Fachbereich Veterinärmedizin



    Funktionelle und molekularbiologische Untersuchungen zum Glukosetransport am proximalen Jejunum des Schweins (2014)

    Schenke, Kristina (WE 2)
    — VI, 110 Seiten
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000097914
    Institut für Veterinär-Physiologie

    Oertzenweg 19 b
    14163 Berlin
    Tel.+49 30 838 62600 Fax.+49 30 838-62610

    Abstract / Zusammenfassung

    Pigs cover their energy demand with meals rich in starch and carbohydrates. That requires highly efficient mechanism of glucose absorption in the small intestine. The aim of the present studies was to characterize glucose uptake at higher luminal concentrations (6-20 mM) in proximal jejunum of pig and to undertake a systematic screening for the presence of mRNA of glucose transport proteins in main organs of glucose absorption, production and conservation. In the glucose uptake studies, apical uptake of radioactively labeled glucose into the epithelial layer of the porcine small intestine was measured. Furthermore, transepithelial short-circuit current and transepithelial conductance were measured by using the Ussing chamber technique. Possible interactions between glucose absorption and other metabolic activities like glycolysis or gluconeogenesis were investigated, as well as the possible involvement of endocytosis in glucose absorption. With comparative studies on uptake und flux of radioactively labeled mannitol, the involvement of paracellular permeation was examined. Finally, the presence of mRNA of glucose transport proteins in main organs of glucose homeostasis like proximal jejunum, liver, kidney and skeletal muscle were investigated via two-step RT-PCR. The competitive SGLT1 inhibitor phlorizin (0.1 mM), as well as GLUT inhibitors phloretin (0.2 mM) and cytochalasin B (0.2 mM), caused significant inhibition of glucose absorption into the epithelium (at 10 mM glucose concentration). 10 or 20 minutes after preincubation with glucose (10 mM), none of these inhibitors achieved a significant inhibition of glucose absorption. The increase of short-circuit current after mucosal glucose addition (10 mM) allowed to calculate the transport capacity of SGLT1 (approx. 7.5 nmol·cm-2·min-1). The inhibiting effect of phlorizin on glucose uptake was multiple times greater. Chlorpromazin (20 μM), an inhibitor of clathrin-mediated endocytosis, caused disappearance of phlorizin and phloretin inhibited glucose uptake, while the increase of short-circuit current after glucose addition was still evident. Serosally administered phloretin (0.2 mM), as well as bilaterally administered N-acetyl-D-glucosamine (20 mM), a hexokinase inhibitor, led at high glucose concentrations (20 mM) to reduced glucose uptake into the epithelium. After more than 3 hours pre-incubation time with glucose (6 mM) and prevention of intracellular lactate accumulation by using a lactate-free buffer and simultaneos inhibition of lactate dehydrogenase by Na-oxamate (20 mM), phlorizin (0.1 mM) still inhibitied glucose uptake, but phloretin (0.2 mM) did not. After more than 3 hours pre-incubation time with glucose (6 mM), avoiding gluconeogenesis from lactate by inhibition of the gluconeogenesis keyenzyme PEPCK via 3-mercaptopicolinic acid (0.2 mM), phlorizin (0.1 mM) but not phloretin (0.2 mM) inhibited glucose uptake, too. Over a period of 20 minutes and in presence of phlorizin (0.5 mM) and phloretin (0.5 mM) glucose and mannitol were taken up into the epithelium at the ratio of 1:0.65 at identical concentrations (20 mM), while the fluxes showed a ratio of 1:1. Fluxes of glucose and mannitol, but not uptakes correlated with transepithelial conductance. The jejunum contained mRNA for SGLT1, GLUT1, GLUT2, GLUT5, GLUT7 and GLUT8, while GLUT3, 4, 10 und 11 were also detectable. The liver contained SGLT1, GLUT1, GLUT2 und GLUT8 mRNA, while GLUT3, 4, 5, 10 and 11 were poorly detectable. The kidney was positive for mRNA of SGLT1, GLUT1, GLUT2, GLUT5, GLUT8 and GLUT11, but traces of GLUT3, 4 and 10 mRNA could also be detected. Skeletal muscle had the strongest signal for GLUT4 mRNA, while GLUT1, 3, 5, 8, 10 and 11 showed weak signals. Altogether, new porcine gene sequences of 3 glucose transport proteins were identified. The SGLT1 and an apical GLUT, probably GLUT2 (Kellett hypothesis) are involved in glucose uptake in porcine jejunal epithelium. SGLT1 induces the apical GLUT. Furthermore intact assembly of clathrin-coated vesicles seems to be necessary for apical insertion of GLUT2. GLUT2 facilitates intracellular glucose accumulation a few minutes after glucose addition and then becomes inactive. Intracellular glucose accumulation by gluconeogenesis or reduced basolateral glucose exit decreases apical glucose uptake. Endocytotic apical glucose and mannitol uptake was not approved and parazellular absorption played a minor role. There are hints for an additional transport mechanism, which is able to transport mannitol in addition to glucose. In main organs of glucose homeostasis like proximal jejunum, liver, kidney and skeletal muscle, mRNA of different glucose transport proteins are expressed with redundancy.