Combined physical exercise reverses the reduced expression of Bmal1 in the liver of aged mice.

Aging can modify the morphology and function of the liver, such as generating a decrease in the mitochondria content, autophagy, and cell senescence. Although exercise training has several beneficial effects on hepatic metabolism, its actions on autophagy processes, mitochondrial function, and cellular senescence need to be more widely explored. The present study verified the effects of aging and exercise on hepatic circadian markers, autophagy, and mitochondria activity in 24-month-old mice with a combined exercise training protocol. In addition, we used public datasets from human livers in several conditions and BMAL1 knockout mice. C57BL/6 mice were distributed into Control (CT, young, 6-month-old mice), sedentary old (Old Sed, sedentary, 24-month-old mice), and exercised old (Old Ex, 24-month-old mice submitted to a combined exercise training protocol). The exercise training protocol consisted of three days of endurance exercise - treadmill running, and two days of resistance exercise - climbing a ladder, for three weeks. At the end of the protocol, the liver was removed and prepared for histological analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunoblotting technique, and oxygen consumption. Heatmaps were built using a human dataset and Bmal1 knockout samples. In summary, the Old Sed had reduced strength, coordination, and balance, as well as a decrease in Bmal1 expression and the presence of degenerated liver cells. Still, this group upregulated the transcription factors related to mitochondrial biogenesis. The Old Ex group had increased strength, coordination, and balance, improved glucose sensitivity, as well as restored Bmal1 expression and the mitochondrial transcription factors. The human datasets indicated that mitochondrial markers and autophagy strongly correlate with specific liver diseases but not aging. We can speculate that mitochondrial and autophagy molecular markers alterations may depend on long-term training.

Effect of vitamin D3 vs. calcifediol on VDR concentration and fiber size in skeletal muscle.

INTRODUCTION: This study sought to examine the effect of vitamin D3 (VD3) 3200 IU/d, calcifediol (HyD) 20mcg/d, or placebo on intramyonuclear vitamin D receptor (VDR) concentration, muscle fiber cross-sectional area (FCSA), and muscle satellite cell activation. MATERIALS AND METHODS: It was conducted on a subset of the VD3 (n = 12), HyD (n = 11), and placebo (n = 13) groups who participated in the 6-month randomized controlled HyD Osteopenia Study in postmenopausal women. Baseline and 6-month vastus lateralis muscle cross sections were probed for VDR, fiber type I and II, and PAX7 (satellite cell marker) using immunofluorescence. RESULTS: Baseline mean ± SD age was 61 ± 4 years and serum 25-hydroxyvitamin D (25OHD) level was 55.1 ± 22.8 nmol/L. Baseline characteristics did not differ significantly by group. Six-month mean ± SD 25OHD levels were 138.7 ± 22.2 nmol/L (VD3), 206.8 ± 68.8 nmol/L (HyD), and 82.7 ± 36.1 nmol/L (placebo), ANOVA P < 0.001. There were no significant group differences in 6-month change in VDR concentration (ANOVA P = 0.227). Mean ± SD percent 6-month changes in type I FCSA were 20.5 ± 32.7% (VD3), - 6.6 ± 20.4% (HyD), and - 0.3 ± 14.0% (placebo, ANOVA P = 0.022). Type II FCSA or PAX7 concentration did not change significantly by group (all P > 0.358). CONCLUSION: This study demonstrated no significant change in intramyonuclear VDR in response to either form of vitamin D vs. placebo. Type I FCSA significantly increased with VD3, but not with HyD at 6 months. As type I fibers are more fatigue resistant than type II, enlargement in type I suggests potential for improved muscle endurance. Although HyD resulted in the highest 25OHD levels, no skeletal muscle benefits were noted at these high levels. CLINICAL TRIAL: NCT02527668.

Exercise alters the circadian rhythm of REV-ERB-α and downregulates autophagy-related genes in peripheral and central tissues.

The transcriptional repressor REV-ERB-α, encoded by Nuclear Receptor Subfamily 1 Group D Member 1 (Nr1d1), has been considered to play an essential role in the skeletal muscle oxidative capacity adaptation and muscle mass control. Also, this molecule regulates autophagy via the repression of autophagy-related genes both in skeletal muscle and brain regions. Classically, training programs based on endurance or strength characteristics enhance skeletal muscle mass content and/or oxidative capacity, leading to autophagy activation in several tissues. Thus, it seems that REV-ERB-α regulates similar responses induced by exercise. However, how this molecule responds to different exercise models/intensities in different tissues is still unclear. Therefore, the main aim was to characterize the responses of REV-ERB-α and autophagy-related genes to different exercise protocols (endurance/interval run/strength) in distinct tissues (gastrocnemius, soleus and hippocampus). Since REV-ERB-α presents a circadian rhythm, the analyses were performed in a time-course manner. The endurance and strength groups attenuated REV-ERB-α transcriptional response during the time course in gastrocnemius and soleus. Conversely, the interval group enhanced the Nr1d1 expression in the hippocampus. All protocols downregulated the REV-ERB-α protein levels in gastrocnemius following the exercise session with concomitant nuclear exclusion. The major autophagy-related genes presented downregulation after the exercise session in all analyzed tissues. Altogether, these results highlight that REV-ERB-α is extremely sensitive to physical exercise stimuli, including different models and intensities in skeletal muscle and the hippocampus.

IL-6 deletion decreased REV-ERBα protein and influenced autophagy and mitochondrial markers in the skeletal muscle after acute exercise.

Interleukin 6 (IL-6) acts as a pro and anti-inflammatory cytokine, has an intense correlation with exercise intensity, and activates various pathways such as autophagy and mitochondrial unfolded protein response. Also, IL-6 is interconnected to circadian clock-related inflammation and can be suppressed by the nuclear receptor subfamily 1, group D, member 1 (Nr1d1, protein product REV-ERBα). Since IL-6 is linked to physical exercise-modulated metabolic pathways such as autophagy and mitochondrial metabolism, we investigated the relationship of IL-6 with REV-ERBα in the adaptations of these molecular pathways in response to acute intense physical exercise in skeletal muscle. The present study was divided into three experiments. In the first one, wild-type (WT) and IL-6 knockout (IL-6 KO) mice were divided into three groups: Basal time (Basal; sacrificed before the acute exercise), 1 hour (1hr post-Ex; sacrificed 1 hour after the acute exercise), and 3 hours (3hr post-Ex; sacrificed 3 hours after the acute exercise). In the second experiment, C2C12 cells received IL-6 physiological concentrations or REV-ERBα agonist, SR9009. In the last experiment, WT mice received SR9009 injections. After the protocols, the gastrocnemius muscle or the cells were collected for reverse transcription-quantitative polymerase chain reaction (RTq-PCR) and immunoblotting techniques. In summary, the downregulation of REV-ERBα, autophagic flux, and most mitochondrial genes was verified in the IL-6 KO mice independent of exercise. The WT and IL-6 KO treated with SR9009 showed an upregulation of autophagic genes. C2C12 cells receiving IL-6 did not modulate the Nr1d1 mRNA levels but upregulated the expression of some mitochondrial genes. However, when treated with SR9009, IL-6 and mitochondrial gene expression were upregulated in C2C12 cells. The autophagic flux in C2C12 suggest the participation of REV-ERBα protein in the IL-6-induced autophagy. In conclusion, the present study verified that the adaptations required through physical exercise (increases in mitochondrial content and improvement of autophagy machinery) might be intermediated by an interaction between IL-6 and REVERBα.

Characterization of cellular senescence in aging skeletal muscle.

Senescence is a cell fate that contributes to multiple aging-related pathologies. Despite profound age-associated changes in skeletal muscle (SkM), whether its constituent cells are prone to senesce has not been methodically examined. Herein, using single cell and bulk RNA-sequencing and complementary imaging methods on SkM of young and old mice, we demonstrate that a subpopulation of old fibroadipogenic progenitors highly expresses p16 Ink4a together with multiple senescence-related genes and, concomitantly, exhibits DNA damage and chromatin reorganization. Through analysis of isolated myofibers, we also detail a senescence phenotype within a subset of old cells, governed instead by p2 Cip1 . Administration of a senotherapeutic intervention to old mice countered age-related molecular and morphological changes and improved SkM strength. Finally, we found that the senescence phenotype is conserved in SkM from older humans. Collectively, our data provide compelling evidence for cellular senescence as a hallmark and potentially tractable mediator of SkM aging.

Carbohydrate intake in recovery from aerobic exercise differentiates skeletal muscle microRNA expression.

Posttranscriptional regulation by microRNA (miRNA) facilitates exercise and diet-induced skeletal muscle adaptations. However, the impact of diet on miRNA expression during postexercise recovery remains unclear. The objective of this study was to examine the effects of consuming carbohydrate or a nutrient-free control on skeletal muscle miRNA expression during 3 h of recovery from aerobic exercise. Using a randomized, crossover design, seven men (means ± SD, age: 21 ± 3 yr; body mass: 83 ± 13 kg; V̇o2peak: 43 ± 2 mL/kg/min) completed two-cycle ergometry glycogen depletion trials followed by 3 h of recovery while consuming either carbohydrate (CHO: 1 g/kg/h) or control (CON: nutrient free). Muscle biopsy samples were obtained under resting fasted conditions at baseline and at the end of the 3-h recovery (REC) period. miRNA expression was determined using unbiased RT-qPCR microarray analysis. Trials were separated by 7 days. Twenty-five miRNAs were different (P < 0.05) between CHO and CON at REC, with Let7i-5p and miR-195-5p being the most predictive of treatment. In vitro overexpression of Let7i-5p and miR-195-p5 in C2C12 skeletal muscle cells decreased (P < 0.05) the expression of protein breakdown (Foxo1, Trim63, Casp3, and Atf4) genes, ubiquitylation, and protease enzyme activity compared with control. Energy sensing (Prkaa1 and Prkab1) and glycolysis (Gsy1 and Gsk3b) genes were lower (P < 0.05) with Let7i-5p overexpression compared with miR-195-5p and control. Fat metabolism (Cpt1a, Scd1, and Hadha) genes were lower (P < 0.05) in miR-195-5p than in control. These data indicate that consuming CHO after aerobic exercise alters miRNA profiles compared with CON, and these differences may govern mechanisms facilitating muscle recovery.NEW & NOTEWORTHY Results provide novel insight into effects of carbohydrate intake on the expression of skeletal muscle microRNA during early recovery from aerobic exercise and reveal that Let7i-5p and miR-195-5p are important regulators of skeletal muscle protein breakdown to aid in facilitating muscle recovery.

Effects of Low Doses of L-Carnitine Tartrate and Lipid Multi-Particulate Formulated Creatine Monohydrate on Muscle Protein Synthesis in Myoblasts and Bioavailability in Humans and Rodents.

The primary objective of this study was to investigate the potential synergy between low doses of L-carnitine tartrate and creatine monohydrate to induce muscle protein synthesis and anabolic pathway activation in primary human myoblasts. In addition, the effects of Lipid multi-particulates (LMP) formulation on creatine stability and bioavailability were assessed in rodents and healthy human subjects. When used individually, L-carnitine tartrate at 50 µM and creatine monohydrate at 0.5 µM did not affect myoblast protein synthesis and signaling. However, when combined, they led to a significant increase in protein synthesis. Increased AKT and RPS6 phosphorylation were observed with 50 µM L-carnitine tartrate 5 µM creatine in combination in primary human myoblasts. When Wistar rats were administered creatine with LMP formulation at either 21 or 51 mg/kg, bioavailability was increased by 27% based on the increase in the area under the curve (AUC) at a 51 mg/kg dose compared to without LMP formulation. Tmax and Cmax were unchanged. Finally, in human subjects, a combination of LMP formulated L-carnitine at 500 mg (from L-carnitine tartrate) with LMP formulated creatine at 100, 200, or 500 mg revealed a significant and dose-dependent increase in plasma creatine concentrations. Serum total L-carnitine levels rose in a similar manner in the three combinations. These results suggest that a combination of low doses of L-carnitine tartrate and creatine monohydrate may lead to a significant and synergistic enhancement of muscle protein synthesis and activation of anabolic signaling. In addition, the LMP formulation of creatine improved its bioavailability. L-carnitine at 500 mg and LMP-formulated creatine at 200 or 500 mg may be useful for future clinical trials to evaluate the effects on muscle protein synthesis.

miR-19b-3p is associated with a diametric response to resistance exercise in older adults and regulates skeletal muscle anabolism via PTEN inhibition.

Understanding paradoxical responses to anabolic stimulation and identifying the mechanisms for this inconsistency in mobility-limited older adults may provide new targets for the treatment of sarcopenia. Our laboratory has discovered that dysregulation in microRNA (miRNA) that target anabolic pathways is a potential mechanism resulting in age-associated decreases in skeletal muscle mass and function (sarcopenia). The objective of the current study was to assess circulating miRNA expression profiles in diametric response of leg lean mass in mobility-limited older individuals after a 6-mo progressive resistance exercise training intervention (PRET) and determine the influence of differentially expressing miRNA on regulation of skeletal muscle mass. Participants were dichotomized by gain (Gainers; mean +561.4 g, n = 33) or loss (Losers; mean -589.8 g, n = 40) of leg lean mass after PRET. Gainers significantly increased fat-free mass 2.4% vs. -0.4% for Losers. Six miRNA (miR-1-3p, miR-19b-3p, miR-92a, miR-126, miR-133a-3p, and miR-133b) were significantly identified to be differentially expressed between Gainers and Losers, with miR-19b-3p being the miRNA most highly associated with increases in fat-free mass. Using an aging mouse model, we then assessed if miR-19b-3p expression was different in young mice with larger muscle mass compared with older mice. Circulating and skeletal muscle miR-19b-3p expression was higher in young compared with old mice and was positively associated with muscle mass and grip strength. We then used a novel integrative approach to determine if differences in circulating miR-19b-3p potentially translate to augmented anabolic response in human skeletal muscle cells in vitro. Results from this analysis identified that overexpression of miR-19b-3p targeted and downregulated PTEN by 64% to facilitate significant ∼50% increase in muscle protein synthetic rate as measured with SUnSET. The combine results of these three models identify miR-19b-3p as a potent regulator of muscle anabolism that may contribute to an inter-individual response to PRET in mobility-limited older adults.

Interleukin-6 ablation does not alter morphofunctional heart characteristics but modulates physiological and inflammatory markers after strenuous exercise.

Interleukin-6 (IL-6) is associated with pathological cardiac hypertrophy and can be dramatically increased in serum after an acute strenuous exercise session. However, IL-6 is also associated with the increased production and release of anti-inflammatory cytokines and the inhibition of tumor necrosis factor-alpha (TNF-α) after chronic moderate exercise. To elucidate the relevance of IL-6 in inflammatory and hypertrophic signaling in the heart in response to an acute strenuous exercise session, we combined transcriptome analysis using the BXD mice database and exercised IL-6 knockout mice (IL-6KO). Bioinformatic analysis demonstrated that low or high-levels of Il6 mRNA in the heart did not change the inflammation- and hypertrophy-related genes in BXD mice strains. On the other hand, bioinformatic analysis revealed a strong positive correlation between Il6 gene expression in skeletal muscle with inflammation-related genes in cardiac tissue in several BXD mouse strains, suggesting that skeletal muscle-derived IL-6 could alter the heart's intracellular signals, particularly the inflammatory signaling. As expected, an acute strenuous exercise session increased IL-6 levels in wild-type, but not in IL-6KO mice. Despite not showing morphofunctional differences in the heart at rest, the IL-6KO group presented a reduction in physical performance and attenuated IL-6, TNF-α, and IL-1beta kinetics in serum, as well as lower p38MAPK phosphorylation, Ampkalpha expression, and higher Acta1 and Tnf gene expressions in the left ventricle in the basal condition. In response to strenuous exercise, IL-6 ablation was linked to a reduction in the pro-inflammatory response and higher activation of classical physiological cardiac hypertrophy proteins.

Moderate, but Not Excessive, Training Attenuates Autophagy Machinery in Metabolic Tissues.

The protective effects of chronic moderate exercise-mediated autophagy include the prevention and treatment of several diseases and the extension of lifespan. In addition, physical exercise may impair cellular structures, requiring the action of the autophagy mechanism for clearance and renovation of damaged cellular components. For the first time, we investigated the adaptations on basal autophagy flux in vivo in mice's liver, heart, and skeletal muscle tissues submitted to four different chronic exercise models: endurance, resistance, concurrent, and overtraining. Measuring the autophagy flux in vivo is crucial to access the functionality of the autophagy pathway since changes in this pathway can occur in more than five steps. Moreover, the responses of metabolic, performance, and functional parameters, as well as genes and proteins related to the autophagy pathway, were addressed. In summary, the regular exercise models exhibited normal/enhanced adaptations with reduced autophagy-related proteins in all tissues. On the other hand, the overtrained group presented higher expression of Sqstm1 and Bnip3 with negative morphological and physical performance adaptations for the liver and heart, respectively. The groups showed different adaptions in autophagy flux in skeletal muscle, suggesting the activation or inhibition of basal autophagy may not always be related to improvement or impairment of performance.

Sphingosine-1-phosphate analog FTY720 reverses obesity but not age-induced anabolic resistance to muscle contraction.

Sarcopenia, the age-associated loss of skeletal muscle mass and function, is coupled with declines in physical functioning leading to subsequent higher rates of disability, frailty, morbidity, and mortality. Aging and obesity independently contribute to muscle atrophy that is assumed to be a result of the activation of mutual physiological pathways. Understanding mechanisms contributing to the induction of skeletal muscle atrophy with aging and obesity is important for determining targets that may have pivotal roles in muscle loss in these conditions. We find that aging and obesity equally induce an anabolic resistance to acute skeletal muscle contraction as observed with decreases in anabolic signaling activation after contraction. Furthermore, treatment with the sphingosine-1-phosphate analog FTY720 for 4 wk increased lean mass and strength, and the anabolic signaling response to contraction was improved in obese but not older animals. To determine the role of chronic inflammation and different fatty acids on anabolic resistance in skeletal muscle cells, we overexpressed IKKß with and without exposure to saturated fatty acid (SFA; palmitic acid), polyunsaturated fatty acid (eicosapentaenoic acid), and monounsaturated fatty acid (oleic acid). We found that IKKß overexpression increased inflammation markers in muscle cells, and this chronic inflammation exacerbated anabolic resistance in response to SFA. Pretreatment with FTY720 reversed the inflammatory effects of palmitic acid in the muscle cells. Taken together, these data demonstrate chronic inflammation can induce anabolic resistance, SFA aggravates these effects, and FTY720 can reverse this by decreasing ceramide accumulation in skeletal muscle.

JNK regulates muscle remodeling via myostatin/SMAD inhibition.

Skeletal muscle has a remarkable plasticity to adapt and remodel in response to environmental cues, such as physical exercise. Endurance exercise stimulates improvements in muscle oxidative capacity, while resistance exercise induces muscle growth. Here we show that the c-Jun N-terminal kinase (JNK) is a molecular switch that when active, stimulates muscle fibers to grow, resulting in increased muscle mass. Conversely, when muscle JNK activation is suppressed, an alternative remodeling program is initiated, resulting in smaller, more oxidative muscle fibers, and enhanced aerobic fitness. When muscle is exposed to mechanical stress, JNK initiates muscle growth via phosphorylation of the transcription factor, SMAD2, at specific linker region residues leading to inhibition of the growth suppressor, myostatin. In human skeletal muscle, this JNK/SMAD signaling axis is activated by resistance exercise, but not endurance exercise. We conclude that JNK acts as a key mediator of muscle remodeling during exercise via regulation of myostatin/SMAD signaling.

Pilot Study Examining the Influence of Potassium Bicarbonate Supplementation on Nitrogen Balance and Whole-Body Ammonia and Urea Turnover Following Short-Term Energy Restriction in Older Men.

With aging there is a chronic low-grade metabolic-acidosis that may exacerbate negative protein balance during weight loss. The objective of this randomized pilot study was to assess the impact of 90 mmol∙day-1 potassium bicarbonate (KHCO3) versus a placebo (PLA) on 24-h urinary net acid excretion (NAE), nitrogen balance (NBAL), and whole-body ammonia and urea turnover following short-term diet-induced weight loss. Sixteen (KHCO3; n = 8, PLA; n = 8) older (64 ± 4 years) overweight (BMI: 28.5 ± 2.1 kg∙day-1) men completed a 35-day controlled feeding study, with a 7-day weight-maintenance phase followed by a 28-day 30% energy-restriction phase. KHCO3 or PLA supplementation began during energy restriction. NAE, NBAL, and whole-body ammonia and urea turnover (15N-glycine) were measured at the end of the weight-maintenance and energy-restriction phases. Following energy restriction, NAE was -9.8 ± 27.8 mmol∙day-1 in KHCO3 and 43.9 ± 27.8 mmol∙day-1 in PLA (p < 0.05). No significant group or time differences were observed in NBAL or ammonia and urea turnover. Ammonia synthesis and breakdown tended (p = 0.09) to be higher in KHCO3 vs. PLA following energy restriction, and NAE was inversely associated (r = -0.522; p < 0.05) with urea synthesis in all subjects. This pilot study suggests some benefit may exist with KHCO3 supplementation following energy restriction as lower NAE indicated higher urea synthesis.

Potential Role of MicroRNA in the Anabolic Capacity of Skeletal Muscle With Aging.

Age-induced loss of skeletal muscle mass and function, termed sarcopenia, may be the result of diminished response to anabolic stimulation. This review will explore the hypothesis that alterations in the expression of microRNA with aging contributes to reduced muscle plasticity resulting in impaired skeletal muscle adaptations to exercise-induced anabolic stimulation.

Effects of Potassium Bicarbonate Supplements on Circulating microRNA Expression.

Several studies suggest that neutralizing acid load in the diet with alkali had favorable effects on intermediate markers of musculoskeletal health. We examined whether alkali supplementation with potassium bicarbonate [(KHCO3); 81 mmol/d; n = 12] vs placebo (n = 12) for 84 days altered serum microRNAs, potential biomarkers associated with innumerable biological processes including bone and muscle metabolism. Serum microRNAs, urinary net acid excretion (UNAE), urinary N-telopeptide (UNTX), urinary calcium (UCa), urinary nitrogen (UN), glomerular filtration rate, serum procollagen type 1 amino-terminal propeptide (P1NP), serum insulin-like growth factor-1 (IGF-1), and its serum binding protein IGFBP3 were measured at baseline and day 84. Baseline characteristics and measurements were similar in the two treatment groups. Eighty-four-day changes in UNAE differed by group (KHCO3, -47 ± 9 mmol; placebo, -5 ± 5 mmol; P < 0.01). KHCO3 significantly reduced UNTX, UCa, and serum P1NP but did not affect UN, serum IGF-1, or IGFBP3 levels compared with placebo over 84 days. Fold change in serum circulating microRNA (c-miR)-133b differed significantly by group (KHCO3, 2.26 ± 0.85; placebo, -1.23 ± 0.69; P < 0.01); there was a similar trend in c-miR-21-5p. Fold changes in c-miR-133b and c-miR-21-5p were inversely associated with changes in UNAE and UNTX; fold change in c-miR-21-5p was inversely associated with change in UCa, with a similar trend with c-miR-133b. In summary, reducing renal acid load with KHCO3 was associated with increased expressions of c-miR-133b and c-miR-21-5p. Furthermore, increases in c-miRNA-133b and c-miR-21-5p were inversely associated with bone resorption markers UNTX and UCa consistent with potential beneficial effects on bone in older adults. However, the broader significance of c-miRNAs as musculoskeletal biomarkers is still under investigation, and larger studies are needed to verify these preliminary results.

Upregulation of circulating myomiR following short-term energy restriction is inversely associated with whole body protein synthesis.

The objective of the present investigation was to determine whether energy restriction (ER) influences expression of skeletal muscle-specific microRNA (miRNA) in circulation (c-myomiR) and whether changes in c-myomiR are associated with rates of whole body protein synthesis. Sixteen older (64 ± 2 yr) overweight (28.5 ± 1.2 kg/m2) men enrolled in this 35-day controlled feeding trial. A 7-day weight maintenance (WM) period was followed by 28 days of 30% ER. Whole body protein turnover was determined from [15N]glycine enrichments in 24-h urine collections, and c-myomiR (miR-1-3p, miR-133a-3p, miR-133b, and miR-206) expression was assessed from serum samples by RT-quantitative PCR upon completion of the WM and ER periods. Participants lost 4.4 ± 0.3 kg body mass during ER (P < 0.05). After 28 days of ER, miR-133a and miR-133b expression was upregulated (P < 0.05) compared with WM. When all four c-myomiR were grouped as c-myomiR score (sum of the median fold change of all myomiR), overall expression of c-myomiR was higher (P < 0.05) at ER than WM. Backward linear regression analysis of whole body protein synthesis and breakdown and carbohydrate, fat, and protein oxidation determined protein synthesis to be the strongest predictor of c-myomiR score. An inverse association (P < 0.05) was observed with ER c-myomiR score and whole body protein synthesis (r = -0.729, r2 = -0.530). Findings from the present investigation provide evidence that upregulation of c-myomiR expression profiles in response to short-term ER is associated with lower rates of whole body protein synthesis.

Circulating MicroRNA Are Predictive of Aging and Acute Adaptive Response to Resistance Exercise in Men.

Circulating microRNA (c-miRNA) have the potential to function as novel noninvasive markers of the underlying physiological state of skeletal muscle. This investigation sought to determine the influence of aging on c-miRNA expression at rest and following resistance exercise in male volunteers (Young: n = 9; Older: n = 9). Primary findings were that fasting c-miRNA expression profiles were significantly predictive of aging, with miR-19b-3p, miR-206, and miR-486 distinguishing between age groups. Following resistance exercise, principal component analysis revealed a divergent response in expression of 10 c-miRNA, where expression profiles were upregulated in younger and downregulated in older participants. Using Ingenuity Pathway Analysis to test c-miRNA-to-mRNA interactions in skeletal muscle, it was found that response of c-miRNA to exercise was indicative of an anabolic response in younger but not older participants. These findings were corroborated with a positive association observed with the phosphorylation status of p-AktSer473 and p-S6K1Thr389 and expression of miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-26b-5p, miR-143-3p, and miR-195-5p. These important findings provide compelling evidence that dysregulation of c-miRNA expression with aging may not only serve as a predictive marker, but also reflect underlying molecular mechanisms resulting in age-associated declines in skeletal muscle mass, increased fat mass, and "anabolic resistance."

Prolonged Calorie Restriction Downregulates Skeletal Muscle mTORC1 Signaling Independent of Dietary Protein Intake and Associated microRNA Expression.

Short-term (5-10 days) calorie restriction (CR) downregulates muscle protein synthesis, with consumption of a high protein-based diet attenuating this decline. Benefit of increase protein intake is believed to be due to maintenance of amino acid-mediated anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1), however, there is limited evidence to support this contention. The purpose of this investigation was to determine the effects of prolonged CR and high protein diets on skeletal muscle mTORC1 signaling and expression of associated microRNA (miR). Twelve-week old male Sprague Dawley rats consumed ad libitum (AL) or calorie restricted (CR; 40%) adequate (10%, AIN-93M) or high (32%) protein milk-based diets for 16 weeks. Body composition was determined using dual energy X-ray absorptiometry and muscle protein content was calculated from muscle homogenate protein concentrations expressed relative to fat-free mass to estimate protein content. Western blot and RT-qPCR were used to determine mTORC1 signaling and mRNA and miR expression in fasted mixed gastrocnemius. Independent of dietary protein intake, muscle protein content was 38% lower (P < 0.05) in CR compared to AL. Phosphorylation and total Akt, mTOR, rpS6, and p70S6K were lower (P < 0.05) in CR vs. AL, and total rpS6 was associated with muscle protein content (r = 0.64, r2 = 0.36). Skeletal muscle miR expression was not altered by either energy or protein intake. This study provides evidence that chronic CR attenuates muscle protein content by downregulating mTORC1 signaling. This response is independent of skeletal muscle miR and dietary protein.

Calorie Restricted High Protein Diets Downregulate Lipogenesis and Lower Intrahepatic Triglyceride Concentrations in Male Rats.

The purpose of this investigation was to assess the influence of calorie restriction (CR) alone, higher-protein/lower-carbohydrate intake alone, and combined CR higher-protein/lower-carbohydrate intake on glucose homeostasis, hepatic de novo lipogenesis (DNL), and intrahepatic triglycerides. Twelve-week old male Sprague Dawley rats consumed ad libitum (AL) or CR (40% restriction), adequate (10%), or high (32%) protein (PRO) milk-based diets for 16 weeks. Metabolic profiles were assessed in serum, and intrahepatic triglyceride concentrations and molecular markers of de novo lipogenesis were determined in liver. Independent of calorie intake, 32% PRO tended to result in lower homeostatic model assessment of insulin resistance (HOMA-IR) values compared to 10% PRO, while insulin and homeostatic model assessment of ß-cell function (HOMA-ß) values were lower in CR than AL, regardless of protein intake. Intrahepatic triglyceride concentrations were 27.4 ± 4.5 and 11.7 ± 4.5 µmol·g(-1) lower (p < 0.05) in CR and 32% PRO compared to AL and 10% PRO, respectively. Gene expression of fatty acid synthase (FASN), stearoyl-CoA destaurase-1 (SCD1) and pyruvate dehydrogenase kinase, isozyme 4 (PDK4) were 45% ± 1%, 23% ± 1%, and 57% ± 1% lower (p < 0.05), respectively, in CR than AL, regardless of protein intake. Total protein of FASN and SCD were 50% ± 1% and 26% ± 1% lower (p < 0.05) in 32% PRO compared to 10% PRO, independent of calorie intake. Results from this investigation provide evidence that the metabolic health benefits associated with CR-specifically reduction in intrahepatic triglyceride content-may be enhanced by consuming a higher-protein/lower-carbohydrate diet.

Diminished anabolic signaling response to insulin induced by intramuscular lipid accumulation is associated with inflammation in aging but not obesity.

The loss of skeletal muscle mass is observed in many pathophysiological conditions, including aging and obesity. The loss of muscle mass and function with aging is defined as sarcopenia and is characterized by a mismatch between skeletal muscle protein synthesis and breakdown. Characteristic metabolic features of both aging and obesity are increases in intramyocellular lipid (IMCL) content in muscle. IMCL accumulation may play a mechanistic role in the development of anabolic resistance and the progression of muscle atrophy in aging and obesity. In the present study, aged and high-fat fed mice were used to determine mechanisms leading to muscle loss. We hypothesized the accumulation of bioactive lipids in skeletal muscle, such as ceramide or diacylglycerols, leads to insulin resistance with aging and obesity and the inability to activate protein synthesis, contributing to skeletal muscle loss. We report a positive association between bioactive lipid accumulation and the loss of lean mass and muscle strength. Obese and aged animals had significantly higher storage of ceramide and diacylglycerol compared with young. Furthermore, there was an attenuated insulin response in components of the mTOR anabolic signaling pathway. We also observed differential increases in the expression of inflammatory cytokines and the phosphorylation of IκBα with aging and obesity. These data challenge the accepted role of increased inflammation in obesity-induced insulin resistance in skeletal muscle. Furthermore, we have now established IκBα with a novel function in aging-associated muscle loss that may be independent of its previously understood role as an NF-κB inhibitor.