein (HRG), NADPH oxidase (NOX1) and sprouty homolog 1 (SPRY), were upregulated by LPS treatment, but significantly blocked by trans-resveratrol pre-treatment (padj< 0.05, after adjusting for Benjamini-Hocheberg procedure). Moreover, genes implicated in synthesis of lipids (z-score= -1.195) and concentration of cholesterol (z-score= -0.109), were markedly downregulated by trans-resveratrol. Other genes involved in fat turnover, but also in cell death and survival function, such as transcription factors Krüppel-like factor 5 (KLF5) and amphiregulin (AREG), were also significantly inhibited by trans-resveratrol pre-treatment. RT-qPCR-data confirmed the microarray results. Special mention deserves acyl-CoA synthetase long-chain family member 3 (ACSL3) and endothelial lipase (LIPG), which were downregulated by this stilbene and have been previously associated with fatty acid synthesis and obesity in other tissues. This study envisages that trans-resveratrol might exert an important anti-lipogenic effect at intestinal level under pro-inflammatory conditions, which has not been previously described.
Autores: Pujol Giménez, Jonai; Pérez de Heredia, F.; Idoate Gastearena, Miguel Ángel; et al.
ISSN 1837-9664  Vol. 6  Nº 2  2015  págs. 139 - 143
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
Autores: Pujol Giménez, Jonai; Pérez, A. A.; Reyes, A. M.; et al.
ISSN 0363-6143  Vol. 308  Nº 12  2015  págs. C1008 - C1022
GLUT12 is a member of the facilitative family of glucose transporters. The goal of this study was to characterize the functional properties of GLUT12, expressed in Xenopus laevis oocytes, using radiotracer and electrophysiological methods. Our results showed that GLUT12 is a facilitative sugar transporter with substrate selectivity: d-glucose ¿ ¿-methyl-d-glucopyranoside (¿-MG) > 2-deoxy-d-glucose(2-DOG) > d-fructose = d-galactose. ¿-MG is a characteristic substrate of the Na(+)/glucose (SGLT) family and has not been shown to be a substrate of any of the GLUTs. In the absence of sugar, (22)Na(+) was transported through GLUT12 at a higher rate (40%) than noninjected oocytes, indicating that there is a Na(+) leak through GLUT12. Genistein, an inhibitor of GLUT1, also inhibited sugar uptake by GLUT12. Glucose uptake was increased by the PKA activator 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) but not by the PKC activator phorbol-12-myristate-13-acetate (PMA). In high K(+) concentrations, glucose uptake was blocked. Addition of glucose to the external solution induced an inward current with a reversal potential of approximately -15 mV and was blocked by Cl(-) channel blockers, indicating the current was carried by Cl(-) ions. The sugar-activated Cl(-) currents were unaffected by genistein. In high external K(+) concentrations, sugar-activated Cl(-) currents were also blocked, indicating that GLUT12 activity is voltage dependent. Furthermore, glucose-induced current w
Autores: Fanjul González, María del Carmen; Barreneche Huici, Jayone; De Pablo-Maiso, L.; et al.
ISSN 0022-0795  Vol. 224  Nº 1  2015  págs. 17 - 23
Leptin is secreted by the gastric mucosa and is able to reach the intestinal lumen and bind to its receptors located in the apical membranes of enterocytes. We have previously demonstrated that apical leptin inhibits uptake of amino acids in rat intestine in vitro and in Caco-2 cells. The aim of the present work was to investigate the effect of leptin on absorption of amino acids using in vivo techniques, which generate situations closer to physiological conditions. In vivo intestinal absorption of amino acids in rats was measured by isolating a jejunal loop and using the single-pass perfusion system. Disappearance of glutamine (Gln), proline (Pro), and ß-alanine (ß-Ala) from the perfusate, in the absence or presence of leptin, was measured using a radioactivity method. Luminal leptin (25¿nM) inhibited the absorption of 2¿mM Pro, 5¿mM ß-Ala, and 5¿mM Gln by approximately 45% after 5-15¿min; the effect remained constant until the end of the experiment (80¿min) and was rapidly and completely reversed when leptin was removed from the perfusion medium. Moreover, leptin was able to regulate the absorption of galactose and Gln in the same animal, indicating a direct action of the hormone on the specific transporters implicated in the uptake of each nutrient. The results of the present work indicate that luminal leptin decreases absorption of amino acids in vivo in a short-term manner and in a reversible way.
Autores: Fanjul González, María del Carmen; Barreneche Huici, Jayone; Lostao Crespo, María del Pilar; et al.
ISSN 1138-7548  Vol. 71  Nº 2  2015  págs. 311 - 317
Leptin is secreted into the digestive tract and contributes to the absorption of dietary molecules by regulating transporters activity. Here, we studied the effect of luminal leptin on the intestinal transport of L-glutamate, an important component of human diet. We examined the effect of leptin on L-glutamate uptake in rat intestine in vitro measuring glutamate-induced short-circuit current (Isc) in Ussing chambers and L-[(3)H (U)]-glutamate uptake in jejunal everted rings. Glutamate-induced Isc was only observed in Na(+)-free conditions. This Isc was concentration (1-60 mmol L(-1)) and pH dependent. Luminal leptin increased glutamate Isc (~100 %). Dose-response curve showed a biphasic pattern, with maximal stimulations observed at 10(-13) and 10(-10) mmol L(-1), that were sensitive to leptin receptor antagonist. In everted rings, two glutamate transport mechanisms were distinguished: a Na(+)-dependent, H(+)-independent, that was inhibited by leptin (~20 %), and a Na(+)-independent but H(+)-dependent, that was enhanced by leptin (~20 %), in line with data obtained in Ussing chambers. Altogether, these data reveal original non-monotonic effect of luminal leptin in the intestine and demonstrate a new role for this hormone in the modulation of L-glutamate transport, showing that luminal active gut peptides can influence absorption of amino acids
Autores: Claudio-Montero, A.; Pinilla-Macua, I.; Fernández-Calotti, P.; et al.
ISSN 1543-8384  Vol. 12  Nº 6  2015  págs. 2158 - 2166
The abundance and function of transporter proteins at the plasma membrane is likely to be crucial in drug responsiveness. The detection of Human Concentrative Nucleoside Transporters (hCNTs) function is of interest to predict drug sensitivity due to their ability to transport most nucleoside-derived drugs. In the present study two fluorescent nucleoside analogues, Uridine-furan and Etheno-cytidine, were evaluated as tools to study in vivo nucleoside transporter-related functions. These two molecules showed high affinity interactions with hCNT1 and hCNT3 being also hCNT substrates. Both fluorescent microscopy and flux cytometry experiments showed that Uridine-furan uptake was better suited for distinguishing cells that express or not hCNT1 or hCNT3. These data highlight the usefulness of fluorescent nucleoside derivatives as long as they fulfil the requirements of confocal microscopy and flow cytometry for in vivo analysis of hCNT-related function. Fluorescent Nucleoside Derivatives as a Tool for the Detection of Concentrative Nucleoside Transporter Activity Using Confocal Microscopy and Flow Cytometry. Available from: [accessed Mar 27, 2017].
Autores: Pujol Giménez, Jonai; Martisová, Eva; Pérez Mediavilla, Alberto; et al.
ISSN 1387-2877  Vol. 42  Nº 1  2014  págs. 97 - 101
Alzheimer's disease (AD) might be conceptualized as a metabolic disease with progressive impairment of the brain's capacity to utilize glucose. One of the last glucose transporters discovered is GLUT12. The aim of the present work was to investigate the expression of GLUT12 in frontal cortex from AD patients. Human samples from young control donors barely expressed GLUT12. The level of expression of GLUT12 was significantly higher in AD compare to aged controls. Expression of GLUT12 and Ox-42, a microglia marker, correlate in controls but not in AD. The implications of these findings in AD are discussed further.
Autores: Pujol Giménez, Jonai; Barreneche Huici, Jayone; González Muniesa, Pedro; et al.
ISSN 1138-7548  Vol. 69  Nº 2  2013  págs. 325 - 333
Autores: Fanjul González, María del Carmen; Barreneche Huici, Jayone; Lostao Crespo, María del Pilar
ISSN 1138-7548  Vol. 69  Nº 3  2013  págs. 507 - 512
Leptin is secreted by gastric mucosa and is able to reach the intestinal lumen where its receptors are located in the apical membrane of the enterocytes. We have previously demonstrated that apical leptin inhibits sugar and amino acids uptake in vitro and glucose absorption in vivo. Since leptin receptors are also expressed in the basolateral membrane of the enterocytes, the aim of the present work was to investigate whether leptin acting from the basolateral side could also regulate amino acid uptake. Tritiated Gln and ß-Ala were used to measure uptake into Caco-2 cells grown on filters, in the presence of basal leptin at short incubation times (5 and 30 min) and after 6 h of preincubation with the hormone. In order to compare apical and basal leptin effect, Gln and ß-Ala uptake was measured in the presence of leptin acting from the apical membrane also in cells grown on filters. Basal leptin (8 mM) inhibited by ~15-30 % the uptake of 0.1 mM Gln and 1 mM ß-Ala quickly, after 5 min exposure, and the effect was maintained after long preincubation periods. Apical leptin had the same effect. Moreover, the inhibition was rapidly and completely reversed when leptin was removed from the apical or basolateral medium. These results extend our previous findings and contribute to the vision of leptin as an important hormonal signal for the regulation of intestinal absorption of nutrients.
Autores: de la Garza Hernández, Ana Laura Isabel; Etxeberría Aramburu, Usune; Lostao Crespo, María del Pilar; et al.
ISSN 0021-8561  Vol. 61  Nº 49  2013  págs. 12012 - 12019
Several plant extracts rich in flavonoids have been reported to improve hyperglycemia by inhibiting digestive enzyme activities and SGLT1-mediated glucose uptake. In this study, helichrysum ( Helichrysum italicum ) and grapefruit ( Citrus × paradisi ) extracts inhibited in vitro enzyme activities. The helichrysum extract showed higher inhibitory activity of ¿-glucosidase (IC50 = 0.19 mg/mL) than ¿-amylase (IC50 = 0.83 mg/mL), whereas the grapefruit extract presented similar ¿-amylase and ¿-glucosidase inhibitory activities (IC50 = 0.42 mg/mL and IC50 = 0.41 mg/mL, respectively). Both extracts reduced maltose digestion in noneverted intestinal sacs (57% with helichrysum and 46% with grapefruit). Likewise, both extracts inhibited SGLT1-mediated methylglucoside uptake in Caco-2 cells in the presence of Na+ (56% of inhibition with helichrysum and 54% with grapefruit). In vivo studies demonstrated that helichrysum decreased blood glucose levels after an oral maltose tolerance test (OMTT), and both extracts reduced postprandial glucose levels after the oral starch tolerance test (OSTT). Finally, both extracts improved hyperinsulinemia (31% with helichrysum and 50% with grapefruit) and HOMA index (47% with helichrysum and 54% with grapefruit) in a dietary model of insulin resistance in rats. In summary, helichrysum and grapefruit extracts improve postprandial glycemic control in rats, possibly by inhibiting ¿-glucosidase and ¿-amylase enzyme activities and decreasing SGLT1-mediated glucose uptake.
Autores: Barreneche Huici, Jayone; Sánchez, O.; Barber Cárcamo, Ana María; et al.
ISSN 1043-4666  Vol. 64  Nº 1  2013  págs. 181 - 187
PURPOSE During intestinal inflammation TNF¿ levels are increased and as a consequence malabsorption of nutrients may occur. We have previously demonstrated that TNF¿ inhibits galactose, fructose and leucine intestinal absorption in animal models. In continuation with our work, the purpose of the present study was to investigate in the human intestinal epithelial cell line Caco-2, the effect of TNF¿ on sugar transport and to identify the intracellular mechanisms involved. METHODS Caco-2 cells were grown on culture plates and pre-incubated during different periods with various TNF¿ concentrations before measuring the apical uptake of galactose, ¿-methyl-glucoside (MG) or fructose for 15min. To elucidate the signaling pathway implicated, cells were pre-incubated for 30min with the PKA inhibitor H-89 or the PKC inhibitor chelerythrine, before measuring the sugar uptake. The expression in the apical membrane of the transporters implicated in the sugars uptake process (SGLT1 and GLUT5) was determined by Western blot. RESULTS TNF¿ inhibited 0.1mM MG uptake after pre-incubation of the cells for 6-48h with the cytokine and in the absence of cytokine pre-incubation. In contrast, 5mM fructose uptake was stimulated by TNF¿ only after long pre-incubation times (24 and 48h). These effects were mediated by the binding of the cytokine to its specific receptor TNFR1, present in the apical membrane of the Caco-2 cells. Analysis of the expression of the MG and fructose transporters at the brush
Autores: Cano-Soldado, P.; Gorraitz Eusa, Edurne; Errasti-Murugarren, E.; et al.
ISSN 0363-6143  Vol. 302  Nº 1  2012  págs. C257-C266
Cano-Soldado P, Gorraitz E, Errasti-Murugarren E, Casado FJ, Lostao MP, Pastor-Anglada M. Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket. Am J Physiol Cell Physiol 302: C257-C266, 2012. First published October 12, 2011; doi: 10.1152/ajpcell.00198.2011.-SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na(+)/1 nucleoside stoichiometry, showed altered Na(+) binding properties associated with a shift in the Hill coefficient, consistent with one Na(+) binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na(+) leak, which was dependent on the concentration of extracellular Na(+) indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.
Autores: Fanjul González, María del Carmen; Barreneche Huici, Jayone; Íñigo Ganuza, Carmen; et al.
ISSN 1748-1708  Vol. 205  Nº 1  2012  págs. 82 - 91
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 alpha-methyl-D-glucoside uptake after 5 or 30 min treatment and decreased SGLT1 protein abundance in the apical membrane. Uptake of 20 mu M 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 B(0)AT1. This inhibition was accompanied by a reduction in the transporters expression at the brush border membrane. Leptin also inhibited 1 mM proline and beta-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 beta-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
Autores: Martinez Becerra, Pablo; Briz, Oscar; Romero, Marta R.; et al.
ISSN 0026-895X  Vol. 79  Nº 3  2011  págs. 596 - 607
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 (Delta Psi) 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 17 beta-D-glucuronide (E(2)17 beta G) 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 Delta Psi [DilC(1)(5)]. The results suggest that activity of these three carriers is sodium/potassium-independent and affected differently by changes in pHo and Delta Psi: Oatp1a1 was confirmed to be an electroneutral anion exchanger, whereas the function of both OATP1B1 and OATP1B3 was markedly affected by the magnitude of Del
Autores: Gorraitz Eusa, Edurne; Pastor-Anglada, M.; Lostao Crespo, María del Pilar
ISSN 0031-6768  Vol. 460  Nº 3  2010  págs. 617 - 632
Human concentrative nucleoside transporter 3 (hCNT3) uses the electrochemical gradient of Na+ and H+ to drive the transport of nucleosides and therapeutic nucleoside analogs into the cells. We employed the two-electrode voltage clamp technique to compare the steady-state and presteady-state kinetics of hCNT3 in the presence of Na+ and H+. We found that H+ supported a higher maximal rate of uridine transport than Na+, but the efficiency of transport was lower. For both cations, maximal uridine-induced current increased with hyperpolarizing potentials and did not saturate within the voltage range tested. Apparent affinity of hCNT3 for uridine in H+ was insensitive to membrane voltage at negative potentials, and decreased with depolarization. In contrast, apparent affinity for uridine in Na+ decreased with hyperpolarization and was independent of voltage at depolarizing potentials. H+-coupled hCNT3 exhibited lower affinity for all natural nucleosides and different substrate selectivity compared to Na+-coupled hCNT3. In H+, lack of the hydroxyl groups at 2' and 5' decreased the affinity, while lack of the nitrogen N-7 or inversion of the configuration of the hydroxyl group at 2' prevented transport. Presteady-state charge movements of hCNT3 did not decrease when extracellular cation concentration (Na+ or H+) was reduced, but the tau (ON)-V and Q-V curves were shifted to more negative potentials. The different effects of uridine and inosine on presteady-state currents in H+ indica
Autores: Ducroc, R.; Sakar, Y.; Fanjul González, María del Carmen; et al.
Revista: American Journal of Physiology, Gastrointestinal and Liver Physiology
ISSN 0193-1857  Vol. 299  Nº 1  2010  págs. G179 - G185
-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; IC50 = similar to 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
Autores: Barreneche Huici, Jayone; Fanjul González, María del Carmen; Ducroc, Robert; et al.
Libro:  Glucose uptake: regulation, signaling pathways and health implications
2013  págs. 215 - 234
Autores: Fanjul González, María del Carmen; Lostao Crespo, María del Pilar
Libro:  Fundamentos de Nutrición y Dietética. Bases Metodológicas y Aplicaciones
2011  págs. 127 - 131