L-leucine Supplementation Reverses Hyperammonemia Induced Skeletal Muscle Mitochondrial Dysfunction

Background. Skeletal muscle hyperammonemia occurs in cirrhosis and contributes to sarcopenia or loss of skeletal muscle mass. Skeletal muscle ammonia disposal occurs in the mitochondria by cataplerosis (loss of tricarboxylic acid- TCA- cycle intermediates) of a-ketoglutarate to generate glutamate and glutamine. Skeletal muscle hyperammonemia also results a unique cellular stress response with partitioning of L-leucine into the mitochondria to assist ammonia disposal, potentially promoting anaple- rosis (entry of TCA cycle intermediates) either directly or indirectly. We have previously reported that skeletal muscle hyperammonemia decreases cellular oxygen consumption, lowers tricarboxylic acid cycle intermediates and increases mitochondrial reactive oxygen species in the muscle. We therefore hypothesized that ammonia induced skeletal muscle mitochondrial dysfunction will be reversed by L-leucine supplementation that is sufficient to satisfy the metabolic demand in the mitochondria. Methods. Differentiated C2C12 murine myotubes were treated with 10mM ammonium acetate or vehicle alone and the response to L leucine 5mM was quantified. Cellular oxygen consumption in the basal state and the response to substrates for components of the electron transport chain (pyruvate and malate for complex I; succinate for complex II) were measured using high sensitivity respirometry in intact and permeabilized myotubes. Concentration of TCA cycle intermediates in cell lysates were quantified using isotope ratio mass spectrometry respectively. Mitochondrial reactive oxygen species were measured by flow cytometry using the fluoroprobe, MitoSox®. ATP content was measured by a fluorometric assay. Complementary studies were performed in myotubes grown in leucine deficient medium that were treated with L-leu- cine. All experiments were done in at least 4 biological replicates. Results. Hyperammonemia decreased intact cell and substrate dependent oxygen consumption in myotubes that were reversed by 5mM L-leucine. Increased reactive oxygen species during hyperammonemia was also decreased by L-Leucine Hyperammonemia resulted in a reduction in TCA cycle intermediates and the intermediate ratios at 6 and 24 h that was reversed by L-leucine. Low cellular ATP content and ATP synthesis were also reversed by L-leucine supplementation. L-leucine deficient medium resulted in a depletion of TCA cycle intermediates that was aggravated by hyperammonemia and reversed by L leucine. Conclusions. We demonstrate for the first time that ammonia induced skeletal muscle mitochondrial dysfunction is reversed by L leucine supplementation and restores the anaplerosis/cataplerosis balance

Serum Tricarboxylic Acid Cycle Intermediates: Potential Biomarker for Hyperammonemic Metabolic Dysregulation in Human Cirrhosis

Introduction. Hyperammonemia is a consistent abnormality in cirrhosis and the skeletal muscle functions as a metabolic partner in ammonia disposal in liver disease. Skeletal muscle ammonia disposal is mediated by conversion of a-ketoglutarate (aKG) a critical intermediate in the tricarboxylic acid (TCA) cycle to glutamate and glutamine. We have previously reported low skeletal muscle TCA cycle intermediates during hyperammonemia. Since TCA cycle intermediates can leak into the circulation, we hypothesized that plasma TCA cycle intermediates will be lower in cirrhosis. We also hypothesized that branched chain amino acids (BCAA) that serve as anaplerotic (entry of TCA cycle intermediates) substrates will reverse this abnormality. Materials and Methods. In 6 well compensated, stable alcoholic cirrhotic patients and 8 controls, serial plasma samples were obtained before and 1h, 3h, 6h after a single oral BCAA mixture enriched with leucine (BCAA/ Leu). Intermediary metabolites extracted using ethyl acetate and Tertbutyldimethylsilyl derivatives generated and quantified by gas chromatography-mass spectrometry using protocols established in our laboratory. All experiments were performed in at least 6 experimental replicates for each sample and the data expressed as percentage mean±SD after normalization with the concentration of control. Results. Surprisingly, plasma concentrations of all TCA cycle intermediates in cirrhotic patients were consistently higher at baseline and remained elevated at 1h, 3h, and 6h after a single oral BCAA mixture enriched with leucine (BCAA/Leu) compared to controls (Table). Conclusion. Elevated plasma TCA cycle intermediates are potential biomarkers for metabolic perturbations in cirrhosis. Identifying the molecular and biochemical mechanisms for elevated plasma TCA cycle intermediates, lack of response to BCAA/Leu and the source of intermediates in circulation will be necessary to restore anaplerosis/cataplerosis homeostasis in cirrhosis