Biochemical and functional characterization of the GLUT5 fructose transporter in rat skeletal muscle.
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.
Darakhshan
F
F
Department of Anatomy & Physiology, The University of Dundee, Dundee DD1 4HN, Scotland, U.K.
Hajduch
E
E
Kristiansen
S
S
Richter
E A
EA
Hundal
H S
HS
D000818:Animals; D005632:Fructose / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D051296:Glucose Transporter Type 5; D007583:Jejunum / Q000378:metabolism; D007668:Kidney; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D012313:RNA; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012508:Sarcolemma / Q000378:metabolism; D013347:Subcellular Fractions; D014018:Tissue Distribution
D051296:Glucose Transporter Type 5; D009004:Monosaccharide Transport Proteins; D005632:Fructose; D012313:RNA