Minimal adaptation of the molecular regulators of mitochondrial dynamics in response to unilateral limb immobilisation and retraining in middle-aged men.

PURPOSE: Mitochondrial dynamics are regulated by the differing molecular pathways variously governing biogenesis, fission, fusion, and mitophagy. Adaptations in mitochondrial morphology are central in driving the improvements in mitochondrial bioenergetics following exercise training. However, there is a limited understanding of mitochondrial dynamics in response to inactivity. METHODS: Skeletal muscle biopsies were obtained from middle-aged males (n = 24, 49.4 ± 3.2 years) who underwent sequential 14-day interventions of unilateral leg immobilisation, ambulatory recovery, and resistance training. We quantified vastus lateralis gene and protein expression of key proteins involved in mitochondrial biogenesis, fusion, fission, and turnover in at baseline and following each intervention. RESULTS: PGC1α mRNA decreased 40% following the immobilisation period, and was accompanied by a 56% reduction in MTFP1 mRNA, a factor involved in mitochondrial fission. Subtle mRNA decreases were also observed in TFAM (17%), DRP1 (15%), with contrasting increases in BNIP3L and PRKN following immobilisation. These changes in gene expression were not accompanied by changes in respective protein expression. Instead, we observed subtle decreases in NRF1 and MFN1 protein expression. Ambulatory recovery restored mRNA and protein expression to pre-intervention levels of all altered components, except for BNIP3L. Resistance training restored BNIP3L mRNA to pre-intervention levels, and further increased mRNA expression of OPA-1, MFN2, MTFP1, and PINK1 past baseline levels. CONCLUSION: In healthy middle-aged males, 2 weeks of immobilisation did not induce dramatic differences in markers of mitochondria fission and autophagy. Restoration of ambulatory physical activity following the immobilisation period restored altered gene expression patterns to pre-intervention levels, with little evidence of further adaptation to resistance exercise training.

Circulatory miRNAs as Correlates of Elevated Intra-Pancreatic Fat Deposition in a Mixed Ethnic Female Cohort: The TOFI_Asia Study.

Ectopic lipid accumulation, including intra-pancreatic fat deposition (IPFD), exacerbates type 2 diabetes risk in susceptible individuals. Dysregulated circulating microRNAs (miRNAs) have been identified as correlating with clinical measures of pancreatitis, pancreatic cancer and type 1 diabetes. The aim of the current study was therefore to examine the association between circulating abundances of candidate miRNAs, IPFD and liver fat deposition as quantified using magnetic resonance imaging (MRI) and spectroscopy (MRS). Asian Chinese (n = 34; BMI = 26.7 ± 4.2 kg/m2) and European Caucasian (n = 34; BMI = 28.0 ± 4.5 kg/m2) females from the TOFI_Asia cohort underwent MRI and MRS analysis of pancreas (MR-%IPFD) and liver fat (MR-%liver fat), respectively, to quantify ectopic lipid deposition. Plasma miRNA abundances of a subset of circulatory miRNAs associated with IPFD and liver fat deposition were quantified by qRT-PCR. miR-21-3p and miR-320a-5p correlated with MR-%IPFD, plasma insulin and HOMA2-IR, but not MR-%liver fat. MR-%IPFD remained associated with decreasing miR-21-3p abundance following multivariate regression analysis. miR-21-3p and miR-320a were demonstrated to be negatively correlated with MR-%IPFD, independent of ethnicity. For miR-21-3p, this relationship persists with the inclusion of MR-%liver fat in the model, suggesting the potential for a wider application as a specific circulatory correlate of IPFD.

Daily protein supplementation attenuates immobilization-induced blunting of postabsorptive muscle mTORC1 activation in middle-aged men.

Disuse-induced muscle atrophy is accompanied by a blunted postprandial response of the mammalian target of rapamycin complex 1 (mTORC1) pathway. Conflicting observations exist as to whether postabsorptive mTORC1 pathway activation is also blunted by disuse and plays a role in atrophy. It is unknown whether changes in habitual protein intake alter mTORC1 regulatory proteins and how they may contribute to the development of anabolic resistance. The primary objective of this study was to characterize the downstream responsiveness of skeletal muscle mTORC1 activation and its upstream regulatory factors, following 14 days of lower limb disuse in middle-aged men (45-60 yr). The participants were further randomized to receive daily supplementation of 20 g/d of protein (n = 12; milk protein concentrate) or isocaloric carbohydrate placebo (n = 13). Immobilization reduced postabsorptive skeletal muscle phosphorylation of the mTORC1 downstream targets, 4E-BP1, P70S6K, and ribosomal protein S6 (RPS6), with phosphorylation of the latter two decreasing to a greater extent in the placebo, compared with the protein supplementation groups (37% ± 13% vs. 14% ± 11% and 38% ± 20% vs. 25% ± 8%, respectively). Sestrin2 protein was also downregulated following immobilization irrespective of supplement group, despite a corresponding increase in its mRNA content. This decrease in Sestrin2 protein was negatively correlated with the immobilization-induced change in the in silico-predicted regulator miR-23b-3p. No other measured upstream proteins were altered by immobilization or supplementation. Immobilization downregulated postabsorptive mTORC1 pathway activation, and 20 g/day of protein supplementation attenuated the decrease in phosphorylation of targets regulating muscle protein synthesis.

ß-Catenin is required for optimal exercise- and contraction-stimulated skeletal muscle glucose uptake.

KEY POINTS: Loss of ß-catenin impairs in vivo and isolated muscle exercise/contraction-stimulated glucose uptake. ß-Catenin is required for exercise-induced skeletal muscle actin cytoskeleton remodelling. ß-Catenin675 phosphorylation during exercise may be intensity dependent. ABSTRACT: The conserved structural protein ß-catenin is an emerging regulator of vesicle trafficking in multiple tissues and supports insulin-stimulated glucose transporter 4 (GLUT4) translocation in skeletal muscle by facilitating cortical actin remodelling. Actin remodelling may be a convergence point between insulin and exercise/contraction-stimulated glucose uptake. Here we investigated whether ß-catenin is involved in regulating exercise/contraction-stimulated glucose uptake. We report that the muscle-specific deletion of ß-catenin induced in adult mice (BCAT-mKO) impairs both exercise- and contraction (isolated muscle)-induced glucose uptake without affecting running performance or canonical exercise signalling pathways. Furthermore, high intensity exercise in mice and contraction of myotubes and isolated muscles led to the phosphorylation of ß-cateninS675 , and this was impaired by Rac1 inhibition. Moderate intensity exercise in control and Rac1 muscle-specific knockout mice did not induce muscle ß-cateninS675 phosphorylation, suggesting exercise intensity-dependent regulation of ß-cateninS675 . Introduction of a non-phosphorylatable S675A mutant of ß-catenin into myoblasts impaired GLUT4 translocation and actin remodelling stimulated by carbachol, a Rac1 and RhoA activator. Exercise-induced increases in cross-sectional phalloidin staining (F-actin marker) of gastrocnemius muscle was impaired in muscle from BCAT-mKO mice. Collectively our findings suggest that ß-catenin is required for optimal glucose transport in muscle during exercise/contraction, potentially via facilitating actin cytoskeleton remodelling.

α1-Antitrypsin A treatment attenuates neutrophil elastase accumulation and enhances insulin sensitivity in adipose tissue of mice fed a high-fat diet.

Neutrophils accumulate in insulin-sensitive tissues during obesity and may play a role in impairing insulin sensitivity. The major serine protease expressed by neutrophils is neutrophil elastase (NE), which is inhibited endogenously by α1-antitrypsin A (A1AT). We investigated the effect of exogenous (A1AT) treatment on diet-induced metabolic dysfunction. Male C57Bl/6j mice fed a chow or a high-fat diet (HFD) were randomized to receive intraperitoneal injections three times weekly of either Prolastin (human A1AT; 2 mg) or vehicle (PBS) for 10 wk. Prolastin treatment did not affect plasma NE concentration, body weight, glucose tolerance, or insulin sensitivity in chow-fed mice. In contrast, Prolastin treatment attenuated HFD-induced increases in plasma and white adipose tissue (WAT) NE without affecting circulatory neutrophil levels or increases in body weight. Prolastin-treated mice fed a HFD had improved insulin sensitivity, as assessed by insulin tolerance test, and this was associated with higher insulin-dependent IRS-1 (insulin receptor substrate) and AktSer473 phosphorylation, and reduced inflammation markers in WAT but not liver or muscle. In 3T3-L1 adipocytes, Prolastin reversed recombinant NE-induced impairment of insulin-stimulated glucose uptake and IRS-1 phosphorylation. Furthermore, PDGF mediated p-AktSer473 activation and glucose uptake (which is independent of IRS-1) was not affected by recombinant NE treatment. Collectively, our findings suggest that NE infiltration of WAT during metabolic overload contributes to insulin resistance by impairing insulin-induced IRS-1 signaling.NEW & NOTEWORTHY Neutrophils accumulate in peripheral tissues during obesity and are critical coordinators of tissue inflammatory responses. Here, we provide evidence that inhibition of the primary neutrophil protease, neutrophil elastase, with α1-antitrypsin A (A1AT) can improve insulin sensitivity and glucose homeostasis of mice fed a high-fat diet. This was attributed to improved insulin-induced IRS-1 phosphorylation in white adipose tissue and provides further support for a role of neutrophils in mediating diet-induced peripheral tissue insulin resistance.

Use of Human Induced Pluripotent Stem Cells and Kidney Organoids To Develop a Cysteamine/mTOR Inhibition Combination Therapy for Cystinosis.

BACKGROUND: Mutations in CTNS-a gene encoding the cystine transporter cystinosin-cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments. METHODS: To address a lack of good human-based cell culture models for studying cystinosis, we generated the first human induced pluripotent stem cell (iPSC) and kidney organoid models of the disorder. We used a variety of techniques to examine hallmarks of cystinosis-including cystine accumulation, lysosome size, the autophagy pathway, and apoptosis-and performed RNA sequencing on isogenic lines to identify differentially expressed genes in the cystinosis models compared with controls. RESULTS: Compared with controls, these cystinosis models exhibit elevated cystine levels, increased apoptosis, and defective basal autophagy. Cysteamine treatment ameliorates this phenotype, except for abnormalities in apoptosis and basal autophagy. We found that treatment with everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes, decreases apoptosis, and activates autophagy, but it does not rescue the defect in cystine loading. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and everolimus corrects all of the observed phenotypic abnormalities. CONCLUSIONS: These observations suggest that combination therapy with a cystine-depleting drug such as cysteamine and an mTOR pathway inhibitor such as everolimus has potential to improve treatment of cystinosis.

MitoQ and CoQ10 supplementation mildly suppresses skeletal muscle mitochondrial hydrogen peroxide levels without impacting mitochondrial function in middle-aged men.

PURPOSE: Excess production of reactive oxygen species (ROS) from the mitochondria can promote mitochondrial dysfunction and has been implicated in the development of a range of chronic diseases. As such there is interest in whether mitochondrial-targeted antioxidant supplementation can attenuate mitochondrial-associated oxidative stress. We investigated the effect of MitoQ and CoQ10 supplementation on oxidative stress and skeletal muscle mitochondrial ROS levels and function in healthy middle-aged men. METHODS: Skeletal muscle and blood samples were collected from twenty men (50 ± 1 y) before and following six weeks of daily supplementation with MitoQ (20 mg) or CoQ10 (200 mg). High-resolution respirometry was used to determine mitochondrial respiration and H2O2 levels, markers of mitochondrial mass and antioxidant defences were measured in muscle samples and oxidative stress markers in urine and blood samples. RESULTS: Both MitoQ and CoQ10 supplementation suppressed mitochondrial net H2O2 levels during leak respiration, while MitoQ also elevated muscle catalase expression. However, neither supplement altered urine F2-isoprostanes nor plasma TBARS levels. Neither MitoQ nor CoQ10 supplementation had a significant impact on mitochondrial respiration or mitochondrial density markers (citrate synthase, mtDNA/nDNA, PPARGC1A, OXPHOS expression). CONCLUSION: Our results suggest that neither MitoQ and CoQ10 supplements impact mitochondrial function, but both can mildly suppress mitochondrial ROS levels in healthy middle-aged men, with some indication that MitoQ may be more effective than CoQ10.

Circulatory microRNAs are not effective biomarkers of muscle size and function in middle-aged men.

Loss of muscle size and strength with aging is a major cause of morbidity. Although muscle size and strength are measured by imaging or fiber cross-sectional staining and exercise testing, respectively, the development of circulatory biomarkers for these phenotypes would greatly simplify identification of muscle function deficits. MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene translation and, thereby, contribute to muscle phenotype. To assess circulatory miRNAs (c-miRNAs) applicability as potential biomarkers of muscular phenotypes, fasting plasma and muscle samples were obtained from 50 middle-aged healthy men [mean (SD); age: 48.8 yr (SD 4.5); BMI: 26.6 kg/m2 (SD 3.3)]. RT-PCR of 38 miRNAs with known regulatory function within skeletal muscle identified four c-miRNAs (miR-221, miR-451a, miR-361, and miR-146a) related to either total body lean mass, leg lean mass, and 50% thigh cross-sectional area (CSA), but not strength. There was no relationship with the expression of these miRNAs in muscle. Six miRNAs within muscle were correlated with whole body lean mass, leg lean mass, and isometric knee extension torque (miR-133a and miR-146a), and 50% thigh CSA (miR-486, miR-208b, miR-133b, and miR-208a). Only miR-23b demonstrated a relationship between tissue and circulatory expression; however, only 10% of the variance was explained. miR-146a in both plasma and muscle was related to phenotype; however, no relationship between plasma and muscle expression was evident. A different subset of miRNAs correlated to muscle phenotype in muscle compared with plasma samples, suggesting that c-miRNA biomarkers of muscle phenotype are likely unrelated to muscle expression in healthy individuals.

The Degree of Aminoacidemia after Dairy Protein Ingestion Does Not Modulate the Postexercise Anabolic Response in Young Men: A Randomized Controlled Trial.

BACKGROUND: Resistance exercise and dietary protein stimulate muscle protein synthesis (MPS). The rate at which proteins are digested and absorbed into circulation alters peak plasma amino acid concentrations and may modulate postexercise MPS. A novel mineral modified milk protein concentrate (mMPC), with identical amino acid composition to standard milk protein concentrate (MPC), was formulated to induce rapid aminoacidemia. OBJECTIVES: The aim of this study was to determine whether rapid aminoacidemia and greater peak essential amino acid (EAA) concentrations induced by mMPC would stimulate greater postresistance exercise MPS, anabolic signaling, and ribosome biogenesis compared to standard dairy proteins, which induce a small but sustained plasma essential aminoacidemia. METHODS: Thirty healthy young men (22.5 ± 3.0 y; BMI 23.8 ± 2.7 kg/m2) received primed constant infusions of l-[ring-13C6]-phenylalanine and completed 3 sets of leg presses and leg extensions at 80% of 1 repetition. Afterwards, participants were randomly assigned in a double-blind fashion to consume 25 g mMPC, MPC, or calcium caseinate (CAS). Vastus lateralis biopsies were collected at rest, and 2 and 4 h post exercise. RESULTS: Plasma EAA concentrations, including leucine, were 19.2-26.6% greater in the mMPC group 45-90 min post ingestion than in MPC and CAS groups (P < 0.001). Myofibrillar fractional synthetic rate from baseline to 4 h was increased by 82.6 ± 64.8%, 137.8 ± 72.1%, and 140.6 ± 52.4% in the MPC, mMPC, and CAS groups, respectively, with no difference between groups (P = 0.548). Phosphorylation of anabolic signaling targets (P70S6KThr389, P70S6KThr421/Ser424, RPS6Ser235/236, RPS6Ser240/244, P90RSKSer380, 4EBP1) were elevated by <3-fold at both 2 and 4 h post exercise in all groups (P < 0.05). CONCLUSIONS: The amplitude of plasma leucine and EAA concentrations does not modulate the anabolic response to resistance exercise after ingestion of 25 g dairy protein in young men. This trial was registered at http://www.anzctr.org.au/ as ACTRN12617000393358.

Identification of human skeletal muscle miRNA related to strength by high-throughput sequencing.

The loss of muscle size, strength, and quality with aging is a major determinant of morbidity and mortality in the elderly. The regulatory pathways that impact the muscle phenotype include the translational regulation maintained by microRNAs (miRNA). Yet the miRNAs that are expressed in human skeletal muscle and relationship to muscle size, strength, and quality are unknown. Using next-generation sequencing, we selected the 50 most abundantly expressed miRNAs and then analyzed them in vastus lateralis muscle, obtained by biopsy from middle-aged males ( n = 48; 50.0 ± 4.3 yr). Isokinetic strength testing and midthigh computed tomography was undertaken for muscle phenotype analysis. Muscle attenuation was measured by computerized tomography and is inversely proportional to myofiber lipid content. miR-486-5p accounted for 21% of total miR sequence reads, with miR-10b-5p, miR-133a-3p, and miR-22-3p accounting for a further 15, 12, and 10%, respectively. Isokinetic knee extension strength and muscle cross-sectional area were positively correlated with miR-100-5p, miR-99b-5p, and miR-191-5p expression. Muscle attenuation was negatively correlated to let-7f-5p, miR-30d-5p, and miR-125b-5p expression. In silico analysis implicates miRNAs related to strength and muscle size in the regulation of mammalian target of rapamycin, while miRNAs related to muscle attenuation may have potential roles regulating the transforming growth factor-ß/SMAD3 pathway.