There is evidence of differences in the insulin signaling pathway that may explain the low glycogen content in these muscles. Extraocular muscles are the effector arm of the ocular motor system and are involved in all voluntary and reflexive eye movements.1,2The functional demands of extraocular muscles are different from those imposed on other skeletal muscles, leading to unique metabolic adaptations as demonstrated by gene expression profiling and by biochemical and functional studies.38For example, extraocular muscles seem to have relatively low adenosine triphosphate (ATP)buffering capacity, evidenced by decreased reliance on glycogenolysis and less creatine kinase activity.912Moreover, extraocular muscles can reduce lactate to pyruvate for entry into the Krebs cycle to sustain their high activity demand.9,11,13Another Fosfomycin calcium potential mechanism for enhancing ATP production in the extraocular muscles is enhanced glucose uptake by the muscle fibers, priming glycolysis and then mitochondrial energy pathways. In limb skeletal muscles, glucose enters muscle fibers by way of specific membrane transporters (GLUT1 and GLUT4) in a process influenced by insulin and contractile activity to match metabolic supply to demand.14GLUT1 transporters mediate insulin- and activity-independent basal glucose uptake. activity. == Methods. == Extraocular muscles from adult male Sprague-Dawley rats were incubated with 100 nM insulin or were electrically stimulated to contract (activity); glucose uptake was measured with 2-deoxy-d[1,2-3H]glucose. The contents of GLUT1, GLUT4, total and phosphorylated protein kinase B (Akt), phosphorylated AMP-activated protein kinase (AMPK), and glycogen synthase kinase 3 (GSK3) underwent Western blot analysis. == Results. == Insulin and activity increased glucose uptake over the basal rate to 108% and 78%, respectively. GLUT1 and GLUT4 were detectable in extraocular muscles. Phosphorylated AKT/total AKT increased by twofold after insulin stimulation, but there was no change with activity. AMPK phosphorylation increased 35% with activity. Phosphorylated-GSK3/total GSK3 did not change Fosfomycin calcium with insulin or activity. == Conclusions. == Glucose uptake in extraocular muscles is regulated by insulin and contractile activity. There is evidence of differences in the insulin signaling pathway that may explain the low glycogen content in these muscles. Extraocular muscles are the effector arm of the ocular motor system and are involved in all voluntary and reflexive eye movements.1,2The functional demands of extraocular muscles are different from those imposed on other skeletal muscles, leading to unique metabolic adaptations as demonstrated by gene expression profiling and by biochemical and functional studies.38For example, extraocular muscles seem to have relatively low adenosine triphosphate (ATP)buffering capacity, evidenced by decreased reliance on glycogenolysis and less creatine kinase activity.912Moreover, extraocular muscles can reduce lactate to pyruvate for entry into the Krebs cycle to sustain their high activity demand.9,11,13Another potential RLPK mechanism for enhancing ATP production in the extraocular muscles is enhanced glucose uptake by the muscle fibers, priming glycolysis and then mitochondrial energy pathways. In limb skeletal muscles, glucose enters muscle fibers by way of specific membrane transporters (GLUT1 and GLUT4) in a process influenced by insulin and contractile activity to match metabolic supply to demand.14GLUT1 transporters mediate insulin- and activity-independent basal glucose uptake. On binding to its membrane receptors, insulin triggers a signaling cascade that activates protein kinase B (AKT), which in turn initiates the translocation of GLUT4-containing vesicles to the membrane, increasing glucose uptake over the basal rate.15,16At the same time, glycogen synthase kinase 3 (GSK3) is activated and phosphorylates glycogen synthase, stimulating glycogen production.17Contractile activity has a similar effect on glucose uptake: it induces the translocation of a separate pool of GLUT4-containing vesicles to the membrane through activation of the adenosine monophosphate (AMP)activated kinase (AMPK) pathway.18,19Given their functional characteristics, extraocular muscles may have very high basal glucose uptake rates, negating the need for further regulation by insulin or contractile activity. Therefore, we tested the hypothesis that glucose uptake into extraocular muscles is not influenced by insulin or contractile activity as in other skeletal muscles. == Materials and Methods == == Materials == All reagents were obtained from Sigma Aldrich (St. Louis, MO) unless otherwise stated. 2D-3H glucose and14C-mannitol were obtained from Perkin Elmer (Boston, MA). SDS-polyacrylamide gels and other electrophoresis-related materials were purchased from Bio-Rad (Hercules, CA). Polyvinylidene difluoride (PVDF) membranes were obtained from Immobilon-FL, Millipore (Billerica MA). Protease and phosphatase inhibitor cocktail (100) was purchased from Thermo Scientific (Halt; Thermo Scientific, Rockford, IL). Rabbit polyclonal GLUT1 and GLUT4 antibodies were purchased from Abcam (Cambridge, MA). Total AKT and phosphorylated-AKT (Ser473) antibodies and phosphorylated AMPK (Thr172) were obtained from Cell Signaling (Danvers, MA). Total GSK-3 antibody was purchased from BD Bioscience (San Jose, CA), whereas antibody against phosphorylated-GSK-3/ (Ser21/9) was obtained from Cell Signaling. Secondary fluorescence-labeled antibodies were obtained from Invitrogen (Carlsbad, CA). == Animals == The use of experimental animals was approved by the Institutional Animal Care and Use Committee at the University of Kentucky and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Twenty-four adult male Sprague-Dawley rats (300500 g; Harlan, Indianapolis, IN) were euthanatized by CO2asphyxia followed by pneumothorax. == In Vitro Glucose Uptake == Whole orbits, including the bony posterior wall, were quickly excised from 12 rats immediately after euthanatization and then immersed in Krebs-Ringer’s bicarbonate buffer (117 mM NaCl, 4.7 mM KCl, 24.6 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM CaCl2, 2.5 mM MgSO4) bubbled with 95% O2and 5% CO2. The orbits were carefully dissected Fosfomycin calcium to leave the intact extraocular muscles (four rectus and superior oblique muscles) attached to a small bone fragment and a ring of sclera and then tied to a glass rod. Extraocular muscles from one side were used to measure.