GLUT12 expression and regulation in murine small intestine and human Caco-2 cells.

GLUT12 was cloned from the mammary cancer cell line MCF-7, but its physiological role still needs to be elucidated. To gain more knowledge of GLUT12 function in the intestine, we investigated GLUT12 subcellular localization in the small intestine and its regulation by sugars, hormones, and intracellular mediators in Caco-2 cells and mice. Immunohistochemical methods were used to determine GLUT12 subcellular localization in human and murine small intestine. Brush border membrane vesicles were isolated for western blot analyses. Functional studies were performed in Caco-2 cells by measuring α-methyl-d-glucose (αMG) uptake in the absence of sodium. GLUT12 is located in the apical cytoplasm, below the brush border membrane, and in the perinuclear region of murine and human enterocytes. In Caco-2 cells, GLUT12 translocation to the apical membrane and α-methyl- d-glucose uptake by the transporter are stimulated by protons, glucose, insulin, tumor necrosis factor-α (TNF-α), protein kinase C, and AMP-activated protein kinase. In contrast, hypoxia decreases GLUT12 expression in the apical membrane. Upregulation of TNF-α and hypoxia-inducible factor-1α ( HIF-1α) genes is found in the jejunal mucosa of diet-induced obese mice. In these animals, GLUT12 expression in the brush border membrane is slightly decreased compared with lean animals. Moreover, an intraperitoneal injection of insulin does not induce GLUT12 translocation to the membrane, as it occurs in lean animals. GLUT12 rapid translocation to the enterocytes' apical membrane in response to glucose and insulin could be related to GLUT12 participation in sugar absorption during postprandial periods. In obesity, in which insulin sensitivity is reduced, the contribution of GLUT12 to sugar absorption is affected.

EPA blocks TNF-α-induced inhibition of sugar uptake in Caco-2 cells via GPR120 and AMPK.

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-α (TNF-α) on sugar transport, and identify the intracellular signaling pathways involved. After pre-incubation of the Caco-2 cells with TNF-α and EPA for 1 hr, EPA prevented the inhibitory effect of the cytokine on α-methyl-d-glucose (αMG) 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-α on both αMG uptake and SGLT1 expression. Interestingly, the AMPK inhibitor, Compound C, abolished the ability of EPA to prevent TNF-α-induced reduction of sugar uptake and transporter expression. The GPR120 antagonist, AH7614, also blocked the preventive effect of EPA on TNF-α-induced decrease of αMG uptake and AMPK phosphorylation. In summary, TNF-α inhibits αMG uptake by decreasing SGLT1 expression in the brush border membrane through the activation of ERK1/2 pathway. EPA prevents the inhibitory effect of TNF-α through the involvement of GPR120 and AMPK activation.

TNFα regulates sugar transporters in the human intestinal epithelial cell line Caco-2.

PURPOSE: During intestinal inflammation TNFα levels are increased and as a consequence malabsorption of nutrients may occur. We have previously demonstrated that TNFα 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 TNFα 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 TNFα concentrations before measuring the apical uptake of galactose, α-methyl-glucoside (MG) or fructose for 15 min. 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: TNFα 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 TNFα only after long pre-incubation times (24 and 48 h). 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 border membrane of the cells, after 24h pre-incubation with the cytokine, revealed decrease on the amount of SGLT1 and increase on the amount of GLUT5 proteins. Short-term inhibition of MG transport by TNFα was not modified by H-89 but was blocked by chelerythrine. CONCLUSIONS: SGLT1 and GLUT5 expression in the plasma membrane is regulated by TNFα in the human epithelial cell line Caco-2 cells, leading to alteration on sugars transport, suggesting that TNFα could be considered as a physiological local regulator of nutrients absorption in response to an intestinal inflammatory status.

Effect of a Diet Supplemented with Sphingomyelin and Probiotics on Colon Cancer Development in Mice.

Previous studies have reported that dietary sphingomyelin could inhibit early stages of colon cancer. Lactic acid-producing bacteria have also been associated with an amelioration of cancer symptoms. However, little is known about the potential beneficial effects of the combined administration of both sphingomyelin and lactic acid-producing bacteria. This article analyzes the effect of a diet supplemented with a combination of the probiotics Lacticaseibacillus casei and Bifidobacterium bifidum (108 CFU/ml) and sphingomyelin (0.05%) on mice with 1,2-dimethylhydrazine (DMH)-induced colon cancer. Thirty-six BALB/c mice were divided into 3 groups: one healthy group (group C) and two groups with DMH-induced cancer, one fed a standard diet (group D) and the other fed a diet supplemented with sphingomyelin and probiotics (DS). The number of aberrant crypt foci, marker of colon cancer development, was lower in the DS. The dietary supplementation with the synbiotic reversed the cancer-induced impairment of galactose uptake in enterocyte brush-border-membrane vesicles. These results confirm the beneficial effects of the synbiotic on the intestinal physiology of colon cancer mice and contribute to the understanding of the possible mechanisms involved.

Azoxymethane-Induced Colorectal Cancer Mice Treated with a Polyphenol-Rich Apple Extract Show Less Neoplastic Lesions and Signs of Cachexia.

Obesity is considered a risk factor for the development of colorectal cancer. In rodents, high-fat (HF) diets are able to increase the formation of azoxymethane (AOM)-induced polyps. Polyphenol-rich apple extracts have antioxidant and anti-inflammatory activities and may induce an amelioration of the manifestations of colorectal cancer. Twenty-seven male Crl:CD-1 mice received AOM during four weeks and were subsequently divided into three groups fed a HF diet (n = 9 each group): a non-supplemented group, a second group supplemented with apple extract at 1%, and a third group supplemented with the same apple extract at 1.5%. Energy metabolism and the respiratory quotient were not affected by the supplementation with the apple extract. Although body weight was not affected by the treatment, the mice supplemented with the apple extract showed less signs of cachexia than the non-treated mice. In the intestine, the mice supplemented with the apple extract showed lower sucrase, dipeptidyl-peptidase IV, and aminopeptidase N activities, and less intestinal lesions (aberrant crypt foci and polyps). Administration of a polyphenol-rich apple extract reduces the number of neoplastic lesions in mice with AOM-induced colorectal cancer and contributes to preserve adipose tissue mass.

Matrix metalloproteinase-10 is upregulated by thrombin in endothelial cells and increased in patients with enhanced thrombin generation.

OBJECTIVE: Thrombin is a multifunctional serine protease that promotes vascular proinflammatory responses whose effect on endothelial MMP-10 expression has not previously been evaluated. METHODS AND RESULTS: Thrombin induced endothelial MMP-10 mRNA and protein levels, through a protease-activated receptor-1 (PAR-1)-dependent mechanism, in a dose- and time-dependent manner. This effect was mimicked by a PAR-1 agonist peptide (TRAP-1) and antagonized by an anti-PAR-1 blocking antibody. MMP-10 induction was dependent on extracellular regulated kinase1/2 (ERK1/2) and c-jun N-terminal kinase (JNK) pathways. By serial deletion analysis, site-directed mutagenesis and electrophoretic mobility shift assay an AP-1 site in the proximal region of MMP-10 promoter was found to be critical for thrombin-induced MMP-10 transcriptional activity. Thrombin and TRAP-1 upregulated MMP-10 in murine endothelial cells in culture and in vivo in mouse aorta. This effect of thrombin was not observed in PAR-1-deficient mice. Interestingly, circulating MMP-10 levels (P<0.01) were augmented in patients with endothelial activation associated with high (disseminated intravascular coagulation) and moderate (previous acute myocardial infarction) systemic thrombin generation. CONCLUSIONS: Thrombin induces MMP-10 through a PAR-1-dependent mechanism mediated by ERK1/2, JNK, and AP-1 activation. Endothelial MMP-10 upregulation could be regarded as a new proinflammatory effect of thrombin whose pathological consequences in thrombin-related disorders and plaque stability deserve further investigation.

Modulation of intestinal L-glutamate transport by luminal leptin.

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.

Leptin resistance and diet-induced obesity: central and peripheral actions of leptin.

Obesity is a chronic disease that represents one of the most serious global health burdens associated to an excess of body fat resulting from an imbalance between energy intake and expenditure, which is regulated by environmental and genetic interactions. The adipose-derived hormone leptin acts via a specific receptor in the brain to regulate energy balance and body weight, although this protein can also elicit a myriad of actions in peripheral tissues. Obese individuals, rather than be leptin deficient, have in most cases, high levels of circulating leptin. The failure of these high levels to control body weight suggests the presence of a resistance process to the hormone that could be partly responsible of disturbances on body weight regulation. Furthermore, leptin resistance can impair physiological peripheral functions of leptin such as lipid and carbohydrate metabolism and nutrient intestinal utilization. The present document summarizes those findings regarding leptin resistance development and the role of this hormone in the development and maintenance of an obese state. Thus, we focused on the effect of the impaired leptin action on adipose tissue, liver, skeletal muscle and intestinal function and the accompanying relationships with diet-induced obesity. The involvement of some inflammatory mediators implicated in the development of obesity and their roles in leptin resistance development are also discussed.

In vivo regulation of intestinal absorption of amino acids by leptin.

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. These results, together with our previous findings, make it evident that leptin can be considered as a hormone which provides the intestine with a control mechanism to handle absorption of nutrients.

Basal leptin regulates amino acid uptake in polarized Caco-2 cells.

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.

Helichrysum and grapefruit extracts inhibit carbohydrate digestion and absorption, improving postprandial glucose levels and hyperinsulinemia in rats.

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 α-glucosidase (IC50 = 0.19 mg/mL) than α-amylase (IC50 = 0.83 mg/mL), whereas the grapefruit extract presented similar α-amylase and α-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 α-glucosidase and α-amylase enzyme activities and decreasing SGLT1-mediated glucose uptake.

Modulation of intestinal L-glutamate transport by luminal leptin

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-[3H (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

Involvement of PKC and PKA in the inhibitory effect of leptin on intestinal galactose absorption.

Studies from our laboratory have demonstrated that leptin inhibits galactose absorption in vitro by acting on the Na(+)/glucose cotransporter SGLT1. Since PKC and PKA are involved in the regulation of SGLT1 and leptin is able to activate these kinases, we have investigated the possible implication of PKC and PKA in the inhibition of sugar absorption by leptin in rat small intestinal rings. Inhibition of 1 mM galactose uptake by 0.2 nM leptin is blocked by 2 microM chelerythrine, a PKC inhibitor, which by itself does not affect galactose uptake. However, 1 microM H-89, a PKA inhibitor, inhibits galactose uptake and does not block leptin inhibition. Biochemical assays show that the inhibitory effect of leptin is accompanied by a approximately 2-fold increase in PKA and PKC activity. These findings indicate that the activation of PKC is more relevant than PKA activation in the inhibition of galactose absorption by leptin.

Basal leptin regulates amino acid uptake in polarized Caco-2 cells

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 β-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 β-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 β-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

Mecanismos reparadores neuronales en la enfermedad de Alzheimer / Neuronal repair mechanisms in Alzheimer's disease

La enfermedad de Alzheimer (EA) es el tipo de demencia más frecuente y afecta a unos 15 millones de personas en el mundo. A pesar de su alta incidencia, aún no disponemos de un método de diagnóstico claro, eficaz y excluyente para esta afección. En la actualidad éste se realiza con precaución tras la observación de una serie de síntomas, entre los que se encuentran la pérdida de memoria, alteraciones en el lenguaje, etc. En cuanto al tratamiento de la EA, en los últimos años están adquiriendo importancia otros abordajes de tipo no farmacológicos como es la rehabilitación neuronal. Este tratamiento es un proceso terapéutico altamente individualizado, específicamente desarrollado para resolver las necesidades del paciente y está basado en la plasticidad del cerebro. De la misma manera, se conoce que la nutrición desempeña un papel muy relevante en el desarrollo de numerosas afecciones, incluidas las enfermedades neurodegenerativas. Son muchas las evidencias que sustentan la idea de la participación del estrés oxidativo en el desarrollo de la EA y en los procesos apoptóticos que se deriven de ella. En este sentido, el consumo de antioxidantes, en la dieta o a través de suplementos dietéticos, parece ser neuroprotector y puede mitigar el declive cognitivo. Ambas terapias (farmacológica y no farmacológica) comparten los mismos objetivos: retrasar el deterioro, recuperar funciones perdidas o mantenerlas conservadas y mejorar la calidad de vida (AU)

Alzheimer's disease (AD) is the most common type of dementia, affecting 15 million persons worldwide. Despite the high incidence of this disease, a clear and effective diagnostic method specific to AD is lacking. Currently, diagnosis of AD is made with caution, after observation of a series of symptoms that include memory loss and speech alterations, among others. In the last few years, non-pharmacological approaches, such as neuronal rehabilitation, have become more important in the treatment of AD. This form of treatment is highly individualized, specifically developed to resolve the needs of the patient and are based on cerebral plasticity. Nutrition plays a major role in the development of numerous diseases included under the heading of neurodegenerative disorders. A large body of evidence supports the role of oxidative stress in the development of AD and in apoptotic processes involved in this disease. Antioxidant intake, whether through the diet or in the form of supplements, seems to confer neuroprotection and could mitigate against cognitive decline. Both forms of treatment (pharmacological and non-pharmacological) share a common goal: to delay impairment, recover lost function or conserve existing function and improve quality of life (AU)