A role for ß-catenin in diet-induced skeletal muscle insulin resistance.

A central characteristic of insulin resistance is the impaired ability for insulin to stimulate glucose uptake into skeletal muscle. While insulin resistance can occur distal to the canonical insulin receptor-PI3k-Akt signaling pathway, the signaling intermediates involved in the dysfunction are yet to be fully elucidated. ß-catenin is an emerging distal regulator of skeletal muscle and adipocyte insulin-stimulated GLUT4 trafficking. Here, we investigate its role in skeletal muscle insulin resistance. Short-term (5-week) high-fat diet (HFD) decreased skeletal muscle ß-catenin protein expression 27% (p = 0.03), and perturbed insulin-stimulated ß-cateninS552 phosphorylation 21% (p = 0.009) without affecting insulin-stimulated Akt phosphorylation relative to chow-fed controls. Under chow conditions, mice with muscle-specific ß-catenin deletion had impaired insulin responsiveness, whereas under HFD, both mice exhibited similar levels of insulin resistance (interaction effect of genotype × diet p < 0.05). Treatment of L6-GLUT4-myc myocytes with palmitate lower ß-catenin protein expression by 75% (p = 0.02), and attenuated insulin-stimulated ß-catenin phosphorylationS552 and actin remodeling (interaction effect of insulin × palmitate p < 0.05). Finally, ß-cateninS552 phosphorylation was 45% lower in muscle biopsies from men with type 2 diabetes while total ß-catenin expression was unchanged. These findings suggest that ß-catenin dysfunction is associated with the development of insulin resistance.

The minor allele of the CREBRF rs373863828 p.R457Q coding variant is associated with reduced levels of myostatin in males: Implications for body composition.

OBJECTIVE: The minor allele (A) of the rs373863828 variant (p.Arg457Gln) in CREBRF is restricted to indigenous peoples of the Pacific islands (including New Zealand Maori and peoples of Polynesia), with a frequency of up to 25% in these populations. This allele associates with a large increase in body mass index (BMI) but with significantly lower risk of type-2 diabetes (T2D). It remains unclear whether the increased BMI is driven by increased adiposity or by increased lean mass. METHODS: We undertook body composition analysis using DXA in 189 young men of Maori and Pacific descent living in Aotearoa New Zealand. Further investigation was carried out in two orthologous Arg458Gln knockin mouse models on FVB/NJ and C57BL/6j backgrounds. RESULTS: The rs373863828 A allele was associated with lower fat mass when adjusted for BMI (p < 0.05) and was associated with significantly lower circulating levels of the muscle inhibitory hormone myostatin (p < 0.05). Supporting the human data, significant reductions in adipose tissue mass were observed in the knockin mice. This was more significant in older mice in both backgrounds and appeared to be the result of reduced age-associated increases in fat mass. The older male knockin mice on C57BL/6j background also had increased grip strength (p < 0.01) and lower levels of myostatin (p < 0.05). CONCLUSION: Overall, these results prove that the rs373863828 A-allele is associated with a reduction of myostatin levels which likely contribute to an age-dependent lowering of fat mass, at least in males.

Effect of immune modulation on the skeletal muscle mitochondrial exercise response: An exploratory study in mice with cancer.

Cancer causes mitochondrial alterations in skeletal muscle, which may progress to muscle wasting and, ultimately, to cancer cachexia. Understanding how exercise adaptations are altered by cancer and cancer treatment is important for the effective design of exercise interventions aimed at improving cancer outcomes. We conducted an exploratory study to investigate how tumor burden and cancer immunotherapy treatment (anti-PD-1) modify the skeletal muscle mitochondrial response to exercise training in mice with transplantable tumors (B16-F10 melanoma and EO771 breast cancer). Mice remained sedentary or were provided with running wheels for ~19 days immediately following tumor implant while receiving no treatment (Untreated), isotype control antibody (IgG2a) or anti-PD-1. Exercise and anti-PD-1 did not alter the growth rate of either tumor type, either alone or in combination therapy. Untreated mice with B16-F10 tumors showed increases in most measured markers of skeletal muscle mitochondrial content following exercise training, as did anti-PD-1-treated mice, suggesting increased mitochondrial content following exercise training in these groups. However, mice with B16-F10 tumors receiving the isotype control antibody did not exhibit increased skeletal muscle mitochondrial content following exercise. In untreated mice with EO771 tumors, only citrate synthase activity and complex IV activity were increased following exercise. In contrast, IgG2a and anti-PD-1-treated groups both showed robust increases in most measured markers following exercise. These results indicate that in mice with B16-F10 tumors, IgG2a administration prevents exercise adaptation of skeletal muscle mitochondria, but adaptation remains intact in mice receiving anti-PD-1. In mice with EO771 tumors, both IgG2a and anti-PD-1-treated mice show robust skeletal muscle mitochondrial exercise responses, while untreated mice do not. Taken together, we postulate that immune modulation may enhance skeletal muscle mitochondrial response to exercise in tumor-bearing mice, and suggest this as an exciting new avenue for future research in exercise oncology.

Mitochondrial-derived peptides and exercise.

Acute exercise, and in particular aerobic exercise, increases skeletal muscle energy demand causing mitochondrial stress, and mitochondrial-related adaptations which are a hallmark of exercise training. Given that mitochondria are central players in the exercise response, it is imperative that they have networks that can communicate their status both intra- and inter-cellularly. Peptides encoded by short open-reading frames within mitochondrial DNA, mitochondrial-derived peptides (MDPs), have been suggested to form a newly recognised branch of this retrograde signalling cascade that contribute to coordinating the adaptive response to regular exercise. Here we summarise the recent evidence that acute high intensity exercise in humans can increase concentrations of the MDPs humanin and MOTS-c in skeletal muscle and plasma, and speculate on the mechanisms controlling MDP responses to exercise stress. Evidence that exercise training results in chronic changes in MDP expression within tissues and the circulation is conflicting and may depend on the mode, duration, intensity of training plan and participant characteristics. Further research is required to define the effect of these variables on MDPs and to determine whether MDPs other than MOTS-c have exercise mimetic properties. MOTS-c treatment of young and aged mice improves exercise capacity/performance and leads to adaptions that are similar to that of being physically active (weight loss, increased antioxidant capacity and improved insulin sensitivity), however, studies utilising a MOTS-c inactivating genetic variant or combination of exercise + MOTS-c treatment in mice suggest that there are distinct and overlapping pathways through which exercise and MOTS-c evoke metabolic benefits. Overall, MOTS-c, and potentially other MDPs, may be exercise-sensitive myokines and further work is required to define inter- and intra-tissue targets in an exercise context.

The CREBRF diabetes-protective rs373863828-A allele is associated with enhanced early insulin release in men of Maori and Pacific ancestry.

AIMS/HYPOTHESIS: The minor A allele of rs373863828 (CREBRF p.Arg457Gln) is associated with increased BMI, but reduced risk of type 2 and gestational diabetes in Polynesian (Pacific peoples and Aotearoa New Zealand Maori) populations. This study investigates the effect of the A allele on insulin release and sensitivity in overweight/obese men without diabetes. METHODS: A mixed meal tolerance test was completed by 172 men (56 with the A allele) of Maori or Pacific ancestry, and 44 (24 with the A allele) had a frequently sampled IVGTT and hyperinsulinaemic-euglycaemic clamp. Mixed linear models with covariates age, ancestry and BMI were used to analyse the association between the A allele of rs373863828 and markers of insulin release and blood glucose regulation. RESULTS: The A allele of rs373863828 is associated with a greater increase in plasma insulin 30 min following a meal challenge without affecting the elevation in plasma glucose or incretins glucagon-like polypeptide-1 or gastric inhibitory polypeptide. Consistent with this point, following an i.v. infusion of a glucose bolus, participants with an A allele had higher early (p < 0.05 at 2 and 4 min) plasma insulin and C-peptide concentrations for a similar elevation in blood glucose as those homozygous for the major (G) allele. Despite increased plasma insulin, rs373863828 genotype was not associated with a significant difference (p > 0.05) in insulin sensitivity index or glucose disposal during hyperinsulinaemic-euglycaemic clamp. CONCLUSIONS/INTERPRETATION: rs373863828-A allele associates with increased glucose-stimulated insulin release without affecting insulin sensitivity, suggesting that CREBRF p.Arg457Gln may increase insulin release to reduce the risk of type 2 diabetes.

Efficacy of Providing the PI3K p110α Inhibitor BYL719 (Alpelisib) to Middle-Aged Mice in Their Diet.

BYL719 (alpelisib) is a small molecule inhibitor of PI3K p110α developed for cancer therapy. Targeted suppression of PI3K has led to lifespan extension in rodents and model organisms. If PI3K inhibitors are to be considered as an aging therapeutic, it is important to understand the potential consequences of long-term exposure, and the most practical way to achieve this is through diet administration. Here, we investigated the pharmacokinetics of BYL719 delivered in diet and the efficacy of BYL719 to suppress insulin signaling when administered in the diet of 8-month-old male and female mice. Compared to oral gavage, diet incorporation resulted in a lower peak plasma BYL719 (3.6 vs. 9.2 µM) concentration but similar half-life (~1.5 h). Consuming BYL719 resulted in decreased insulin signaling in liver and muscle within 72 h, and mice still showed impaired glucose tolerance and insulin sensitivity following 6 weeks of access to a diet containing 0.3 g/kg BYL719. However, consuming BYL719 did not affect food intake, body mass, muscle function (rotarod and hang time performance) or cognitive behaviors. This provides evidence that BYL719 has long-term efficacy without major toxicity or side effects, and suggests that administering BYL719 in diet is suitable for studying the effect of pharmacological suppression of PI3K p110α on aging and metabolic function.

Prolonged treatment with a PI3K p110α inhibitor causes sex- and tissue-dependent changes in antioxidant content, but does not affect mitochondrial function.

Genetic inhibition of the p110α isoform of phosphatidylinositol-3-kinase (PI3K) can increase murine lifespan, enhance mitochondrial function and alter tissue-specific oxidative balance. Here, we investigated whether pharmacological inhibition of the p110α isoform of PI3K induces similar enhancement of mitochondrial function in middle-aged mice. Eight-month-old male and female mice were fed a diet containing 0.3 g/kg of the p110α-selective inhibitor BYL-719 (BYL) or a vehicle diet (VEH) for 6 weeks. Mice consuming BYL-719 had higher blood glucose and insulin, and tended towards decreased body weight. After 72 h, gene expression of the mitochondrial biogenesis mediators Pgc1α, Tfam and Nrf1 was greater in liver of BYL-719 males only, but unchanged in skeletal muscle of either sex. Six weeks of BYL-719 treatment did not affect mitochondrial content or function in the liver or skeletal muscle of either sex. In livers of males only, the expression of the antioxidant genes Nfe2l2, Cat, Sod1 and Sod2 increased within 72 h of BYL-719 treatment, and remained higher after 6 weeks. This was associated with an increase in hepatic GSH content and catalase protein expression, and lower H2O2 levels. Our results suggest that pharmacological inhibition of p110α in adult mice does not affect liver or skeletal muscle mitochondrial function, but does show sex- and tissue-specific effects on up-regulation of antioxidant response.

Mitochondrial-derived peptides in energy metabolism.

Mitochondrial-derived peptides (MDPs) are small bioactive peptides encoded by short open-reading frames (sORF) in mitochondrial DNA that do not necessarily have traditional hallmarks of protein-coding genes. To date, eight MDPs have been identified, all of which have been shown to have various cyto- or metaboloprotective properties. The 12S ribosomal RNA (MT-RNR1) gene harbors the sequence for MOTS-c, whereas the other seven MDPs [humanin and small humanin-like peptides (SHLP) 1-6] are encoded by the 16S ribosomal RNA gene. Here, we review the evidence that endogenous MDPs are sensitive to changes in metabolism, showing that metabolic conditions like obesity, diabetes, and aging are associated with lower circulating MDPs, whereas in humans muscle MDP expression is upregulated in response to stress that perturbs the mitochondria like exercise, some mtDNA mutation-associated diseases, and healthy aging, which potentially suggests a tissue-specific response aimed at restoring cellular or mitochondrial homeostasis. Consistent with this, treatment of rodents with humanin, MOTS-c, and SHLP2 can enhance insulin sensitivity and offer protection against a range of age-associated metabolic disorders. Furthermore, assessing how mtDNA variants alter the functions of MDPs is beginning to provide evidence that MDPs are metabolic signal transducers in humans. Taken together, MDPs appear to form an important aspect of a retrograde signaling network that communicates mitochondrial status with the wider cell and to distal tissues to modulate adaptative responses to metabolic stress. It remains to be fully determined whether the metaboloprotective properties of MDPs can be harnessed into therapies for metabolic disease.

High-intensity interval exercise increases humanin, a mitochondrial encoded peptide, in the plasma and muscle of men.

Humanin is a small regulatory peptide encoded within the 16S ribosomal RNA gene (MT-RNR2) of the mitochondrial genome that has cellular cyto- and metabolo-protective properties similar to that of aerobic exercise training. Here we investigated whether acute high-intensity interval exercise or short-term high-intensity interval training (HIIT) impacted skeletal muscle and plasma humanin levels. Vastus lateralis muscle biopsies and plasma samples were collected from young healthy untrained men (n = 10, 24.5 ± 3.7 yr) before, immediately following, and 4 h following the completion of 10 × 60 s cycle ergometer bouts at V̇o2peak power output (untrained). Resting and postexercise sampling was also performed after six HIIT sessions (trained) completed over 2 wk. Humanin protein abundance in muscle and plasma were increased following an acute high-intensity exercise bout. HIIT trended (P = 0.063) to lower absolute humanin plasma levels, without effecting the response in muscle or plasma to acute exercise. A similar response in the plasma was observed for the small humanin-like peptide 6 (SHLP6), but not SHLP2, indicating selective regulation of peptides encoded by MT-RNR2 gene. There was a weak positive correlation between muscle and plasma humanin levels, and contraction of isolated mouse EDL muscle increased humanin levels ~4-fold. The increase in muscle humanin levels with acute exercise was not associated with MT-RNR2 mRNA or humanin mRNA levels (which decreased following acute exercise). Overall, these results suggest that humanin is an exercise-sensitive mitochondrial peptide and acute exercise-induced humanin responses in muscle are nontranscriptionally regulated and may partially contribute to the observed increase in plasma concentrations.NEW & NOTEWORTHY Small regulatory peptides encoded within the mitochondrial genome (mitochondrial derived peptides) have been shown to have cellular cyto- and metabolo-protective roles that parallel those of exercise. Here we provide evidence that humanin and SHLP6 are exercise-sensitive mitochondrial derived peptides. Studies to determine whether mitochondrial derived peptides play a role in regulating exercise-induced adaptations are warranted.

Increased expression of the mitochondrial derived peptide, MOTS-c, in skeletal muscle of healthy aging men is associated with myofiber composition.

Mitochondria putatively regulate the aging process, in part, through the small regulatory peptide, mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) that is encoded by the mitochondrial genome. Here we investigated the regulation of MOTS-c in the plasma and skeletal muscle of healthy aging men. Circulating MOTS-c reduced with age, but older (70-81 y) and middle-aged (45-55 y) men had ~1.5-fold higher skeletal muscle MOTS-c expression than young (18-30 y). Plasma MOTS-c levels only correlated with plasma in young men, was associated with markers of slow-type muscle, and associated with improved muscle quality in the older group (maximal leg-press load relative to thigh cross-sectional area). Using small mRNA assays we provide evidence that MOTS-c transcription may be regulated independently of the full length 12S rRNA gene in which it is encoded, and expression is not associated with antioxidant response element (ARE)-related genes as previously seen in culture. Our results suggest that plasma and muscle MOTS-c are differentially regulated with aging, and the increase in muscle MOTS-c expression with age is consistent with fast-to-slow type muscle fiber transition. Further research is required to determine the molecular targets of endogenous MOTS-c in human muscle but they may relate to factors that maintain muscle quality.

The CSF1 receptor inhibitor pexidartinib (PLX3397) reduces tissue macrophage levels without affecting glucose homeostasis in mice.

BACKGROUND AND OBJECTIVES: Excessive adipose tissue macrophage accumulation in obesity has been implicated in mediating inflammatory responses that impair glucose homeostasis and promote insulin resistance. Colony-stimulating factor 1 (CSF1) controls macrophage differentiation, and here we sought to determine the effect of a CSF1 receptor inhibitor, PLX3397, on adipose tissue macrophage levels and understand the impact on glucose homeostasis in mice. METHODS: A Ten-week-old mice were fed a chow or high-fat diet for 10 weeks and then treated with PLX3397 via oral gavage (50 mg/kg) every second day for 3 weeks, with subsequent monitoring of glucose tolerance, insulin sensitivity and assessment of adipose tissue immune cells. RESULTS: PLX3397 treatment substantially reduced macrophage numbers in adipose tissue of both chow and high-fat diet fed mice without affecting total myeloid cell levels. Despite this, PLX3397 did not greatly alter glucose homeostasis, did not affect high-fat diet-induced increases in visceral fat cytokine expression (Il-6 and Tnfa) and had limited effect on the phosphorylation of the stress kinases JNK and ERK and macrophage polarization. CONCLUSIONS: Our results indicate that macrophage infiltration of adipose tissue induced by a high-fat diet may not be the trigger for impairments in whole body glucose homeostasis, and that anti-CSF1 therapies are not likely to be useful as treatments for insulin resistance.

The Maori and Pacific specific CREBRF variant and adult height.

BACKGROUND: The CREBRF missense variant (p.Arg457Gln) is paradoxically associated with lower risk of type 2 diabetes, yet higher body mass index (BMI). Here we sought to determine whether this CREBRF variant might be associated with adult height. METHODS: Linear regression was used to analyse the association of the CREBRF minor (A) allele with height in 2286 Maori and Pacific adults living in Aotearoa/New Zealand. A potential type 2 diabetes index event was corrected to account for a bias that may be the cause of paradoxical association between the CREBRF diabetes-protective allele and higher BMI and height. RESULTS: The CREBRF protective allele was associated with increased adult height (ß = 1.25 cm, P = 3.9 × 10-6), with the effect being more pronounced in males. The lower odds of diabetes remained similar when analyses were adjusted for height (OR = 0.67-0.65). We found no evidence of a diabetes index event bias to explain the paradoxical effect of CREBRF with either BMI or height and diabetes. The orthologous CREBRF p.Arg457Gln variant was created in knock-in mice to independently assess the effect of the variant, and length was found to be greater in male mice at 8 weeks of age. CONCLUSION: These data taken together indicate that CREBRF p.Arg457Gln is associated with taller stature in Maori and Pacific adults.

Nuclear factor erythroid-derived 2-like 2 (NFE2L2, Nrf2) mediates exercise-induced mitochondrial biogenesis and the anti-oxidant response in mice.

KEY POINTS: Reactive oxygen species (ROS) and nitric oxide (NO) regulate exercise-induced nuclear factor erythroid 2-related factor 2 (NFE2L2) expression in skeletal muscle. NFE2L2 is required for acute exercise-induced increases in skeletal muscle mitochondrial biogenesis genes, such as nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A, and anti-oxidant genes, such as superoxide dismutase (SOD)1, SOD2 and catalase. Following exercise training mice with impaired NFE2L2 expression have reduced exercise performance, energy expenditure, mitochondrial volume and anti-oxidant activity. In muscle cells, ROS and NO can regulate mitochondrial biogenesis via a NFE2L2/NRF-1-dependent pathway. ABSTRACT: Regular exercise induces adaptations to skeletal muscle, which can include mitochondrial biogenesis and enhanced anti-oxidant reserves. These adaptations and others are at least partly responsible for the improved health of physically active individuals. Reactive oxygen species (ROS) and nitric oxide (NO) are produced during exercise and may mediate the adaptive response to exercise in skeletal muscle. However, the mechanisms through which they act are unclear. In the present study, we aimed to determine the role of the redox-sensitive transcription factor nuclear factor erythroid-derived 2-like 2 (NFE2L2) in acute exercise- and training-induced mitochondrial biogenesis and the anti-oxidant response. We report that ROS and NO regulate acute exercise-induced expression of NFE2L2 in mouse skeletal muscle and muscle cells, and that deficiency in NFE2L2 prevents normal acute treadmill exercise-induced increases in mRNA of the mitochondrial biogenesis markers, nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (mtTFA), and the anti-oxidants superoxide dismutase (SOD) 1 and 2, as well as catalase, in mouse gastrocnemius muscle. Furthermore, after 5 weeks of treadmill exercise training, mice deficient in NFE2L2 had reduced exercise capacity and whole body energy expenditure, as well as skeletal muscle mitochondrial mass and SOD activity, compared to wild-type littermates. In C2C12 myoblasts, acute treatment with exogenous H2 O2 (ROS)- and diethylenetriamine/NO adduct (NO donor) induced increases in mtTFA, which was prevented by small interfering RNA and short hairpin RNA knockdown of either NFE2L2 or NRF-1. Our results suggest that, during exercise, ROS and NO can act via NFE2L2 to functionally regulate skeletal muscle mitochondrial biogenesis and anti-oxidant defence gene expression.

Do antioxidant supplements interfere with skeletal muscle adaptation to exercise training?

A popular belief is that reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced during exercise by the mitochondria and other subcellular compartments ubiquitously cause skeletal muscle damage, fatigue and impair recovery. However, the importance of ROS and RNS as signals in the cellular adaptation process to stress is now evident. In an effort to combat the perceived deleterious effects of ROS and RNS it has become common practice for active individuals to ingest supplements with antioxidant properties, but interfering with ROS/RNS signalling in skeletal muscle during acute exercise may blunt favourable adaptation. There is building evidence that antioxidant supplementation can attenuate endurance training-induced and ROS/RNS-mediated enhancements in antioxidant capacity, mitochondrial biogenesis, cellular defence mechanisms and insulin sensitivity. However, this is not a universal finding, potentially indicating that there is redundancy in the mechanisms controlling skeletal muscle adaptation to exercise, meaning that in some circumstances the negative impact of antioxidants on acute exercise response can be overcome by training. Antioxidant supplementation has been more consistently reported to have deleterious effects on the response to overload stress and high-intensity training, suggesting that remodelling of skeletal muscle following resistance and high-intensity exercise is more dependent on ROS/RNS signalling. Importantly there is no convincing evidence to suggest that antioxidant supplementation enhances exercise-training adaptions. Overall, ROS/RNS are likely to exhibit a non-linear (hormetic) pattern on exercise adaptations, where physiological doses are beneficial and high exposure (which would seldom be achieved during normal exercise training) may be detrimental.

Mitohormesis in exercise training.

Hormesis is a process whereby exposure to a low dose of a potentially harmful stressor promotes adaptive changes to the cell that enables it to better tolerate subsequent stress. In recent years this concept has been applied specifically to the mitochondria (mitohormesis), suggesting that in response to a perturbation the mitochondria can initiate and transduce a signal to the nucleus that coordinates a transcriptional response resulting in both mitochondrial and non-mitochondrial adaptations that return and maintain cellular homeostasis. In this review we summarize the evidence that mitohormesis is a significant adaptive-response signaling pathway, and suggest that it plays a role in mediating exercise-induced adaptations. We discuss potential mitochondrial emitters of retrograde signals that may activate known exercise-sensitive transcription factors to modulate transcription responses to exercise, and draw on evidence from mitochondrial dysfunction animal models to support a role for mitohormesis in mitochondrial biogenesis. Studies directly linking mitohormesis to the exercise training response are lacking, however mounting evidence suggests numerous signals are emitted from the mitochondria during exercise and have the potential to induce a nuclear transcription response, with reactive oxygen species (ROS) being the primary candidate.

D-Glucosamine supplementation extends life span of nematodes and of ageing mice.

D-Glucosamine (GlcN) is a freely available and commonly used dietary supplement potentially promoting cartilage health in humans, which also acts as an inhibitor of glycolysis. Here we show that GlcN, independent of the hexosamine pathway, extends Caenorhabditis elegans life span by impairing glucose metabolism that activates AMP-activated protein kinase (AMPK/AAK-2) and increases mitochondrial biogenesis. Consistent with the concept of mitohormesis, GlcN promotes increased formation of mitochondrial reactive oxygen species (ROS) culminating in increased expression of the nematodal amino acid-transporter 1 (aat-1) gene. Ameliorating mitochondrial ROS formation or impairment of aat-1-expression abolishes GlcN-mediated life span extension in an NRF2/SKN-1-dependent fashion. Unlike other calorie restriction mimetics, such as 2-deoxyglucose, GlcN extends life span of ageing C57BL/6 mice, which show an induction of mitochondrial biogenesis, lowered blood glucose levels, enhanced expression of several murine amino-acid transporters, as well as increased amino-acid catabolism. Taken together, we provide evidence that GlcN extends life span in evolutionary distinct species by mimicking a low-carbohydrate diet.

N-Acetylcysteine infusion does not affect glucose disposal during prolonged moderate-intensity exercise in humans.

There is evidence that reactive oxygen species (ROS) signalling is required for normal increases in glucose uptake during contraction of isolated mouse skeletal muscle, and that AMP-activated protein kinase (AMPK) is involved. The aim of this study was to determine whether ROS signalling is involved in the regulation of glucose disposal and AMPK activation during moderate-intensity exercise in humans. Nine healthy males completed 80 min of cycle ergometry at 62 +/- 1% of peak oxygen consumption ( V(O(2)peak).A 6,6-(2)H-glucose tracer was infused at rest and during exercise, and in a double-blind randomised cross-over design, N-acetylcysteine (NAC) or saline (CON) was co-infused. NAC was infused at 125 mg kg(1) h(1) for 15 min and then at 25 mg kg(1) h(1) for 20 min before and throughout exercise. NAC infusion elevated plasma NAC and cysteine, and muscle NAC and cysteine concentrations during exercise. Although neither NAC infusion nor exercise significantly affected muscle reduced or oxidised glutathione (GSH or GSSG) concentration (P > 0.05), S-glutathionylation (an indicator of oxidative stress) of a protein band of approximately 270 kDa was increased approximately 3-fold with contraction and this increase was prevented by NAC infusion. Despite this, exercised-induced increases in tracer determined glucose disposal, plasma lactate, plasma non-esterified fatty acids (NEFAs), and decreases in plasma insulin were not affected by NAC infusion. In addition, skeletal muscle AMPKalpha and acetyl-CoA carboxylase-beta (ACCbeta) phosphorylation increased during exercise by approximately 3- and approximately 6-fold (P < 0.05), respectively, and this was not affected by NAC infusion. Unlike findings in mouse muscle ex vivo, NAC does not attenuate skeletal muscle glucose disposal or AMPK activation during moderate-intensity exercise in humans.

Skeletal muscle glucose uptake during contraction is regulated by nitric oxide and ROS independently of AMPK.

Reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in the regulation of skeletal muscle glucose uptake during contraction, and there is evidence that they do so via interaction with AMP-activated protein kinase (AMPK). In this study, we tested the hypothesis that ROS and NO regulate skeletal muscle glucose uptake during contraction via an AMPK-independent mechanism. Isolated extensor digitorum longus (EDL) and soleus muscles from mice that expressed a muscle-specific kinase dead AMPKalpha2 isoform (AMPK-KD) and wild-type litter mates (WT) were stimulated to contract, and glucose uptake was measured in the presence or absence of the antioxidant N-acetyl-l-cysteine (NAC) or the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-l-arginine (l-NMMA). Contraction increased AMPKalpha2 activity in WT but not AMPK-KD EDL muscles. However, contraction increased glucose uptake in the EDL and soleus muscles of AMPK-KD and WT mice to a similar extent. In EDL muscles, NAC and l-NMMA prevented contraction-stimulated increases in oxidant levels (dichloroflourescein fluorescence) and NOS activity, respectively, and attenuated contraction-stimulated glucose uptake in both genotypes to a similar extent. In soleus muscles of AMPK-KD and WT mice, NAC prevented contraction-stimulated glucose uptake and l-NMMA had no effect. This is likely attributed to the relative lack of neuronal NOS in the soleus muscles compared with EDL muscles. Contraction increased AMPKalpha Thr(172) phosphorylation in EDL and soleus muscles of WT but not AMPK-KD mice, and this was not affected by NAC or l-NMMA treatment. In conclusion, ROS and NO are involved in regulating skeletal muscle glucose uptake during contraction via an AMPK-independent mechanism.