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

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

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 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

Glucose and insulin tolerance in WT and IL-6^−/− mice fed a HFD.

<p>Glucose curves and the quantified area under the curves for glucose (A, B) and insulin (C, D) tolerance tests. Data are presented as means +SE for 8–10 mice per group. * P<0.05 versus WT group at the same time point in A and C. * P<0.05 versus WT group in B and D.</p

Descriptive characteristics of WT and IL-6^−/− mice fed a high fat diet (60% Kcals from fat) for 10 weeks (N = 9–10/group).

<p>Descriptive characteristics of WT and IL-6^−/− mice fed a high fat diet (60% Kcals from fat) for 10 weeks (N = 9–10/group).</p

Mitochondrial content and respiration are not altered in epididymal adipose tissue from IL-6^−/− mice fed a HFD.

<p>A) Mitochondrial marker proteins, B) mtDNA content and C) mitochondrial respiration are similar between genotypes. Data are presented as means + SE for 8–10 animals per group. Western blot images are given above the quantified data in A. Relative mtDNA content is normalized to genomic MCT1 (monocarboxylate transporter 1) DNA content and expressed as fold differences compared to the WT group in B. Mitochondrial respiration was measured by high-resolution O2 consumption. GM (glutamate, malate), GMD (glutamate, malate, ADP), GMDPS (glutamate, malate, ADP, pyruvate, succinate).</p

IL-6 exerts depot specific differences in gene expression in mouse adipose tissue.

<p>IL-6 treatment (6 hours, 75 ng/ml) increased the expression of A) SOCS3, PGC-1α and PRC mRNA in cultured epididymal adipose tissue (eWAT). B) A 12 hr IL-6 (75 ng/ml) treatment increased the expression of COXIV and CPT-1 in cultured eWAT. C) Cultured subcutaneous adipose tissue (sWAT) was not responsive to IL-6 treatment (75 ng/ml, 6 hours) and this was associated with D) reductions in the protein content of IL-6 receptor alpha and GP130. Data are presented as means + SE for 7 cultures, each from an individual mouse, per group and is expressed relative to the vehicle treated control culture from the same animal. For the Western blot data in D) data are presented as means + SE for 8–10 mice per group. Representative Western blots are presented above the quantified data. * P<0.05.</p