Site-specific acetylation of adenine nucleotide translocase 1 at lysine 23 in human muscle.

Evidence suggests acetylation of human adenine nucleotide translocase 1 (ANT1) at lysine 23 (Lys23) reduces binding of ADP. Lys23 contributes to the positive charge that facilitates this interaction. This study was undertaken to characterize ANT1 abundance and acetylation by a novel method using small amounts of human skeletal muscle biopsies. Lysates of whole muscle or mitochondria from the same tissue were prepared from needle biopsies of vastus lateralis muscle of healthy volunteers. Lysed proteins were resolved on gels, the section containing ANT1 (surrounding 30 Kd) was excised, digested with trypsin, spiked with labeled unacetylated and acetylated synthetic standard peptides and analyzed by mass spectrometry. Natural logarithm transformation of data linearized ion intensities over a 10-fold range of peptide mass. Coefficients of variation ranged from 7 to 30% for ANT1 abundance and Lys23 acetylation. In three volunteers, ANT1 content was 8.36 ± 0.33 nmol/g wet weight muscle and 0.64 ± 0.05 nmol/mg mitochondria, so mitochondrial content was 13.3 ± 2.4 mg mitochondria per gram muscle. Acetylation of Lys23 averaged 14.3 ± 4.2% and 4.87 ± 1.84% in whole muscle and mitochondria, respectively. This assay makes it possible to assess effects of acetylation on the function of ANT1 in human muscle.

Fuel Selection in Skeletal Muscle Exercising at Low Intensity; Reliance on Carbohydrate in Very Sedentary Individuals.

Background: Resting skeletal muscle in insulin resistance prefers to oxidize carbohydrate rather than lipid, exhibiting metabolic inflexibility. Although this is established in resting muscle, complexities involved in directly measuring fuel oxidation using indirect calorimetry across a muscle bed have limited studies of this phenomenon in working skeletal muscle. During mild exercise and at rest, whole-body indirect calorimetry imperfectly estimates muscle fuel oxidation. We provide evidence that a method termed "ΔRER" can reasonably estimate fuel oxidation in skeletal muscle activated by exercise. Methods: Completely sedentary volunteers (n = 20, age 31 ± 2 years, V̇O2peak 24.4 ± 1.5 mL O2 per min/kg) underwent glucose clamps to determine insulin sensitivity and graded exercise consisting of three periods of mild steady-state cycle ergometry (15, 30, 45 watts, or 10%, 20%, and 30% of maximum power) with measurements of whole-body gas exchange. ΔRER, the RER in working muscle, was calculated as (V̇CO2exercise -V̇CO2rest)/(V̇O2exercise - V̇O2rest), from which the fraction of fuel accounted for by lipid was estimated. Results: Lactate levels were low and stable during steady-state exercise. Muscle biopsies were used to estimate mitochondrial content. The rise of V̇O2 at onset of exercise followed a monoexponential function, with a time constant of 51 ± 7 sec, typical of skeletal muscle; the average O2 cost of work was about 12 mL O2/watt/min, representing a mechanical efficiency of about 24%. At work rates of 30 or 45 watts, active muscle relied predominantly on carbohydrate, independent of insulin sensitivity within this group of very sedentary volunteers. Conclusions: The fraction of muscle fuel oxidation from fat was predicted by power output (P < 0.001) and citrate synthase activity (P < 0.05), indicating that low mitochondrial content may be the main driver of fuel choice in sedentary people, independent of insulin sensitivity.

Oxidative phosphorylation K0.5ADP in vitro depends on substrate oxidative capacity: Insights from a luciferase-based assay to evaluate ADP kinetic parameters.

The K0.5ADP of oxidative phosphorylation (OxPhos) identifies the cytosolic ADP concentration which elicits one-half the maximum OxPhos rate. This kinetic parameter is commonly measured to assess mitochondrial metabolic control sensitivity. Here we describe a luciferase-based assay to evaluate the ADP kinetic parameters of mitochondrial ATP production from OxPhos, adenylate kinase (AK), and creatine kinase (CK). The high sensitivity, reproducibility, and throughput of the microplate-based assay enabled a comprehensive kinetic assessment of all three pathways in mitochondria isolated from mouse liver, kidney, heart, and skeletal muscle. Carboxyatractyloside titrations were also performed with the assay to estimate the flux control strength of the adenine nucleotide translocase (ANT) over OxPhos in human skeletal muscle mitochondria. ANT flux control coefficients were 0.91 ± 0.07, 0.83 ± 0.06, and 0.51 ± 0.07 at ADP concentrations of 6.25, 12.5, and 25 µM, respectively, an [ADP] range which spanned the K0.5ADP. The oxidative capacity of substrate combinations added to drive OxPhos was found to dramatically influence ADP kinetics in mitochondria from several tissues. In mouse skeletal muscle ten different substrate combinations elicited a 7-fold range of OxPhos Vmax, which correlated positively (R2 = 0.963) with K0.5ADP values ranging from 2.3 ± 0.2 µM to 11.9 ± 0.6 µM. We propose that substrate-enhanced capacity to generate the protonmotive force increases the OxPhos K0.5ADP because flux control at ANT increases, thus K0.5ADP rises toward the dissociation constant, KdADP, of ADP-ANT binding. The findings are discussed in the context of top-down metabolic control analysis.

Impact of trauma dispatch algorithm software on the rate of missions of emergency medical services.

BACKGROUND: Trauma still stands atop of the list of emergencies. Transfer of these patients via Emergency Medical Services (EMS) dispatch is critical with regard to importance of timing. This aspect has achieved greater importance due to population increase and telephone triage. OBJECTIVES: We aimed to decrease unnecessary Emergency Medical Services (EMS) missions via a computer program designed for an algorithmic approach for trauma care by nurses involved in EMS, to help them evaluate the case more accurately. We named our program "Trauma Dispatch Algorithm". MATERIALS AND METHODS: First, the most common chief complaints regarding traumatic events were chosen from searching all the calls in December 2008 recorded in Tehran, Iran's EMS center; and then an algorithm approach was written for them. These algorithms were revised by three traumatologists and emergency medicine specialists, after their approval the algorithms were evaluated by EMS dispatch center for their practicality. Finally all data were turned into computer software. The program was used at the Tehran EMS center; 100 recorded calls assessed with each system were selected randomly. They were evaluated by another traumatologist whether it was necessary to send a team to the site or not. RESULTS: The age average was 26 years in both groups. The "trauma dispatch algorithm" was significantly effective in reducing the unnecessary missions of EMS by 16% (from 42% to 26%) (P = 0.005). CONCLUSIONS: This program was effective in reducing unnecessary missions. We propose the usage of this system in all EMS centers.