Biochemical and functional characterization of the GLUT5 fructose transporter in rat skeletal muscle

Previous work has demonstrated that human skeletal muscle and adipose tissu e both express the GLUTS fructose transporter, but to date the issue of whe ther this protein is also expressed in skeletal muscle and adipose tissue o f rodents has remained unresolved. In the present study we have used a comb ination of biochemical and molecular approaches to ascertain whether rat sk eletal muscle expresses GLUTS protein and, if so, whether it possesses the capacity to transport fructose. An isoform-specific antibody against rat GL UTS reacted positively with crude membranes prepared from rat skeletal musc le. A single immunoreactive band of approx. 50 kDa was visualized on immuno blots which was lost when using anti-(rat GLUTS) serum that had been pre-ad sorbed with the antigenic peptide. Subcellular fractionation of skeletal mu scle 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 equivalen t molecular mass to that seen in muscle. Reverse transcription-PCR analyses , using rat-specific GLUTS primers, of muscle and jejunal RNA revealed a si ngle PCR fragment of the expected size in jejunum and in four different ske letal 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 unaffect ed. To determine whether fructose could regulate GLUTS expression in skelet al muscle, rats were maintained on a fructose-enriched diet for 4 days. Thi s procedure increased jejunal and renal GLUTS protein expression by approx. 4- and 2-fold respectively, but had no detectable effects on the abundance of GLUTS protein in skeletal muscle or on fructose uptake in rat adipocyte s. The present results show that GLUTS is expressed in the sarcolemma of ra t skeletal muscle and that it is likely to mediate fructose uptake in this tissue. Furthermore, unlike the situation in absorptive and re-absorptive e pithelia, GLUTS expression in insulin-sensitive tissues is not regulated by increased substrate supply.