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Characterization of the rabbit intestinal fructose transporter (GLUT5).

ABSTRACT: 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.

SUBMITTER: Miyamoto K

PROVIDER: S-EPMC1137628 | biostudies-other | 1994 Nov

REPOSITORIES: biostudies-other

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Project description:Previous work has demonstrated that human skeletal muscle and adipose tissue both express the GLUT5 fructose transporter, but to date the issue of whether this protein is also expressed in skeletal muscle and adipose tissue of rodents has remained unresolved. In the present study we have used a combination of biochemical and molecular approaches to ascertain whether rat skeletal muscle expresses GLUT5 protein and, if so, whether it possesses the capacity to transport fructose. An isoform-specific antibody against rat GLUT5 reacted positively with crude membranes prepared from rat skeletal muscle. A single immunoreactive band of approx. 50 kDa was visualized on immunoblots which was lost when using anti-(rat GLUT5) serum that had been pre-adsorbed with the antigenic peptide. Subcellular fractionation of skeletal muscle localized this immunoreactivity to a single membrane fraction that was enriched with sarcolemma. Plasma membranes, but not low-density microsomes, from rat adipose tissue also displayed a single protein band of equivalent molecular mass to that seen in muscle. Reverse transcription-PCR analyses, using rat-specific GLUT5 primers, of muscle and jejunal RNA revealed a single PCR fragment of the expected size in jejunum and in four different skeletal muscle types. Sarcolemmal vesicles from rat muscle displayed fructose and glucose uptake. Vesicular uptake of glucose was inhibited by nearly 90% in the presence of cytochalasin B, whereas that of fructose was unaffected. To determine whether fructose could regulate GLUT5 expression in skeletal muscle, rats were maintained on a fructose-enriched diet for 4 days. This procedure increased jejunal and renal GLUT5 protein expression by approx. 4- and 2-fold respectively, but had no detectable effects on the abundance of GLUT5 protein in skeletal muscle or on fructose uptake in rat adipocytes. The present results show that GLUT5 is expressed in the sarcolemma of rat skeletal muscle and that it is likely to mediate fructose uptake in this tissue. Furthermore, unlike the situation in absorptive and re-absorptive epithelia, GLUT5 expression in insulin-sensitive tissues is not regulated by increased substrate supply.

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Characterization of the rabbit intestinal fructose transporter (GLUT5).

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