Prijenosnik glukoze ovisan o natriju 1 (SGLT1) prenosi glukozu kroz četkastu membranu u tankom crijevu, dok je za izlazak glukoze iz enterocita na bazolateralnoj membrani odgovoran prijenosnik glukoze neovisan o natriju 2 (GLUT2). Natrij-kalijeva crpka (Na/K-ATPaza), smještena na bazolateralnoj membrani, aktivno izbacuje iz stanice višak natrijevih iona apsorbiranih zajedno s glukozom. Izraženost i raspodjela Na/K-ATPaze, SGLT1 i GLUT2 u tankom crijevu opisana je većinom u laboratorijskih glodavaca, dok podataka za svinje manjka. Spolne razlike u izraženosti Na/K-ATPaze, SGLT1 i GLUT2 do sada se nisu uspjele utvrditi u probavnom sustavu laboratorijskih životinja. Istraživanje je provedeno na 20 svinja pasmine švedski landras podijeljenih u četiri skupine: mužjaci, ženke, kastrirani mužjaci i kastrirane ženke. Uzorci tkiva uzeti od pojedinih dijelova tankog crijeva, duodenuma, jejunumu i ileuma, analizirani su imunohistokemijskom metodom, western-analizom i lančanom reakcijom polimeraze. Potvrđen je opisani stanični smještaj za Na/K-ATPazu i GLUT2 na bazolateralnim membranama te za SGLT1 na četkastim membranama enterocita. Izraženost Na/K-ATPaze na razini proteina pokazuje spolne razlike (mužjaci ˂ ženke), dok postojanje spolnih razlika na razini mRNK nije dokazano. Izraženost SGLT1 na razini proteina ne pokazuje spolne razlike, dok je postojanje spolnih razlika uočeno na razini mRNK (mužjaci ˃ ženke). Izraženost GLUT2 na razini proteina, slično kao i Na/K-ATPaza pokazuje spolne razlike (mužjaci ˂ ženke), dok je postojanje spolnih razlika na razini mRNK uočeno samo u ileumu. Uočene spolne razlike u izraženosti proteina Na/K-ATPaze i GLUT2 nastaju zbog inhibitornog učinka muških spolnih hormona na posttranskripcijskoj razini u tankom crijevu svinje, dok SGLT1 pokazuje drugačije molekularne značajke, bez spolnih razlika u izraženosti. Izraženost funkcionalno povezane Na/K-ATPaze i SGLT1 raste od duodenuma prema ileumu, dok GLUT2 ima najvišu izraženost u jejunumu. Ovakva raspodjela prijenosnika uključenih u prijenos glukoze upućuje na to da se većina glukoze apsorbira u jejunumu i ileumu svinja.
Sodium-glucose cotransporter 1 (SGLT1) transfers glucose across the brush border membrane of the small intestine, while the basolateral sodium-independent glucose transporter 2 (GLUT2) transfers the glucose out of the enterocyte. Sodium-potassium pump (Na/K-ATPase), located on the basolateral membrane, actively transfers excessive sodium ions out of the cell co-absorbed with glucose. The expression and distribution of Na/K-ATPase, SGLT1 and GLUT2 in the small intestine were predominantly investigated in laboratory rodents, while data for pigs are still lacking. Although gender differences in the expression of Na/K-ATPase, SGLT1 and GLUT2 were described in kidneys, lungs and brain of laboratory rodents, so far there are no reports of differences in the digestive system. As the anatomy and physiology of the porcine digestive system is similar to the humans’, the porcine model gains on importance. The aim of this study is to investigate gender differences in the expression, effect of castration and distribution of Na/K-ATPase, SGLT1 and GLUT2 in the small intestine of pigs.
MATERIAL AND METHODS
The study was conducted on 20 pigs Swedish Landrace breed divided into 4 groups (males, females, castrated males, castrated females) with 5 animals in each group. At the age of 10 months the pigs were slaughtered for commercial use and tissue samples of small intestine parts, duodenum, jejunum and ileum, were taken and analyzed by immunostaining of tissue cryosections, Western blotting and polymerase chain reaction (PCR). Tissue cyosections went first through the antigen retrieval technique to maximize the antibody-binding sites. Immunostaining following these procedures included incubation of cryosections in specific primary Na/K-ATPase, SGLT1 and GLUT2 antibodies and matching secondary antibodies.
The stained sections were examined and photographed by fluorescence microscope. Isolation of total cell membranes (TCM) by differential centrifugation method and measurement of TCM proteins by the Bradford assay was performed prior Western blotting. Proteins of the prepared samples of TCM were first separated by electrophoresis and then electrophoretically wet-transferred to a blotting membrane. The blotting membranes were incubated with specific primary Na/K-ATPase, SGLT1 and GLUT2 antibodies and matching secondary antibodies. The protein bands on the blotting membrane were visualized by colorimetric assay and the protein bands were densitometrically measured. Total cellular RNA was isolated from the tissue samples. First-strand cDNA synthesis was performed by reverse transcription of RNA. PCR was performed using first-strand cDNA and specific Na/K-ATPase, SGLT1 and GLUT2 primers. PCR products were detected in agarose gel and the obtained bands were densitometrically measured.
The previous described cellular localization for Na/K-ATPase and GLUT2 on basolateral membranes and for SGLT1 on the brush-border membrane of the enterocytes were confirmed. The expression of Na/K-ATPase at the protein level shows gender differences (males ˂ females), while the existence of gender differences at the mRNA level was not detected. The expression of SGLT1 at the protein level does not show gender differences, while the existence of gender differences is observed at mRNA level (male ˃ females). The expression of GLUT2 at the protein level, similar to Na/K-ATPase, shows gender differences (males ˂ females), whereas the existence of gender differences at mRNA level was observed only in ileum. The distribution of functionally related transporters Na/K-ATPase and SGLT1 showed an increase in expression from the duodenum to the ileum, while GLUT2 had the highest expression in jejunum and the lowest in duodenum of the pig.
Results of this study showed that the observed gender differences in expression of Na/K-ATPase and GLUT2 at the protein level are due to the inhibitory effect of male sex hormones
at posttranscriptional level of gene regulation in the small intestine of the pig, whereas SGLT1 shows different molecular characteristics without gender differences in expression at the protein level. The observed distribution of transporters involved in glucose transport along the small intestine indicates that jejunum and ileum are the main site of glucose absorption in the small intestine of the pig.