The effects of insulin stimulation and muscle contraction on the subcellular distribution of GLUT4 in skeletal muscle have been studied on a preparation of single whole fibers from the rat soleus. The fibers were labeled for GLUT4 by a preembedding technique and observed as whole mounts by immunofluorescence microscopy, or after sectioning, by immunogold electron miscroscopy. The results, supported by subcellular fractionation experiments, suggest that TfR-positive depots are only recruited by contractions. We do not find evidence for stimulation-induced unmasking of resident surface membrane GLUT4 transporters or for dilation of the T tubule system. Previous studies have indicated a role for calmodulin in hypoxia- and insulin-stimulated glucose transport. However, since calmodulin interacts with multiple protein targets, it is unknown which of these targets is involved in the regulation of glucose transport. We have used the calcium-dependent calmodulin protein kinase II (CAMKII) inhibitor 1-[N,O-bis (5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4- phenyl- piperazine (KN-62) to investigate the possible role of this enzyme in the regulation of glucose transport in isolated rate soleus and epitrochlearis muscles. The results of the study suggest that CAMKII might have a distict role in insulin- and hypoxia-stimulated glucose transport, possibly in the vesicular trafficking of GLUT4. We have previously reported that exercise training is associated with enhanced insulin-stimulated glucose transport activity and inhibited hypoxia-stimulated glucose transport activity in rat epitrochlearis muscle. We have examined the potential role of muscle glycogen in the inhibited glucose transport response to hypoxia. The findings demonstrate a strong inverse relationship between glycogen and hypoxia- stimulated glucose transport activity, and that high levels of glycogen contribute to the inhibited glucose transport response of hypoxia. Furthermore, failure of the over expression of GLUT4 after exercise training to enhance the glucose transport response to contraction/ hypoxia suggests selective targeting of the additional GLUT4 to the insulin-responsive pool. The effects of detraining on the regulation of glucose transport in skeletal muscle have been examined after short-term swim training (5 days), long-term swim training (5 wk), and treadmill run training (5 wk). The results demonstrate that increases in GLUT4 mRNA and GLUT4 protein occur during the first week of exercise training and are rapidly lost after training cessation. We believe that the transient enhancement in GLUT4 protein after exercise training is due to a short GLUT4 half-life, a process that is primarily regulated by pretranslational mechanisms. Reduced calorie intake [calorie restriction (CR); 60% of ad libitum (AL)] leads to enhanced glucose transport without altering total GLUT4 glucose transporter abundance in skeletal muscle. Therefore, we tested the hypothesis that CR (20 days) alters the subcellular distribution of GLUT4. The data demonstrate that, despite IRS-1-associated PI3K activity similar to AL, CR specifically increases insulin's activation of glucose transport by enhancing the steady-state proportion of GLUT4 residing on the cell surface.
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