Representative Western blots on isolated subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria and assessment of mitochondrial respiratory coupling.

<p>Isolated mitochondria display an absence of MCT1, Cav1 and SERCA2, but the presence of COXIV, suggesting the preparation recovered mitochondria devoid of plasma membrane and sarcoplasmic reticulum contamination (A). Mitochondria were also utilized to assess coupling efficiencies as an index of mitochondrial damage. Specifically, the P/O ratios and RCR (reported in B) values suggest mitochondria were coupled, and therefore not damaged during the isolation procedure (B). Values are reported as the means ± SEM (n = 4).</p

Effect of acute AICAR administration on mitochondrial FAT/CD36 content.

<p>An intraperitoneal injection of AICAR decreased blood glucose (A) and increased skeletal muscle AMPK Thr172 phosphorylation without altering total AMPK protein content (B). The FAT/CD36 antibody in mice is polyclonal, and therefore we confirmed that an ~88 kDa band was present in giant sarcolemmal vesicle (GSV) and mitochondrial samples from FAT/CD36 WT mice and absent in mitochondria isolated from FAT/CD36 knock out (KO) mice. A representative blot is provided (C). AICAR increased FAT/CD36 protein content in subsarcolemmal (SS) but not in intermyofibrillar (IMF) mitochondria. COXIV was utilized as a loading control. Muscle was taken 1 hour after the intraperitoneal injection of AICAR. Values are reported as the means ± SEM (n = 6). * significantly (P<0.05) different from saline.</p

Effects of acute treadmill running on FAT/CD36 mitochondrial content in WT and AMPKα2 KD mice.

<p>ACC Ser79 phosphorylation was examined in whole muscle (A), while subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria were isolated from WT (B) and AMPKα2 KD (C) mice run at 12m/min at a 5% grade for an hour or from sedentary controls. Mitochondria were utilized to determine FAT/CD36 protein content, while COXIV was utilized as a loading control. Values are reported as the means ± SEM (n = 4 for ACC Westerns, n = 8 for mitochondrial Westerns). *significantly (P<0.05) different from control.</p

Effects of AICAR and acute treadmill running on ERK1/2 phosphorylation.

<p>Treadmill running for 30 minutes increased ERK1/2 Thr202/Tyr204 phosphorylation in an intensity-dependent manner in WT mice (A). ERK1/2 Thr202/Tyr204 phosphorylation increased 30 minutes following an intraperitoneal injection of AICAR in the absence of alterations in total protein content in WT mice (B). Treadmill running for 30 minutes increased ERK1/2 Thr202/Tyr204 phosphorylation in AMPKα2 KD mice (C). Values are reported as the means ± SEM (n = 5). * significantly (P<0.05) different from control (saline or sedentary) and † significantly (P<0.05) different from low-intensity running.</p

Effects of acute treadmill running to exhaustion on FAT/CD36 mitochondrial content in WT mice.

<p>Subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria were isolated from WT (A) mice run at 25 m/min at a 5% grade for an hour or from sedentary controls. Mitochondria were utilized to determine FAT/CD36 protein content, while COXIV was utilized as a loading control. Values are reported as the means ± SEM (n = 6). *significantly (P<0.05) different from control.</p

Temporal response of muscle contraction-induced FAT/CD36 accumulation on sarcolemmal, subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial membranes.

<p>Sciatic nerve stimulation was used to determine FAT/CD36 accumulation, while plasma membrane fatty acid binding protein (FABPpm), which does not respond to contraction, was utilized as a control. While FAT/CD36 accumulated on the plasma membrane after 7.5 minutes, SS and IMF mitochondrial FAT/CD36 protein was not increased until 22.5 minutes. Values are reported as the means ± SEM (n = 7). * significantly (P<0.05) different from the contralateral control muscles (ie. time 0), † significantly (P<0.05) different from 7.5 minutes of stimulation, ‡ significantly (P<0.05) different from 15 minutes of stimulation, and ¥ significantly (P<0.05) different from 22.5 minutes of stimulation.</p

Transport rate of glucose across sarcolemmal vesicles.

<p>Data displayed as mean and standard deviation. Untrained Rested n = 4, Untrained Exercised n = 3, Trained Rested n = 3, Trained Exercised n = 4, Run Exercised n = 6. a: significantly different from Untrained Rested, p = 0.0029; b: significantly different from Untrained Exercised, p = 0.035; c: significantly different from Trained Rested, p = 0.029.</p

Plasma membraneGLUT4 abundance.

<p>Transporter was quantified using Western blot, and abundance is expressed as arbitrary optical density units. Data displayed as mean and standard deviation. Untrained Rested n = 6, Untrained Exercised n = 8, Trained Rested n = 7, Trained Exercised n = 8, Run Exercised n = 7. a: significantly different from Untrained Rested, p = 0.015; b: significantly different from Untrained Exercised, p = 0.0025.</p

FAT/CD36 abundance in whole muscle homogenates.

<p>Transporter was quantified using Western blot, and abundance is expressed as arbitrary optical density units. Data displayed as mean and standard deviation. Untrained Rested n = 5, Untrained Exercised n = 8, Trained Rested n = 8, Trained Exercised n = 8, Run Exercised n = 7.</p

Plasma membraneFAT/CD36 abundance.

<p>Transporter was quantified using Western blot, and abundance is expressed as arbitrary optical density units. Data displayed as mean and standard deviation. Untrained Rested n = 6, Untrained Exercised n = 8, Trained Rested n = 6, Trained Exercised n = 8, Run Exercised n = 7. a: significantly different from Untrained Rested, p = 0.030; b: significantly different from Untrained Exercised, p = 0.0048.</p