Characterization of the rabbit intestinal fructose transporter (GLUT5).
Recent studies suggest that the jejunal/kidney-type facilitative glucose transporter (GLUT5) functions as a high-affinity D-fructose transporter. However, its precise role in the small intestine is not clear. In an attempt to identify the fructose transporter in the small intestine, we measured fructose uptake in Xenopus oocytes expressing jejunal mRNA from five species (rat, mouse, rabbit, hamster and guinea-pig). Only jejunal mRNA from the rabbit significantly increased fructose uptake. We also cloned a rabbit GLUT5 cDNA from a jejunal library The predicted amino acid sequence of the 487-residue rabbit GLUT5 showed 72.3 and 67.1% identity with human and rat GLUT5 respectively. Northern-blot analysis revealed GLUT5 transcripts in rabbit duodenum, jejunum and, to a lesser extent, kidney. After separation of rabbit jejunal mRNA on a sucrose density gradient, the fractions that conferred D-fructose transport activity in oocytes also hybridized with rabbit GLUT5 cDNA. Hybrid depletion of jejunal mRNA with a GLUT5 antisense oligonucleotide markedly inhibited the mRNA-induced fructose uptake in oocytes. Immunoblot analysis indicated that GLUT5 (49 kDa) is located in the brush-border membrane of rabbit intestinal epithelial cells. Xenopus oocytes injected with rabbit GLUT5 cRNA exhibited fructose uptake activity with a Km of 11 mM for D-fructose. D-Fructose transport by GLUT5 was significantly inhibited by D-glucose and D-galactose. D-Fructose uptake in brush-border membrane vesicles shows a Km similar to that of GLUT5, but was not inhibited by D-glucose or D-galactose. Finally, cytochalasin B photolabelled a 49 kDa protein in rabbit brush-border-membrane preparations that was immunoprecipitated by antibodies to GLUT5. Our results suggest that GLUT5 functions as a fructose transporter in rabbit small intestine. However, biochemical properties of fructose transport in Xenopus oocytes injected with GLUT5 cRNA differed from those in rabbit jejunal vesicles.
Department of Nutrition, School of Medicine, University of Tokushima, Japan.
K
K
Miyamoto
S
S
Tatsumi
A
A
Morimoto
H
H
Minami
H
H
Yamamoto
K
K
Sone
Y
Y
Taketani
Y
Y
Nakabou
T
T
Oka
E
E
Takeda
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D006224:Cricetinae; D003571:Cytochalasin B; D004247:DNA; D005260:Female; D005632:Fructose / Q000378:metabolism; D051296:Glucose Transporter Type 5; D006168:Guinea Pigs; D006801:Humans; D007583:Jejunum / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014018:Tissue Distribution; D014982:Xenopus laevis
D051296:Glucose Transporter Type 5; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D005632:Fructose; D003571:Cytochalasin B; D004247:DNA