Sarcopenia or skeletal muscle loss is a frequent, potentially reversible complication in cirrhosis that adversely affects clinical outcomes. Hyperammonemia is a consistent abnormality in cirrhosis ...that results in impaired skeletal muscle protein synthesis and breakdown (proteostasis). Despite the availability of effective ammonia‐lowering therapies, whether lowering ammonia restores proteostasis and increases muscle mass is unknown. Myotube diameter, protein synthesis, and molecular responses in C2C12 murine myotubes to withdrawal of ammonium acetate following 24‐hour exposure to 10 mM ammonium acetate were complemented by in vivo studies in the hyperammonemic portacaval anastomosis rat and sham‐operated, pair‐fed Sprague‐Dawley rats treated with ammonia‐lowering therapy by l‐ornithine l‐aspartate and rifaximin orally for 4 weeks. We observed reduced myotube diameter, impaired protein synthesis, and increased autophagy flux in response to hyperammonemia, which were partially reversed following 24‐hour and 48‐hour withdrawal of ammonium acetate. Consistently, 4 weeks of ammonia‐lowering therapy resulted in significant lowering of blood and skeletal muscle ammonia, increase in lean body mass, improved grip strength, higher skeletal muscle mass and diameter, and an increase in type 2 fibers in treated compared to untreated portacaval anastomosis rats. The increased skeletal muscle myostatin expression, reduced mammalian target of rapamycin complex 1 function, and hyperammonemic stress response including autophagy markers normally found in portacaval anastomosis rats were reversed by treatment with ammonia‐lowering therapy. Despite significant improvement, molecular and functional readouts were not completely reversed by ammonia‐lowering measures. Conclusion: Ammonia‐lowering therapy results in improvement in skeletal muscle phenotype and function and molecular perturbations of hyperammonemia; these preclinical studies complement previous studies on ammonia‐induced skeletal muscle loss and lay the foundation for prolonged ammonia‐lowering therapy to reverse sarcopenia of cirrhosis. (Hepatology 2017;65:2045‐2058).
Key points
Hyperammonaemia occurs in hepatic, cardiac and pulmonary diseases with increased muscle concentration of ammonia.
We found that ammonia results in reduced skeletal muscle mitochondrial ...respiration, electron transport chain complex I dysfunction, as well as lower NAD+/NADH ratio and ATP content.
During hyperammonaemia, leak of electrons from complex III results in oxidative modification of proteins and lipids.
Tricarboxylic acid cycle intermediates are decreased during hyperammonaemia, and providing a cell‐permeable ester of αKG reversed the lower TCA cycle intermediate concentrations and increased ATP content.
Our observations have high clinical relevance given the potential for novel approaches to reverse skeletal muscle ammonia toxicity by targeting the TCA cycle intermediates and mitochondrial ROS.
Ammonia is a cytotoxic metabolite that is removed primarily by hepatic ureagenesis in humans. Hyperammonaemia occurs in advanced hepatic, cardiac and pulmonary disease, and in urea cycle enzyme deficiencies. Increased skeletal muscle ammonia uptake and metabolism are the major mechanism of non‐hepatic ammonia disposal. Non‐hepatic ammonia disposal occurs in the mitochondria via glutamate synthesis from α‐ketoglutarate resulting in cataplerosis. We show skeletal muscle mitochondrial dysfunction during hyperammonaemia in a comprehensive array of human, rodent and cellular models. ATP synthesis, oxygen consumption, generation of reactive oxygen species with oxidative stress, and tricarboxylic acid (TCA) cycle intermediates were quantified. ATP content was lower in the skeletal muscle from cirrhotic patients, hyperammonaemic portacaval anastomosis rat, and C2C12 myotubes compared to appropriate controls. Hyperammonaemia in C2C12 myotubes resulted in impaired intact cell respiration, reduced complex I/NADH oxidase activity and electron leak occurring at complex III of the electron transport chain. Consistently, lower NAD+/NADH ratio was observed during hyperammonaemia with reduced TCA cycle intermediates compared to controls. Generation of reactive oxygen species resulted in increased content of skeletal muscle carbonylated proteins and thiobarbituric acid reactive substances during hyperammonaemia. A cell‐permeable ester of α‐ketoglutarate reversed the low TCA cycle intermediates and ATP content in myotubes during hyperammonaemia. However, the mitochondrial antioxidant MitoTEMPO did not reverse the lower ATP content during hyperammonaemia. We provide for the first time evidence that skeletal muscle hyperammonaemia results in mitochondrial dysfunction and oxidative stress. Use of anaplerotic substrates to reverse ammonia‐induced mitochondrial dysfunction is a novel therapeutic approach.
Key points
Hyperammonaemia occurs in hepatic, cardiac and pulmonary diseases with increased muscle concentration of ammonia.
We found that ammonia results in reduced skeletal muscle mitochondrial respiration, electron transport chain complex I dysfunction, as well as lower NAD+/NADH ratio and ATP content.
During hyperammonaemia, leak of electrons from complex III results in oxidative modification of proteins and lipids.
Tricarboxylic acid cycle intermediates are decreased during hyperammonaemia, and providing a cell‐permeable ester of αKG reversed the lower TCA cycle intermediate concentrations and increased ATP content.
Our observations have high clinical relevance given the potential for novel approaches to reverse skeletal muscle ammonia toxicity by targeting the TCA cycle intermediates and mitochondrial ROS.
Skeletal muscle loss (sarcopenia) is a major clinical complication in alcoholic cirrhosis with no effective therapy. Skeletal muscle autophagic proteolysis and myostatin expression (inhibitor of ...protein synthesis) are increased in cirrhosis and believed to contribute to anabolic resistance. A prospective study was performed to determine the mechanisms of sarcopenia in alcoholic cirrhosis and potential reversal by leucine. In six well‐compensated, stable, alcoholic patients with cirrhosis and eight controls, serial vastus lateralis muscle biopsies were obtained before and 7 hours after a single oral branched chain amino acid mixture enriched with leucine (BCAA/LEU). Primed‐constant infusion of l‐ring‐2H5‐phenylalanine was used to quantify whole‐body protein breakdown and muscle protein fractional synthesis rate using liquid chromatography/mass spectrometry. Muscle expression of myostatin, mammalian target of rapamycin (mTOR) targets, autophagy markers, protein ubiquitination, and the intracellular amino acid deficiency sensor general control of nutrition 2 were quantified by immunoblots and the leucine exchanger (SLC7A5) and glutamine transporter (SLC38A2), by real‐time polymerase chain reaction. Following oral administration, plasma BCAA concentrations showed a similar increase in patients with cirrhosis and controls. Skeletal muscle fractional synthesis rate was 9.63 ± 0.36%/hour in controls and 9.05 ± 0.68%/hour in patients with cirrhosis (P = 0.54). Elevated whole‐body protein breakdown in patients with cirrhosis was reduced with BCAA/LEU (P = 0.01). Fasting skeletal muscle molecular markers showed increased myostatin expression, impaired mTOR signaling, and increased autophagy in patients with cirrhosis compared to controls (P < 0.01). The BCAA/LEU supplement did not alter myostatin expression, but mTOR signaling, autophagy measures, and general control of nutrition 2 activation were consistently reversed in cirrhotic muscle (P < 0.01). Expression of SLC7A5 was higher in the basal state in patients with cirrhosis than controls (P < 0.05) but increased with BCAA/LEU only in controls (P < 0.001). Conclusions: Impaired mTOR1 signaling and increased autophagy in skeletal muscle of patients with alcoholic cirrhosis is acutely reversed by BCAA/LEU. (Hepatology 2015;61:2018‐2029)
Hyperammonemia results in reduced muscle function independent of muscle mass McDaniel, John; Davuluri, Gangarao; Hill, Elizabeth Ann ...
American journal of physiology. Gastrointestinal and liver physiology/American journal of physiology: Gastrointestinal and liver physiology,
02/2016, Volume:
310, Issue:
3
Journal Article
Peer reviewed
Open access
The mechanism of the nearly universal decreased muscle strength in cirrhosis is not known. We evaluated whether hyperammonemia in cirrhosis causes contractile dysfunction independent of reduced ...skeletal muscle mass. Maximum grip strength and muscle fatigue response were determined in cirrhotic patients and controls. Blood and muscle ammonia concentrations and grip strength normalized to lean body mass were measured in the portacaval anastomosis (PCA) and sham-operated pair-fed control rats (n = 5 each). Ex vivo contractile studies in the soleus muscle from a separate group of Sprague-Dawley rats (n = 7) were performed. Skeletal muscle force of contraction, rate of force development, and rate of relaxation were measured. Muscles were also subjected to a series of pulse trains at a range of stimulation frequencies from 20 to 110 Hz. Cirrhotic patients had lower maximum grip strength and greater muscle fatigue than control subjects. PCA rats had a 52.7 ± 13% lower normalized grip strength compared with control rats, and grip strength correlated with the blood and muscle ammonia concentrations (r(2) = 0.82). In ex vivo muscle preparations following a single pulse, the maximal force, rate of force development, and rate of relaxation were 12.1 ± 3.5 g vs. 6.2 ± 2.1 g; 398.2 ± 100.4 g/s vs. 163.8 ± 97.4 g/s; -101.2 ± 22.2 g/s vs. -33.6 ± 22.3 g/s in ammonia-treated compared with control muscle preparation, respectively (P < 0.001 for all comparisons). Tetanic force, rate of force development, and rate of relaxation were depressed across a range of stimulation from 20 to 110 Hz. These data provide the first direct evidence that hyperammonemia impairs skeletal muscle strength and increased muscle fatigue and identifies a potential therapeutic target in cirrhotic patients.
Exercise seems to enhance the beneficial effect of bariatric (Roux-en-Y gastric bypass RYGB) surgery on insulin resistance. We hypothesized that skeletal muscle extracellular matrix (ECM) remodeling ...may underlie these benefits. Women were randomized to either a combined aerobic and resistance exercise training program following RYGB (RYGB + ET) or standard of care (RYGB). Insulin sensitivity was assessed by oral glucose tolerance test. Muscle biopsy specimens were obtained at baseline and 3 and 9 months after surgery and subjected to comprehensive phenotyping, transcriptome profiling, molecular pathway identification, and validation in vitro. Exercise training improved insulin sensitivity beyond surgery alone (e.g., Matsuda index: RYGB 123% vs. RYGB + ET 325%;
≤ 0.0001). ECM remodeling was reduced by surgery alone, with an additive benefit of surgery and exercise training (e.g., collagen I: RYGB -41% vs. RYGB + ET -76%;
≤ 0.0001). Exercise and RYGB had an additive effect on enhancing insulin sensitivity, but surgery alone did not resolve insulin resistance and ECM remodeling. We identified candidates modulated by exercise training that may become therapeutic targets for treating insulin resistance, in particular, the transforming growth factor-β1/SMAD 2/3 pathway and its antagonist follistatin. Exercise-induced increases in insulin sensitivity after bariatric surgery are at least partially mediated by muscle ECM remodeling.
Obesity is a leading cause of preventable death worldwide. Despite this, current strategies for the treatment of obesity remain ineffective at achieving long‐term weight control. This is due, in ...part, to difficulties in identifying tolerable and efficacious small molecules or biologics capable of regulating systemic nutrient homeostasis. Here, we demonstrate that BAM15, a mitochondrially targeted small molecule protonophore, stimulates energy expenditure and glucose and lipid metabolism to protect against diet‐induced obesity. Exposure to BAM15 in vitro enhanced mitochondrial respiratory kinetics, improved insulin action, and stimulated nutrient uptake by sustained activation of AMPK. C57BL/6J mice treated with BAM15 were resistant to weight gain. Furthermore, BAM15‐treated mice exhibited improved body composition and glycemic control independent of weight loss, effects attributable to drug targeting of lipid‐rich tissues. We provide the first phenotypic characterization and demonstration of pre‐clinical efficacy for BAM15 as a pharmacological approach for the treatment of obesity and related diseases.
Synopsis
This study presents a novel therapy for treatment of obesity‐related diseases. Oral delivery of the mitochondrial protonophore BAM15 markedly reduced weight gain and fat accrual while improving glycemic control.
BAM15 displays extended mitochondrial activity compared to previous generation protonophores.
BAM15 protects against diet induced obesity.
BAM15 modulates body composition and glycemic control independently of its weight‐reducing effects.
AMPK is required to sustain the metabolic benefit of BAM15, which occurs primarily in adipose tissue.
This study presents a novel therapy for treatment of obesity‐related diseases. Oral delivery of the mitochondrial protonophore BAM15 markedly reduced weight gain and fat accrual while improving glycemic control.
Signaling and metabolic perturbations contribute to dysregulated skeletal muscle protein homeostasis and secondary sarcopenia in response to a number of cellular stressors including ethanol exposure. ...Using an innovative multiomics-based curating of unbiased data, we identified molecular and metabolic therapeutic targets and experimentally validated restoration of protein homeostasis in an ethanol-fed mouse model of liver disease.
Studies were performed in ethanol-treated differentiated C2C12 myotubes and physiological relevance established in an ethanol-fed mouse model of alcohol-related liver disease (mALD) or pair-fed control C57BL/6 mice. Transcriptome and proteome from ethanol treated-myotubes and gastrocnemius muscle from mALD and pair-fed mice were analyzed to identify target pathways and molecules. Readouts including signaling responses and autophagy markers by immunoblots, mitochondrial oxidative function and free radical generation, and metabolic studies by gas chromatography-mass spectrometry and sarcopenic phenotype by imaging.
Multiomics analyses showed that ethanol impaired skeletal muscle mTORC1 signaling, mitochondrial oxidative pathways, including intermediary metabolite regulatory genes, interleukin-6, and amino acid degradation pathways are β-hydroxymethyl-butyrate targets. Ethanol decreased mTORC1 signaling, increased autophagy flux, impaired mitochondrial oxidative function with decreased tricarboxylic acid cycle intermediary metabolites, ATP synthesis, protein synthesis and myotube diameter that were reversed by HMB. Consistently, skeletal muscle from mALD had decreased mTORC1 signaling, reduced fractional and total muscle protein synthesis rates, increased autophagy markers, lower intermediary metabolite concentrations, and lower muscle mass and fiber diameter that were reversed by β-hydroxymethyl-butyrate treatment.
An innovative multiomics approach followed by experimental validation showed that β-hydroxymethyl-butyrate restores muscle protein homeostasis in liver disease.
Mitochondria are the site of iron utilization, wherein imported iron is incorporated into heme or iron–sulfur clusters. Previously, we showed that a cytosolic siderophore, which resembles a bacterial ...siderophore, facilitates mitochondrial iron import in eukaryotes, including zebrafish. An evolutionarily conserved 3-hydroxy butyrate dehydrogenase, 3-hydroxy butyrate dehydrogenase 2 (Bdh2), catalyzes a rate-limiting step in the biogenesis of the eukaryotic siderophore. We found that inactivation of bdh2 in developing zebrafish embryo results in heme deficiency and delays erythroid maturation. The basis for this erythroid maturation defect is not known. Here we show that bdh2 inactivation results in mitochondrial dysfunction and triggers their degradation by mitophagy. Thus, mitochondria are prematurely lost in bdh2-inactivated erythrocytes. Interestingly, bdh2-inactivated erythroid cells also exhibit genomic alterations as indicated by transcriptome analysis. Reestablishment of bdh2 restores mitochondrial function, prevents premature mitochondrial degradation, promotes erythroid development, and reverses altered gene expression. Thus, mitochondrial communication with the nucleus is critical for erythroid development.
The signals that initiate cell invasion are not well understood, but there is increasing evidence that extracellular physical signals play an important role. Here we show that epithelial cell ...invasion in the intestine of zebrafish meltdown (mlt) mutants arises in response to unregulated contractile tone in the surrounding smooth muscle cell layer. Physical signaling in mlt drives formation of membrane protrusions within the epithelium that resemble invadopodia, matrix-degrading protrusions present in invasive cancer cells. Knockdown of Tks5, a Src substrate that is required for invadopodia formation in mammalian cells blocked formation of the protrusions and rescued invasion in mlt. Activation of Src-signaling induced invadopodia-like protrusions in wild type epithelial cells, however the cells did not migrate into the tissue stroma, thus indicating that the protrusions were required but not sufficient for invasion in this in vivo model. Transcriptional profiling experiments showed that genes responsive to reactive oxygen species (ROS) were upregulated in mlt larvae. ROS generators induced invadopodia-like protrusions and invasion in heterozygous mlt larvae but had no effect in wild type larvae. Co-activation of oncogenic Ras and Wnt signaling enhanced the responsiveness of mlt heterozygotes to the ROS generators. These findings present the first direct evidence that invadopodia play a role in tissue cell invasion in vivo. In addition, they identify an inducible physical signaling pathway sensitive to redox and oncogenic signaling that can drive this process.
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