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

Resistin-Like Molecule-β Inhibits SGLT-1 Activity and Enhances GLUT2-Dependent Jejunal Glucose Transport

International audienceOBJECTIVE: An increased expression of RELM-beta (resistin-like molecule-beta), a gut-derived hormone, is observed in animal models of insulin resistance/obesity and intestinal inflammation. Intestinal sugar absorption is modulated by dietary environment and hormones/cytokines. The aim of this study was to investigate the effect of RELM-beta on intestinal glucose absorption. RESEARCH DESIGN AND METHODS: Oral glucose tolerance test was performed in mice and rats in the presence and the absence of RELM-beta. The RELM-beta action on glucose transport in rat jejunal sacs, everted rings, and mucosal strips was explored as well as downstream kinases modulating SGLT-1 and GLUT2 glucose transporters. RESULTS: Oral glucose tolerance test carried out in rodents showed that oral administration of RELM-beta increased glycemia. Studies in rat jejunal tissue indicated that mucosal RELM-beta promoted absorption of glucose from the gut lumen. RELM-beta had no effect on paracellular mannitol transport, suggesting a transporter-mediated transcellular mechanism. In studies with jejunal mucosa mounted in Ussing chamber, luminal RELM-beta inhibited SGLT-1 activity in line with a diminished SGLT-1 abundance in brush border membranes (BBMs). Further, the potentiating effect of RELM-beta on jejunal glucose uptake was associated with an increased abundance of GLUT2 at BBMs. The effects of RELM-beta were associated with an increased amount of protein kinase C betaII in BBMs and an increased phosphorylation of AMP-activated protein kinase (AMPK). CONCLUSIONS: The regulation of SGLT-1 and GLUT2 by RELM-beta expands the role of gut hormones in short-term AMPK/protein kinase C mediated control of energy balance

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)

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

Role of protease activated receptor-2 in lymph node metastasis of uterine cervical cancers

<p>Abstract</p> <p>Background</p> <p>Protease activated receptor-2 (PAR-2) has been implicated in cellular proliferation, invasion and metastasis in various tumors. Lymph node metastasis is an important patient prognostic factor for uterine cervical cancers. This prompted us to study the role of PAR-2 in lymph node metastasis of uterine cervical cancers.</p> <p>Methods</p> <p>Thirty patients underwent surgery for uterine cervical cancers. PAR-2 histoscores and mRNA levels were determined by immunohistochemistry and real-time reverse transcription-polymerase chain reaction, respectively. Patient prognosis was analyzed with a 48-month survival rate.</p> <p>Results</p> <p>PAR-2 histoscores and mRNA levels significantly (<it>P </it>< 0.05) increased in 12 of 30 metastatic lymph node lesions from the corresponding primary tumor. The 48-month survival rate of the 12 patients with increased PAR-2 levels in metastatic lymph nodes was 42%, while the rate of the other 18 patients with no change in PAR-2 levels was 82%, regardless of histopathological type.</p> <p>Conclusion</p> <p>PAR-2 might work on lymph node metastasis of uterine cervical cancers, and is considered to be a novel prognostic indicator for uterine cervical cancers.</p

Combined NADPH Oxidase 1 and Interleukin 10 Deficiency Induces Chronic Endoplasmic Reticulum Stress and Causes Ulcerative Colitis-Like Disease in Mice

Ulcerative colitis (UC) is a chronic inflammatory bowel disease affecting the rectum which progressively extents. Its etiology remains unknown and the number of treatments available is limited. Studies of UC patients have identified an unbalanced endoplasmic reticulum (ER) stress in the non-inflamed colonic mucosa. Animal models with impaired ER stress are sensitive to intestinal inflammation, suggesting that an unbalanced ER stress could cause inflammation. However, there are no ER stress-regulating strategies proposed in the management of UC partly because of the lack of relevant preclinical model mimicking the disease. Here we generated the IL10/Nox1(dKO) mouse model which combines immune dysfunction (IL-10 deficiency) and abnormal epithelium (NADPH oxidase 1 (Nox1) deficiency) and spontaneously develops a UC-like phenotype with similar complications (colorectal cancer) than UC. Our data identified an unanticipated combined role of IL10 and Nox1 in the fine-tuning of ER stress responses in goblet cells. As in humans, the ER stress was unbalanced in mice with decreased eIF2 alpha phosphorylation preceding inflammation. In IL10/Nox1(dKO) mice, salubrinal preserved eIF2 alpha phosphorylation through inhibition of the regulatory subunit of the protein phosphatase 1 PP1R15A/GADD34 and prevented colitis. Thus, this new experimental model highlighted the central role of epithelial ER stress abnormalities in the development of colitis and defined the defective eIF2 alpha pathway as a key pathophysiological target for UC. Therefore, specific regulators able to restore the defective eIF2 alpha pathway could lead to the molecular remission needed to treat UC