Characterization of Substrate Uptake by Avian Skeletal Muscle

The goal of this work was to characterize avian skeletal muscle (SKM) glucose and fatty acid uptake. English sparrows (Passer domesticus) were used for the following studies: 1. Characterization of glucose uptake, 2. Identification and localization of glucose transporters, 3. Characterization of free fatty acid uptake, and 4. Reciprocal inhibition of glucose and free fatty acids. The results are summarized as follows. Isolated SKM incubated for 60 minutes with insulin, IGF-1, caffeine or AICAR demonstrated no increase in glucose transport. Interestingly, uptake was decreased in the presence of incremental unlabeled glucose suggesting the presence of glucose transporters (GLUT) and by phloretin, an inhibitor of transport proteins, decreased transport. The SKM glycogen content was low, which is supportive of the observed minimal glucose uptake. These findings suggest that GLUT expression may differ in birds as compared to mammals. GLUT1 and GLUT3 gene expression, but not GLUT4, were found in all tissues examined and share a high degree of homology with published chicken sequences. In addition, GLUT3 and GLUT4 proteins were not detected, whereas GLUT1 protein was abundant in blood-tissue barriers. Sparrows have high plasma ketone body levels suggestive of a high rate of free fatty acid (FFA) oxidation. In vitro uptake of radiolabeled oleic acid (OA) was maximal at 60 minutes and competitively inhibited by unlabeled OA suggesting a facilitative process. Radiolabeled OA uptake was not increased by IGF-1, caffeine and AICAR, whereas insulin increased uptake at 60 minutes. Inhibitors of protein-mediated substrate transport increased OA uptake by 60 minutes (DIDS and phloretin) whereas a specific inhibitor of long chain FFA transport, sulfo-N-succinimidyl oleate, decreased its uptake at 2.5 min. In reciprocal inhibition studies, 20mM unlabeled glucose and OA inhibited the uptake of their radiolabeled counterparts. Glucose (20mM) significantly decreased labeled OA uptake 36% and 20mM OA significantly decreased labeled glucose transport by 49%. These data begin to elucidate why avian skeletal muscle may not take up glucose to an appreciable extent and further, why avian skeletal muscle is insulin resistant