The mitochondrial tRNA-derived fragment, mt-tRF-LeuTAA, couples mitochondrial metabolism to insulin secretion.

OBJECTIVE: The contribution of the mitochondrial electron transfer system to insulin secretion involves more than just energy provision. We identified a small RNA fragment (mt-tRF-LeuTAA) derived from the cleavage of a mitochondrially-encoded tRNA that is conserved between mice and humans. The role of mitochondrially-encoded tRNA-derived fragments remains unknown. This study aimed to characterize the impact of mt-tRF-LeuTAA, on mitochondrial metabolism and pancreatic islet functions. METHODS: We used antisense oligonucleotides to reduce mt-tRF-LeuTAA levels in primary rat and human islet cells, as well as in insulin-secreting cell lines. We performed a joint transcriptome and proteome analysis upon mt-tRF-LeuTAA inhibition. Additionally, we employed pull-down assays followed by mass spectrometry to identify direct interactors of the fragment. Finally, we characterized the impact of mt-tRF-LeuTAA silencing on the coupling between mitochondrial metabolism and insulin secretion using high-resolution respirometry and insulin secretion assays. RESULTS: Our study unveils a modulation of mt-tRF-LeuTAA levels in pancreatic islets in different Type 2 diabetes models and in response to changes in nutritional status. The level of the fragment is finely tuned by the mechanistic target of rapamycin complex 1. Located within mitochondria, mt-tRF-LeuTAA interacts with core subunits and assembly factors of respiratory complexes of the electron transfer system. Silencing of mt-tRF-LeuTAA in islet cells limits the inner mitochondrial membrane potential and impairs mitochondrial oxidative phosphorylation, predominantly by affecting the Succinate (via Complex II)-linked electron transfer pathway. Lowering mt-tRF-LeuTAA impairs insulin secretion of rat and human pancreatic ß-cells. CONCLUSIONS: Our findings indicate that mt-tRF-LeuTAA interacts with electron transfer system complexes and is a pivotal regulator of mitochondrial oxidative phosphorylation and its coupling to insulin secretion.

Case-control study of environmental toxins and risk of amyotrophic lateral sclerosis involving the national ALS registry.

Objective: Neurotoxic chemicals are suggested in the etiology of amyotrophic lateral sclerosis (ALS). We examined the association of environmental and occupational risk factors including persistent organochlorine pesticides (OCPs) and ALS risk among cases from the Centers for Disease Control and Prevention National ALS Registry and age, sex, and county-matched controls. Methods: Participants completed a risk factor survey and provided a blood sample for OCP measurement. ALS cases were confirmed through the Registry. Conditional logistic regression assessed associations between ALS and risk factors including OCP levels. Results: 243 matched case-control pairs (61.7% male, mean [SD] age = 62.9 [10.1]) were included. Fifteen of the 29 OCPs examined had sufficient detectable levels for analysis. Modest correlations of self-reported years of exposure to residential pesticide mixtures and OCP serum levels were found (p<.001). Moreover, occupational exposure to lead including soldering and welding with lead/metal dust and use of lead paint/gasoline were significantly related to ALS risk (OR = 1.77, 95% CI: 1.11-2.83). Avocational gardening was a significant risk factor for ALS (OR = 1.57, 95% CI: 1.04-2.37). ALS risk increased for each 10 ng/g of α-Endosulfan (OR = 1.42, 95% CI: 1.14-1.77) and oxychlordane (OR = 1.24, 95% CI: 1.01-1.53). Heptachlor (detectable vs. nondetectable) was also associated with ALS risk (OR = 3.57, 95% CI: 1.50-8.52). Conclusion: This national case-control study revealed both survey and serum levels of OCPs as risk factors for ALS. Despite the United States banning many OCPs in the 1970s and 1980s, their use abroad and long half-lives continue to exert possible neurotoxic health effects.

Functional hypoxia reduces mitochondrial calcium uptake.

Mitochondrial respiration extends beyond ATP generation, with the organelle participating in many cellular and physiological processes. Parallel changes in components of the mitochondrial electron transfer system with respiration render it an appropriate hub for coordinating cellular adaption to changes in oxygen levels. How changes in respiration under functional hypoxia (i.e., when intracellular O2 levels limit mitochondrial respiration) are relayed by the electron transfer system to impact mitochondrial adaption and remodeling after hypoxic exposure remains poorly defined. This is largely due to challenges integrating findings under controlled and defined O2 levels in studies connecting functions of isolated mitochondria to humans during physical exercise. Here we present experiments under conditions of hypoxia in isolated mitochondria, myotubes and exercising humans. Performing steady-state respirometry with isolated mitochondria we found that oxygen limitation of respiration reduced electron flow and oxidative phosphorylation, lowered the mitochondrial membrane potential difference, and decreased mitochondrial calcium influx. Similarly, in myotubes under functional hypoxia mitochondrial calcium uptake decreased in response to sarcoplasmic reticulum calcium release for contraction. In both myotubes and human skeletal muscle this blunted mitochondrial adaptive responses and remodeling upon contractions. Our results suggest that by regulating calcium uptake the mitochondrial electron transfer system is a hub for coordinating cellular adaption under functional hypoxia.

Partial MCT1 invalidation protects against diet-induced non-alcoholic fatty liver disease and the associated brain dysfunction.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) has been associated with mild cerebral dysfunction and cognitive decline, although the exact pathophysiological mechanism remains ambiguous. Using a diet-induced model of NAFLD and monocarboxylate transporter-1 (Mct1+/-) haploinsufficient mice, which resist high-fat diet-induced hepatic steatosis, we investigated the hypothesis that NAFLD leads to an encephalopathy by altering cognition, behaviour, and cerebral physiology. We also proposed that global MCT1 downregulation offers cerebral protection. METHODS: Behavioural tests were performed in mice following 16 weeks of control diet (normal chow) or high-fat diet with high fructose/glucose in water. Tissue oxygenation, cerebrovascular reactivity, and cerebral blood volume were monitored under anaesthesia by multispectral optoacoustic tomography and optical fluorescence. Cortical mitochondrial oxygen consumption and respiratory capacities were measured using ex vivo high-resolution respirometry. Microglial and astrocytic changes were evaluated by immunofluorescence and 3D reconstructions. Body composition was assessed using EchoMRI, and liver steatosis was confirmed by histology. RESULTS: NAFLD concomitant with obesity is associated with anxiety- and depression-related behaviour. Low-grade brain tissue hypoxia was observed, likely attributed to the low-grade brain inflammation and decreased cerebral blood volume. It is also accompanied by microglial and astrocytic morphological and metabolic alterations (higher oxygen consumption), suggesting the early stages of an obesogenic diet-induced encephalopathy. Mct1 haploinsufficient mice, despite fat accumulation in adipose tissue, were protected from NAFLD and associated cerebral alterations. CONCLUSIONS: This study provides evidence of compromised brain health in obesity and NAFLD, emphasising the importance of the liver-brain axis. The protective effect of Mct1 haploinsufficiency points to this protein as a novel therapeutic target for preventing and/or treating NAFLD and the associated brain dysfunction. IMPACT AND IMPLICATIONS: This study is focused on unravelling the pathophysiological mechanism by which cerebral dysfunction and cognitive decline occurs during NAFLD and exploring the potential of monocarboxylate transporter-1 (MCT1) as a novel preventive or therapeutic target. Our findings point to NAFLD as a serious health risk and its adverse impact on the brain as a potential global health system and economic burden. These results highlight the utility of Mct1 transgenic mice as a model for NAFLD and associated brain dysfunction and call for systematic screening by physicians for early signs of psychological symptoms, and an awareness by individuals at risk of these potential neurological effects. This study is expected to bring attention to the need for early diagnosis and treatment of NAFLD, while having a direct impact on policies worldwide regarding the health risk associated with NAFLD, and its prevention and treatment.

Acute Effects of Neuromuscular Electrical Stimulation on Contralateral Plantar Flexor Neuromuscular Function.

Contralateral facilitation, i.e., the increase in contralateral maximal voluntary strength that is observed when neuromuscular electrical stimulation (NMES) is applied to the ipsilateral homonymous muscle, has previously been reported for the knee extensors but the neurophysiological mechanisms remain to be investigated. The aim of this study was to compare plantar flexor contralateral facilitation between a submaximal voluntary contraction (~10% MVC torque) and two evoked contractions (conventional and wide-pulse high-frequency NMES) of the ipsilateral plantar flexors, with respect to a resting condition. Contralateral MVC torque and voluntary activation level were measured in 22 healthy participants while the ipsilateral plantar flexors were at rest, voluntarily contracted or stimulated for 15 s. Additional neurophysiological parameters (soleus H-reflex and V-wave amplitude and tibialis anterior coactivation level) were quantified in a subgroup of 12 participants. Conventional and wide-pulse high-frequency NMES of the ipsilateral plantar flexors did not induce any contralateral facilitation of maximal voluntary strength and activation with respect to the resting condition. Similarly, no alteration of neurophysiological parameters was observed in the different conditions. This absence of contralateral facilitation contrasts with some results previously obtained on the knee extensors but is consistent with the absence of neurophysiological changes on the contralateral soleus.

Abnormal brain oxygen homeostasis in an animal model of liver disease.

Background & Aims: Increased plasma ammonia concentration and consequent disruption of brain energy metabolism could underpin the pathogenesis of hepatic encephalopathy (HE). Brain energy homeostasis relies on effective maintenance of brain oxygenation, and dysregulation impairs neuronal function leading to cognitive impairment. We hypothesised that HE is associated with reduced brain oxygenation and we explored the potential role of ammonia as an underlying pathophysiological factor. Methods: In a rat model of chronic liver disease with minimal HE (mHE; bile duct ligation [BDL]), brain tissue oxygen measurement, and proton magnetic resonance spectroscopy were used to investigate how hyperammonaemia impacts oxygenation and metabolic substrate availability in the central nervous system. Ornithine phenylacetate (OP, OCR-002; Ocera Therapeutics, CA, USA) was used as an experimental treatment to reduce plasma ammonia concentration. Results: In BDL animals, glucose, lactate, and tissue oxygen concentration in the cerebral cortex were significantly lower than those in sham-operated controls. OP treatment corrected the hyperammonaemia and restored brain tissue oxygen. Although BDL animals were hypotensive, cortical tissue oxygen concentration was significantly improved by treatments that increased arterial blood pressure. Cerebrovascular reactivity to exogenously applied CO2 was found to be normal in BDL animals. Conclusions: These data suggest that hyperammonaemia significantly decreases cortical oxygenation, potentially compromising brain energy metabolism. These findings have potential clinical implications for the treatment of patients with mHE. Lay summary: Brain dysfunction is a serious complication of cirrhosis and affects approximately 30% of these patients; however, its treatment continues to be an unmet clinical need. This study shows that oxygen concentration in the brain of an animal model of cirrhosis is markedly reduced. Low arterial blood pressure and increased ammonia (a neurotoxin that accumulates in patients with liver failure) are shown to be the main underlying causes. Experimental correction of these abnormalities restored oxygen concentration in the brain, suggesting potential therapeutic avenues to explore.

Acute RyR1 Ca2+ leak enhances NADH-linked mitochondrial respiratory capacity.

Sustained ryanodine receptor (RyR) Ca2+ leak is associated with pathological conditions such as heart failure or skeletal muscle weakness. We report that a single session of sprint interval training (SIT), but not of moderate intensity continuous training (MICT), triggers RyR1 protein oxidation and nitrosylation leading to calstabin1 dissociation in healthy human muscle and in in vitro SIT models (simulated SIT or S-SIT). This is accompanied by decreased sarcoplasmic reticulum Ca2+ content, increased levels of mitochondrial oxidative phosphorylation proteins, supercomplex formation and enhanced NADH-linked mitochondrial respiratory capacity. Mechanistically, (S-)SIT increases mitochondrial Ca2+ uptake in mouse myotubes and muscle fibres, and decreases pyruvate dehydrogenase phosphorylation in human muscle and mouse myotubes. Countering Ca2+ leak or preventing mitochondrial Ca2+ uptake blunts S-SIT-induced adaptations, a result supported by proteomic analyses. Here we show that triggering acute transient Ca2+ leak through RyR1 in healthy muscle may contribute to the multiple health promoting benefits of exercise.

Modulation of torque evoked by wide-pulse, high-frequency neuromuscular electrical stimulation and the potential implications for rehabilitation and training.

The effectiveness of neuromuscular electrical stimulation (NMES) for rehabilitation is proportional to the evoked torque. The progressive increase in torque (extra torque) that may develop in response to low intensity wide-pulse high-frequency (WPHF) NMES holds great promise for rehabilitation as it overcomes the main limitation of NMES, namely discomfort. WPHF NMES extra torque is thought to result from reflexively recruited motor units at the spinal level. However, whether WPHF NMES evoked force can be modulated is unknown. Therefore, we examined the effect of two interventions known to change the state of spinal circuitry in opposite ways on evoked torque and motor unit recruitment by WPHF NMES. The interventions were high-frequency transcutaneous electrical nerve stimulation (TENS) and anodal transcutaneous spinal direct current stimulation (tsDCS). We show that TENS performed before a bout of WPHF NMES results in lower evoked torque (median change in torque time-integral: - 56%) indicating that WPHF NMES-evoked torque might be modulated. In contrast, the anodal tsDCS protocol used had no effect on any measured parameter. Our results demonstrate that WPHF NMES extra torque can be modulated and although the TENS intervention blunted extra torque production, the finding that central contribution to WPHF NMES-evoked torques can be modulated opens new avenues for designing interventions to enhance WPHF NMES.

Key Nutritional Considerations for Youth Winter Sports Athletes to Optimize Growth, Maturation and Sporting Development.

Despite a wealth of sport nutrition guidelines for adult athletes, there are currently no nutrition guidelines for youth winter sports athletes. Whilst it may be pragmatic to apply nutrition guidelines for adult athletes to youth winter sports athletes, it is inappropriate. Due to a paucity of research on youth athletes, it is impossible to provide evidence-based guidelines for this population, so careful extrapolation from the theoretical and practical considerations that apply to other athletic groups is necessary. Youth winter sport athletes undergo rapid biological growth and maturation which influences their nutritional requirements. A varied and balanced diet that ensures sufficient energy availability for optimal growth and maturation as well as sporting performance is the cornerstone of youth athlete nutrition and should also allow for youth athletes to meet their micronutrient requirements. In some cases, micronutrient status (e.g., vitamin D and iron) should be monitored and optimized if appropriate by a medical professional. Dietary supplement use is prevalent amongst youth athletes, however is often unnecessary. Education of youth athletes, their parents and coaches on best nutritional practices as well as the risks associated with dietary supplements is vital for their long-term athletic development. Further research in youth winter sports athletes across different stages of growth and maturation competing in a variety of sports is urgently required in order to inform nutritional guidelines for this population.

Toxic doses of caffeine are needed to increase skeletal muscle contractility.

Discrepant results have been reported regarding an intramuscular mechanism underlying the ergogenic effect of caffeine on neuromuscular function in humans. Here, we reevaluated the effect of caffeine on muscular force production in humans and combined this with measurements of the caffeine dose-response relationship on force and cytosolic free [Ca2+] ([Ca2+]i) in isolated mouse muscle fibers. Twenty-one healthy and physically active men (29 ± 9 yr, 178 ± 6 cm, 73 ± 10 kg, mean ± SD) took part in the present study. Nine participants were involved in two experimental sessions during which supramaximal single and paired electrical stimulations (at 10 and 100 Hz) were applied to the femoral nerve to record evoked forces. Evoked forces were recorded before and 1 h after ingestion of 1) 6 mg caffeine/kg body mass or 2) placebo. Caffeine plasma concentration was measured in 12 participants. In addition, submaximal tetanic force and [Ca2+]i were measured in single mouse flexor digitorum brevis (FDB) muscle fibers exposed to 100 nM up to 5 mM caffeine. Six milligrams of caffeine per kilogram body mass (plasma concentration ~40 µM) did not increase electrically evoked forces in humans. In superfused FDB single fibers, millimolar caffeine concentrations (i.e., 15- to 35-fold above usual concentrations observed in humans) were required to increase tetanic force and [Ca2+]i. Our results suggest that toxic doses of caffeine are required to increase muscle contractility, questioning the purported intramuscular ergogenic effect of caffeine in humans.

Adding Fish Oil to Whey Protein, Leucine, and Carbohydrate Over a Six-Week Supplementation Period Attenuates Muscle Soreness Following Eccentric Exercise in Competitive Soccer Players.

Soccer players often experience eccentric exercise-induced muscle damage given the physical demands of soccer match-play. Since long chain n-3 polyunsaturated fatty acids (n-3PUFA) enhance muscle sensitivity to protein supplementation, dietary supplementation with a combination of fish oil-derived n-3PUFA, protein, and carbohydrate may promote exercise recovery. This study examined the influence of adding n-3PUFA to a whey protein, leucine, and carbohydrate containing beverage over a six-week supplementation period on physiological markers of recovery measured over three days following eccentric exercise. Competitive soccer players were assigned to one of three conditions (2 × 200 mL): a fish oil supplement beverage (FO; n = 10) that contained n-3PUFA (1100 mg DHA/EPA-approximately 550 mg DHA, 550 mg EPA), whey protein (15 g), leucine (1.8 g), and carbohydrate (20 g); a protein supplement beverage (PRO; n = 10) that contained whey protein (15 g), leucine (1.8 g), and carbohydrate (20 g); and a carbohydrate supplement beverage (CHO; n = 10) that contained carbohydrate (24 g). Eccentric exercise consisted of unilateral knee extension/flexion contractions on both legs separately. Maximal force production was impaired by 22% during the 72-hour recovery period following eccentric exercise (p < 0.05). Muscle soreness, expressed as area under the curve (AUC) during 72-hour recovery, was less in FO (1948 ± 1091 mm × 72 h) than PRO (4640 ± 2654 mm × 72 h, p < 0.05) and CHO (4495 ± 1853 mm × 72 h, p = 0.10). Blood concentrations of creatine kinase, expressed as AUC, were ~60% lower in FO compared to CHO (p < 0.05) and tended to be lower (~39%, p = 0.07) than PRO. No differences in muscle function, soccer performance, or blood c-reactive protein concentrations were observed between groups. In conclusion, the addition of n-3PUFA to a beverage containing whey protein, leucine, and carbohydrate ameliorates the increase in muscle soreness and blood concentrations of creatine kinase following eccentric exercise in competitive soccer players.

Nerve growth factor-induced formation of axonal filopodia and collateral branches involves the intra-axonal synthesis of regulators of the actin-nucleating Arp2/3 complex.

Nerve growth factor (NGF) induces collateral branching along sensory axons by promoting the formation of axonal filopodia dependent on the actin-nucleating Arp2/3 complex. This study shows that chicken embryonic sensory axons contain mRNAs for the actin-nucleating Arp2/3 complex activator WAVE1 and the complex stabilizer cortactin. NGF increases the axonal levels of WAVE1 and cortactin through localized protein synthesis even in axons isolated from the cell body. Inhibition of protein synthesis in severed axons impairs NGF-induced branching, the formation of axonal filopodia, and the initiation of Arp2/3-dependent axonal actin patches, which serve as precursors to the emergence of filopodia. Overexpression of WAVE1 or cortactin in axons not treated with NGF increased the rate of actin patch formation and the frequency of the emergence of filopodia from actin patches, respectively. Antisense inhibition of cortactin mRNA translation in isolated axons blocked NGF-induced filopodia. NGF also activated the Rac1 GTPase, which drives WAVE1 activity, in a protein synthesis-independent manner. Similarly, inhibition of protein synthesis did not impair the effects of NGF on the axonal microtubule cytoskeleton during branching. The effects of NGF on Rac1 activity and increases in axonal levels of WAVE1 and cortactin were both dependent on phosphoinositide 3-kinase (PI3K) signaling. Collectively, the data indicate that NGF promotes sensory axon branching through regulation of the actin cytoskeleton using both canonical signaling mechanisms and intra-axonal protein synthesis downstream of PI3K signaling. Finally, we present experimental evidence of axonal mRNA translation in sensory axons in the living embryonic spinal cord.