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

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)

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

Matrix metalloproteinase-10 effectively reduces infarct size in experimental stroke by enhancing fibrinolysis via a thrombin-activatable fibrinolysis inhibitor-mediated mechanism

BACKGROUND: The fibrinolytic and matrix metalloproteinase (MMP) systems cooperate in thrombus dissolution and extracellular matrix proteolysis. The plasminogen/plasmin system activates MMPs, and some MMPs have been involved in the dissolution of fibrin by targeting fibrin(ogen) directly or by collaborating with plasmin. MMP-10 has been implicated in inflammatory/thrombotic processes and vascular integrity, but whether MMP-10 could have a profibrinolytic effect and represent a promising thrombolytic agent is unknown. METHODS AND RESULTS: The effect of MMP-10 on fibrinolysis was studied in vitro and in vivo, in MMP-10-null mice (Mmp10(-/-)), with the use of 2 different murine models of arterial thrombosis: laser-induced carotid injury and ischemic stroke. In vitro, we showed that MMP-10 was capable of enhancing tissue plasminogen activator-induced fibrinolysis via a thrombin-activatable fibrinolysis inhibitor inactivation-mediated mechanism. In vivo, delayed fibrinolysis observed after photochemical carotid injury in Mmp10(-/-) mice was reversed by active recombinant human MMP-10. In a thrombin-induced stroke model, the reperfusion and the infarct size in sham or tissue plasminogen activator-treated animals were severely impaired in Mmp10(-/-) mice. In this model, administration of active MMP-10 to wild-type animals significantly reduced blood reperfusion time and infarct size to the same extent as tissue plasminogen activator and was associated with shorter bleeding time and no intracranial hemorrhage. This effect was not observed in thrombin-activatable fibrinolysis inhibitor-deficient mice, suggesting thrombin-activatable fibrinolysis inhibitor inactivation as one of the mechanisms involved in the MMP-10 profibrinolytic effect. CONCLUSIONS: A novel profibrinolytic role for MMP-10 in experimental ischemic stroke is described, opening new pathways for innovative fibrinolytic strategies in arterial thrombosis

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

Leptin regulation of intestinal amino acids absorption: in vitro and in vivo studies

Hormone regulation of digestive secretions and gastrointestinal tract motility is well established, nevertheless, little is known about endocrine regulation of nutrients intestinal absorption and information on this regard is not included in the text books yet. For example, serotonin and CCK inhibit sugar absorption (Arruebo et al., 1989, Hirsh et al., 1996; Barber et al., 1997) whereas glucagon-37 shows a stimulatory effect (Stümpel et al., 1998). Serotonin also decreases leucine absorption (Salvador et al., 1996). Shortly after leptin identification, we demonstrated that leptin inhibits sugar absorption in rat intestine in vitro by short-term regulation of the Na+/glucose cotransporter SGLT1 (Lostao et al. 1998). In the same year, another group reported that the stomach chief cells secrete leptin into the gastric lumen after a meal (Bado et al. 1998). We later described that leptin receptors are expressed in both apical and basolateral membrane of human and murine enterocytes (Barrenetxe et al. 2002). In the present work, we decided to extend our studies and investigate the possible effect of leptin on amino acids intestinal absorption in rat and in Caco-2 cells, using in vivo and in vitro techniques. In summary, leptin acting from the apical membrane of the enterocytes modulates, in a short-term manner in vivo and in vitro, amino acids transport in rat intestine and human Caco-2 cells. The mechanisms implicated include direct effects on transporters expression in the apical membrane and indirect processes that leads to modulation of the activity of the implicated transporters. Leptin also rapidly inhibits amino acids uptake acting from the basolateral membrane. In both conditions, the inhibition is reversible. Interestingly, the activity of Na+-independent glutamate transporters was increased by leptin. Taken together, these results contribute to the vision of leptin as an important hormonal signal for the regulation of intestinal absorption of nutrients

Leptin regulation of intestinal amino acids absorption: in vitro and in vivo studies

Hormone regulation of digestive secretions and gastrointestinal tract motility is well established, nevertheless, little is known about endocrine regulation of nutrients intestinal absorption and information on this regard is not included in the text books yet. For example, serotonin and CCK inhibit sugar absorption (Arruebo et al., 1989, Hirsh et al., 1996; Barber et al., 1997) whereas glucagon-37 shows a stimulatory effect (Stümpel et al., 1998). Serotonin also decreases leucine absorption (Salvador et al., 1996). Shortly after leptin identification, we demonstrated that leptin inhibits sugar absorption in rat intestine in vitro by short-term regulation of the Na+/glucose cotransporter SGLT1 (Lostao et al. 1998). In the same year, another group reported that the stomach chief cells secrete leptin into the gastric lumen after a meal (Bado et al. 1998). We later described that leptin receptors are expressed in both apical and basolateral membrane of human and murine enterocytes (Barrenetxe et al. 2002). In the present work, we decided to extend our studies and investigate the possible effect of leptin on amino acids intestinal absorption in rat and in Caco-2 cells, using in vivo and in vitro techniques. In summary, leptin acting from the apical membrane of the enterocytes modulates, in a short-term manner in vivo and in vitro, amino acids transport in rat intestine and human Caco-2 cells. The mechanisms implicated include direct effects on transporters expression in the apical membrane and indirect processes that leads to modulation of the activity of the implicated transporters. Leptin also rapidly inhibits amino acids uptake acting from the basolateral membrane. In both conditions, the inhibition is reversible. Interestingly, the activity of Na+-independent glutamate transporters was increased by leptin. Taken together, these results contribute to the vision of leptin as an important hormonal signal for the regulation of intestinal absorption of nutrients

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

12 Pág. Departamento de Tecnología de Alimentos​​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.This research was funded by the Government of Spain grant number (AGL 2006-1029-C02-01/ALI), CIBER (grant number: CB12/03/30002), and the Department of Innovation, Business and Employment of the Government of Navarra.Peer reviewe