miRNA in the Regulation of Skeletal Muscle Adaptation to Acute Endurance Exercise in C57Bl/6J Male Mice

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNA species involved in post-transcriptional gene regulation. In vitro studies have identified a small number of skeletal muscle-specific miRNAs which play a crucial role in myoblast proliferation and differentiation. In skeletal muscle, an acute bout of endurance exercise results in the up-regulation of transcriptional networks that regulate mitochondrial biogenesis, glucose and fatty acid metabolism, and skeletal muscle remodelling. The purpose of this study was to assess the expressional profile of targeted miRNA species following an acute bout of endurance exercise and to determine relationships with previously established endurance exercise responsive transcriptional networks. C57Bl/6J wild-type male mice (N = 7/group) were randomly assigned to either sedentary or forced-endurance exercise (treadmill run @ 15 m/min for 90 min) group. The endurance exercise group was sacrificed three hours following a single bout of exercise. The expression of miR- 181, 1, 133, 23, and 107, all of which have been predicted to regulate transcription factors and co-activators involved in the adaptive response to exercise, was measured in quadriceps femoris muscle. Endurance exercise significantly increased the expression of miR-181, miR-1, and miR-107 by 37%, 40%, and 56%, respectively, and reduced miR-23 expression by 84% (P≤0.05 for all), with no change in miR-133. Importantly, decreased expression of miRNA-23, a putative negative regulator of PGC-1α was consistent with increased expression of PGC-1α mRNA and protein along with several downstream targets of PGC-1α including ALAS, CS, and cytochrome c mRNA. PDK4 protein content remains unaltered despite an increase in its putative negative regulator, miR-107, and PDK4 mRNA expression. mRNA expression of miRNA processing machinery (Drosha, Dicer, and DGCR8) remained unchanged. We conclude that miRNA-mediated post-transcriptional regulation is potentially involved in the complex regulatory networks that govern skeletal muscle adaptation to endurance exercise in C57Bl/6J male mice

Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies

The reproducibility of 20-min time-trial performance on a virtual cycling platform.

This study aimed to analyse the reproducibility of mean power output during 20-min cycling time-trials, in a remote home-based setting, using the virtual-reality cycling software, Zwift. Forty-four cyclists (11 women, 33 men; 37 ± 8 years old, 180 ± 8 cm, 80.1 ± 13.2 kg) performed 3 x 20-min time-trials on Zwift, using their own setup. Intra-class correlation coefficient (ICC), coefficient of variation (CV) and typical error (TE) were calculated for the overall sample, split into 4 performance groups based on mean relative power output (25% quartiles) and sex. Mean ICC, TE and CV of mean power output between time-trials were 0.97 [0.95-0.98], 9.36 W [8.02-11.28 W], and 3.7% [3.2-4.5], respectively. Women and men had similar outcomes (ICC: 0.96 [0.89-0.99] vs 0.96 [0.92-0.98]; TE: 8.30 W [6.25-13.10] vs. 9.72 W [8.20-12.23]; CV: 3.8% [2.9-6.1] vs. 3.7% [3.1-4.7], respectively), although cyclists from the first quartile showed a lower CV in comparison to the overall sample (Q1: 2.6% [1.9-4.1] vs. overall: 3.7% [3.2-4.5]). Our results indicate that power output during 20-minute cycling time-trials on Zwift are reproducible and provide sports scientists, coaches and athletes, benchmark values for future interventions in a virtual-reality environment. [Abstract copyright: Thieme. All rights reserved.

Real-time PCR primer sequences.

<p>Real-time PCR primer sequences.</p

PDK4 content and miR-107 expression following exercise.

<p>(A) PDK4 mRNA expression and (B) protein content, and (C) miR-107 expression in the <i>quadriceps</i> of C57Bl/6J mice (N = 7/group) 3-hour following an acute bout of END exercise vs. SED group. PDK4 mRNA expression, protein content and miR-107 expression are normalized to β-2 microglobulin, actin and <i>Rnu6</i>, respectively. Asterisks denote significant changes (P≤0.05).</p

PGC-1α content and miR-23 expression following exercise.

<p>PGC-1α (A) mRNA expression and (B) protein content, and (C) miR-23 expression in the <i>quadriceps</i> of C57Bl/6J mice (N = 7/group) 3-hour following an acute bout of END exercise vs. SED group. (D) PGC-1α protein content negatively correlates (R = 0.62) with miR-23 content. PGC-1α mRNA expression, protein content and miR-23 expression are normalized to β-2 microglobulin, actin and <i>Rnu6</i>, respectively. Asterisks denote significant changes (P≤0.05).</p

miR-1 and miR-181 expression following exercise.

<p>miR-1 and miR-181 expression in the <i>quadriceps</i> of C57Bl/6J mice (N = 7/group) 3-hour following an acute bout of END exercise vs. SED group. miR-1 and miR-181 expression are normalized to <i>Rnu6</i>. Asterisks denote significant changes (P≤0.05).</p

A Novel Multi-Ingredient Supplement Activates a Browning Program in White Adipose Tissue and Mitigates Weight Gain in High-Fat Diet-Fed Mice

We investigated the effects of a novel multi-ingredient supplement comprised of polyphenol antioxidants and compounds known to facilitate mitochondrial function and metabolic enhancement (ME) in a mouse model of obesity. In this study, 6-week-old male C57/BL6J mice were placed on a high-fat diet (HFD; ~60% fat) for 6 weeks, with subsequent allocation into experimentalgroups for 4 weeks: HFD control, HFD + ME10 (10 components), HFD + ME7 (7 components), HFD + ME10 + EX, HFD + EX (where ‘+EX’ animals exercised 3 days/week), and chow-fed control. After the intervention, HFD control animals had significantly greater body weight and fat mass. Despite the continuation of HFD, animals supplemented with multi-ingredient ME or who performed exercise training showed an attenuation of fat mass and preservation of lean body mass, which was further enhanced when combined (ME+EX). ME supplementation stimulated the upregulation of white and brown adipose tissue mRNA transcripts associated with mitochondrial biogenesis, browning, fatty acid transport, and fat metabolism. In WAT depots, this was mirrored by mitochodrial oxidative phosphorylation (OXPHOS) protein expression, and increased in vivo fat oxidation measured via CLAMS. ME supplementation also decreased systemic and local inflammation markers. Herein, we demonstrated that novel multi-ingredient nutritional supplements induced significant fat loss independent of physical activity while preserving muscle mass in obese mice. Mechanistically, these MEs appear to act by inducing a browning program in white adipose tissue and decreasing other pathophysiological impairments associated with obesity, including mitochondrial respiration alterations induced by HFD

Elevated Mitochondrial Oxidative Stress Impairs Metabolic Adaptations to Exercise in Skeletal Muscle

<div><p>Mitochondrial oxidative stress is a complex phenomenon that is inherently tied to energy provision and is implicated in many metabolic disorders. Exercise training increases mitochondrial oxidative capacity in skeletal muscle yet it remains unclear if oxidative stress plays a role in regulating these adaptations. We demonstrate that the chronic elevation in mitochondrial oxidative stress present in <i>Sod2</i>^<i>+/-</i> mice impairs the functional and biochemical mitochondrial adaptations to exercise. Following exercise training <i>Sod2</i>^<i>+/-</i> mice fail to increase maximal work capacity, mitochondrial enzyme activity and mtDNA copy number, despite a normal augmentation of mitochondrial proteins. Additionally, exercised <i>Sod2</i>^<i>+/-</i> mice cannot compensate for their higher amount of basal mitochondrial oxidative damage and exhibit poor electron transport chain complex assembly that accounts for their compromised adaptation. Overall, these results demonstrate that chronic skeletal muscle mitochondrial oxidative stress does not impact exercise induced mitochondrial biogenesis, but impairs the resulting mitochondrial protein function and can limit metabolic plasticity.</p> </div

A Five-Ingredient Nutritional Supplement and Home-Based Resistance Exercise Improve Lean Mass and Strength in Free-Living Elderly

Old age is associated with lower physical activity levels, suboptimal protein intake, and desensitization to anabolic stimuli, predisposing for age-related muscle loss (sarcopenia). Although resistance exercise (RE) and protein supplementation partially protect against sarcopenia under controlled conditions, the efficacy of home-based, unsupervised RE (HBRE) and multi-ingredient supplementation (MIS) is largely unknown. In this randomized, placebo-controlled and double-blind trial, we examined the effects of HBRE/MIS on muscle mass, strength, and function in free-living, older men. Thirty-two sedentary men underwent twelve weeks of home-based resistance band training (3 d/week), in combination with daily intake of a novel five-nutrient supplement (&lsquo;Muscle5&rsquo;; M5, n = 16, 77.4 &plusmn; 2.8 y) containing whey, micellar casein, creatine, vitamin D, and omega-3 fatty acids, or an isocaloric/isonitrogenous placebo (PLA; n = 16, 74.4 &plusmn; 1.3 y), containing collagen and sunflower oil. Appendicular and total lean mass (ASM; +3%, TLM; +2%), lean mass to fat ratios (ASM/% body fat; +6%, TLM/% body fat; +5%), maximal strength (grip; +8%, leg press; +17%), and function (5-Times Sit-to-Stand time; &minus;9%) were significantly improved in the M5 group following HBRE/MIS therapy (pre vs. post tests; p &lt; 0.05). Fast-twitch muscle fiber cross-sectional areas of the quadriceps muscle were also significantly increased in the M5 group post intervention (Type IIa; +30.9%, Type IIx, +28.5%, p &lt; 0.05). Sub-group analysis indicated even greater gains in total lean mass in sarcopenic individuals following HBRE/MIS therapy (TLM; +1.65 kg/+3.4%, p &lt; 0.05). We conclude that the Muscle5 supplement is a safe, well-tolerated, and effective complement to low-intensity, home-based resistance exercise and improves lean mass, strength, and overall muscle quality in old age