The autophagy inhibitor NSC185058 suppresses mTORC1-mediated protein anabolism in cultured skeletal muscle.

The mammalian target of rapamycin (mTOR), and specifically the mTOR complex 1 (mTORC1) is the central regulator of anabolism in skeletal muscle. Among the many functions of this kinase complex is the inhibition of the catabolic process of autophagy; however, less work has been done in investigating the role of autophagy in regulating mTORC1 signaling. Using an in vitro model to better understand the pathways involved, we activated mTORC1 by several different means (growth factors, leucine supplementation, or muscle contraction), alone or with the autophagy inhibitor NSC185058. We found that inhibiting autophagy with NSC185058 suppresses mTORC1 activity, preventing any increase in cellular protein anabolism. These decrements were the direct result of action on the mTORC1 kinase, which we demonstrate, for the first time, cannot function when autophagy is inhibited by NSC185058. Our results indicate that, far from being a matter of unidirectional action, the relationship between mTORC1 and the autophagic cascade is more nuanced, with autophagy serving as an mTORC1 input, and mTORC1 inhibition of autophagy as a form of homeostatic feedback to regulate anabolic signaling. Future studies of cellular metabolism will have to consider this fundamental intertwining of protein anabolism and catabolism, and how it ultimately serves to regulate muscle proteostasis.

The Effect of Choline and Resistance Training on Strength and Lean Mass in Older Adults.

Choline plays many important roles, including the synthesis of acetylcholine, and may affect muscle responses to exercise. We previously observed correlations between low choline intake and reduced gains in strength and lean mass following a 12-week resistance exercise training (RET) program for older adults. To further explore these findings, we conducted a randomized controlled trial. Three groups of 50-to-69-year-old healthy adults underwent a 12-week RET program (3x/week, 3 sets, 8-12 reps, 70% of maximum strength (1RM)) and submitted >48 diet logs (>4x/week for 12 weeks). Participants' diets were supplemented with 0.7 mg/kg lean/d (low, n = 13), 2.8 mg/kg lean/d (med, n = 11), or 7.5 mg/kg lean/d (high, n = 13) of choline from egg yolk and protein powder. The ANCOVA tests showed that low choline intake, compared with med or high choline intakes, resulted in significantly diminished gains in composite strength (leg press + chest press 1RM; low, 19.4 ± 8.2%; med, 46.8 ± 8.9%; high, 47.4 ± 8.1%; p = 0.034) and thigh-muscle quality (leg press 1RM/thigh lean mass; low, 12.3 ± 9.6%; med/high, 46.4 ± 7.0%; p = 0.010) after controlling for lean mass, protein, betaine, and vitamin B12. These data suggest that low choline intake may negatively affect strength gains with RET in older adults.

Combined effects of heavy ion exposure and simulated Lunar gravity on skeletal muscle.

BACKGROUND: The limitations to prolonged spaceflight include unloading-induced atrophy of the musculoskeletal system which may be enhanced by exposure to the space radiation environment. Previous results have concluded that partial gravity, comparable to the Lunar surface, may have detrimental effects on skeletal muscle. However, little is known if these outcomes are exacerbated by exposure to low-dose rate, high-energy radiation common to the space environment. Therefore, the present study sought to determine the impact of highly charge, high-energy (HZE) radiation on skeletal muscle when combined with partial weightbearing to simulate Lunar gravity. We hypothesized that partial unloading would compromise skeletal muscle and these effects would be exacerbated by radiation exposure. METHODS: For month old female BALB/cByJ mice were -assigned to one of 2 groups; either full weight bearing (Cage Controls, CC) or partial weight bearing equal to 1/6th bodyweight (G/6). Both groups were then divided to receive either a single whole body absorbed dose of 0.5 Gy of 300 MeV 28Si ions (RAD) or a sham treatment (SHAM). Radiation exposure experiments were performed at the NASA Space Radiation Laboratory (NSRL) located at Brookhaven National Laboratory on Day 0, followed by 21 d of CC or G/6 loading. Muscles of the hind limb were used to measure protein synthesis and other histological measures. RESULTS: Twenty-one days of Lunar gravity (G/6) resulted in lower soleus, plantaris, and gastrocnemius muscle mass. Radiation exposure did not further impact muscle mass. 28Si exposure in normal ambulatory animals (RAD+CC) did not impact gastrocnemius muscle mass when compared to SHAM+CC (p>0.05), but did affect the soleus, where mass was higher following radiation compared to SHAM (p<0.05). Mixed gastrocnemius muscle protein synthesis was lower in both unloading groups. Fiber type composition transitioned towards a faster isoform with partial unloading and was not further impacted by radiation. The combined effects of partial loading and radiation partially mitigated fiber cross-sectional area when compared to partial loading alone. Radiation and G/6 reduced the total number of myonuclei per fiber while leading to elevated BrdU content of skeletal muscle. Similarly, unloading and radiation resulted in higher collagen content of muscle when compared to controls, but the effects of combined exposure were not additive. CONCLUSIONS: The results of this study confirm that partial weightbearing causes muscle atrophy, in part due to reductions of muscle protein synthesis in the soleus and gastrocnemius as well as reduced peripheral nuclei per fiber. Additionally, we present novel data illustrating 28Si exposure reduced nuclei in muscle fibers despite higher satellite cell fusion, but did not exacerbate muscle atrophy, CSA changes, or collagen content. In conclusion, both partial loading and HZE radiation can negatively impact muscle morphology.

Influence of Mechanistic Target of Rapamycin (mTOR)-Regulated Anabolic Pathways on Equine Skeletal Muscle Health.

Skeletal muscle is a highly dynamic organ that is essential for locomotion as well as endocrine regulation in all populations of horses. However, despite the importance of adequate muscle development and maintenance, the mechanisms underlying protein anabolism in horses on different diets, exercise programs, and at different life stages remain obscure. Mechanistic target of rapamycin (mTOR) is a key component of the protein synthesis pathway and is regulated by biological factors such as insulin and amino acid availability. Providing a diet ample in vital amino acids, such as leucine and glutamine, is essential in activating sensory pathways that recruit mTOR to the lysosome and assist in the translation of important downstream targets. When the diet is well balanced, mitochondrial biogenesis and protein synthesis are activated in response to increased exercise bouts in the performing athlete. It is important to note that the mTOR kinase pathways are multifaceted and very complex, with several binding partners and targets that lead to specific functions in protein turnover of the cell, and ultimately, the capacity to maintain or grow muscle mass. Further, these pathways are likely altered across the lifespan, with an emphasis of growth in young horses while decreases in musculature with aged horses appears to be attributable to degradation or other regulators of protein synthesis rather than alterations in the mTOR pathway. Previous work has begun to pinpoint ways in which the mTOR pathway is influenced by diet, exercise, and age; however, future research is warranted to quantify the functional outcomes related to changes in mTOR. Promisingly, this could provide direction on appropriate management techniques to support skeletal muscle growth and maximize athletic potential in differing equine populations.

Myokines derived from contracting skeletal muscle suppress anabolism in MCF7 breast cancer cells by inhibiting mTOR.

There is strong evidence that physical activity has a profound protective effect against multiple types of cancer. Here, we show that this effect may be mediated by factors released from skeletal muscle during simulated exercise, in situ, which suppress canonical anabolic signaling in breast cancer. We report attenuated growth of MCF7 breast cancer cells in the presence of a rodent-derived exercise conditioned perfusate, independent of prior exercise training. This reduction was concomitant with increased levels of DEPTOR protein and reduced mTOR activity.

Muscle miR-16 deletion results in impaired insulin sensitivity and contractile function in a sex-dependent manner.

microRNAs (miRs) are linked to various human diseases including type 2 diabetes mellitus (T2DM) and emerging evidence suggests that miRs may serve as potential therapeutic targets. Lower miR-16 content is consistent across different models of T2DM; however, the role of miR-16 in muscle metabolic health is still elusive. Therefore, the purpose of this study was to investigate how deletion of miR-16 in mice affects skeletal muscle metabolic health and contractile function in both sexes. This study was conducted using both 1) in vitro and 2) in vivo experiments. In in vitro experiments, we used C2C12 myoblasts to test if inhibition or overexpression of miR-16 affected insulin-mediated glucose handling. In in vivo experiments, we generated muscle-specific miR-16 knockout (KO) mice fed a high-fat diet (HFD) to assess how miR-16 content impacts metabolic and contractile properties including glucose tolerance, insulin sensitivity, muscle contractile function, protein anabolism, and mitochondrial network health. In in vitro experiments, although inhibition of miR-16 induced impaired insulin signaling (P = 0.002) and glucose uptake (P = 0.014), overexpression of miR-16 did not attenuate lipid overload-induced insulin resistance using the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol. In in vivo experiments, miR-16 deletion induced both impaired muscle contractility (P = 0.031-0.033), and mitochondrial network health (P = 0.008-0.018) in both sexes. However, although males specifically exhibited impaired insulin sensitivity following miR-16 deletion (P = 0.030), female KO mice showed pronounced glucose intolerance (P = 0.046), corresponding with lower muscle weights (P = 0.015), and protein hyperanabolism (P = 0.023). Our findings suggest distinct sex differences in muscle adaptation in response to miR-16 deletion and miR-16 may serve as a key regulator for metabolic dysregulation in T2DM.NEW & NOTEWORTHY We set to investigate the role of miR-16 in skeletal muscle during diet-induced insulin resistance. Our data provide novel evidence that the lack of miR-16 induced multiple aberrations in insulin sensitivity, muscle contractility, mitochondrial network health, and protein turnover in a sex-dependent manner. Interestingly, miR-16 deletion leads to insulin resistance in males and exacerbated glucose intolerance in females, suggesting different mechanisms of metabolic dysregulation with a lack of miR-16 between sexes.

Culprits or consequences: Understanding the metabolic dysregulation of muscle in diabetes.

The prevalence of type 2 diabetes (T2D) continues to rise despite the amount of research dedicated to finding the culprits of this debilitating disease. Skeletal muscle is arguably the most important contributor to glucose disposal making it a clear target in insulin resistance and T2D research. Within skeletal muscle there is a clear link to metabolic dysregulation during the progression of T2D but the determination of culprits vs consequences of the disease has been elusive. Emerging evidence in skeletal muscle implicates influential cross talk between a key anabolic regulatory protein, the mammalian target of rapamycin (mTOR) and its associated complexes (mTORC1 and mTORC2), and the well-described canonical signaling for insulin-stimulated glucose uptake. This new understanding of cellular signaling crosstalk has blurred the lines of what is a culprit and what is a consequence with regard to insulin resistance. Here, we briefly review the most recent understanding of insulin signaling in skeletal muscle, and how anabolic responses favoring anabolism directly impact cellular glucose disposal. This review highlights key cross-over interactions between protein and glucose regulatory pathways and the implications this may have for the design of new therapeutic targets for the control of glucoregulatory function in skeletal muscle.

Use of deuterium oxide (2H2O) to assess muscle protein synthesis in juvenile red drum (Sciaenops ocellatus) fed complete, and valine-deficient diets.

The use of 2H2O in tank water to assess protein synthesis rates in fish is a relatively novel methodology that could allow for a better understanding of the effects of particular nutritional and environmental variables on rates of protein accretion. As such, this study involved an assessment and comparison of protein synthesis rates in the muscle of juvenile red drum fed a control diet (nutritionally complete) versus a valine (Val)-deficient diet. Six groups of 12 juvenile red drum, initially weighing ~ 4.5 g/fish, were stocked in six separate 38-L aquaria operating as a recirculating system. Fish were acclimatized to experimental conditions for 2 weeks while being fed the control diet. Just prior to initiating the protein synthesis assay, one aquarium of fish was fed the control diet while a second aquarium of fish was fed the Val-deficient diet. Immediately after consuming the experimental diets, each group of fish was moved to an independent aquarium containing 2H2O, and the fractional synthetic rate (FSR) of protein synthesis was obtained at 12, 24, 36 and 48 h after feeding by collecting two fish per treatment at each time point. This protein synthesis assay procedure was performed in three separate sessions, and considered as replicates over time (n = 3) for fish fed the control or Val-deficient diets immediately before initiating the session. Results indicated that a one-time feeding of a diet deficient in Val significantly reduced protein synthesis rates in the muscle of red drum. In addition, a significant effect of time after feeding was found, where observed FSR values peaked at 12 h after feeding and decreased as time progressed. In conclusion, deuterium methodologies were applicable to red drum, and this approach had the sensitivity to assess differences in protein synthesis rates when dietary perturbations were introduced.

Interorgan Metabolism of Amino Acids in Human Health and Disease.

Amino acids are integral for human health, influencing an array of physiological processes from gene expression to vasodilation to the immune response. In accordance with this expansive range of unique functions, the tissues of the body engage in a complex interplay of amino acid exchange and metabolism to respond to the organism's dynamic needs for a range of nitrogenous products. Interorgan amino acid metabolism is required for numerous metabolic pathways, including the synthesis of functional amino acids like arginine, glutamate, glutamine, and glycine. This physiological process requires the cooperative handling of amino acids by organs (e.g., the small intestine, skeletal muscle, kidneys, and liver), as well as the complete catabolism of nutritionally essential amino acids such as the BCAAs, with their α-ketoacids shuttled from muscle to liver. These exchanges are made possible by several mechanisms, including organ location, as well as the functional zonation of enzymes and the cell-specific expression of amino acid transporters. The cooperative handling of amino acids between the various organs does not appear to be under the control of any centralized regulation, but is instead influenced by factors such as fluctuations in nutrient availability, hormones, changes associated with development, and altered environmental factors. While the normal function of these pathways is associated with health and homeostasis, affected by physical activity, diet and body composition, dysregulation is observed in numerous disease states, including cardiovascular disease and cancer cachexia, presenting potential avenues for the manipulation of amino acid consumption as part of the therapeutic approach to these conditions in individuals.

Regulation of cellular anabolism by mTOR: or how I learned to stop worrying and love translation.

The process and regulation of cellular metabolism are extremely complex and accomplished through multiple signalling pathways that operate in parallel, and often experience significant overlap in upstream and downstream a signal transduction. Despite this complexity, single pathway or even single protein activations are commonly used to extrapolate broad characterizations of cellular metabolism. Furthermore, multiple routes for peptide-chain translation initiation exist, some of which may be either exclusive or overlapping depending on the state and environment of the cell. While it may be highly impractical to account for every aspect of metabolic regulation and permutation of mRNA translation, it is important to acknowledge that investigations relating to these pathways are often incomplete and not necessarily indicative of the overall metabolic status. This becomes urgent when considering the role that cellular anabolism plays in both healthy cellular functions and the aetiology of several disease's altered metabolisms. This review describes recent advances in the understanding of cellular metabolic regulation, with specific focus given to the complexity of 'downstream' mRNA translation initiation through both mTOR-dependent and mTOR-independent signallings.

Regulation of mitochondrial quality following repeated bouts of hindlimb unloading.

Muscle disuse impairs muscle quality and is associated with increased mortality. Little is known regarding additive effects of multiple bouts of disuse, which is a common occurrence in patients experiencing multiple surgeries. Mitochondrial quality is vital to muscle health and quality; however, to date mitochondrial quality control has not been investigated following multiple bouts of disuse. Therefore, the purpose of this study was to investigate mitochondrial quality controllers during multiple bouts of disuse by hindlimb unloading. Male rats (n ∼ 8/group) were assigned to the following groups: hindlimb unloading for 28 days, hindlimb unloading with 56 days of reloading, 2 bouts of hindlimb unloading separated by a recovery phase of 56 days of reloading, 2 bouts of hindlimb unloading and recovery after each disuse, or control animals with no unloading. At designated time points, tissues were collected for messenger RNA and protein analysis of mitochondrial quality. Measures of mitochondrial biogenesis, such as proliferator-activated receptor gamma coactivator 1 alpha, decreased 30%-40% with unloading with no differences noted between unloading conditions. Measures of mitochondrial translation were 40%-50% lower in unloading conditions, with no differences noted between bouts of unloading. Measures of mitophagy were 40%-50% lower with reloading, with no differences noted between reloading conditions. In conclusion, disuse causes alterations in measures of mitochondrial quality; however, multiple bouts of disuse does not appear to have additive effects. Novelty Disuse atrophy causes multiple alterations to mitochondrial quality control. With sufficient recovery most detriments to mitochondrial quality control are fixed. In general, multiple bouts of disuse do not produce additive effects.

Effect of combined fish oil & Curcumin on murine skeletal muscle morphology and stress response proteins during mechanical unloading.

Skeletal muscle is a highly adaptable tissue capable of remodeling when dynamic stress is altered, including changes in mechanical loading and stretch. When muscle is subjected to an unloaded state (e.g., bedrest, immobilization, spaceflight) the resulting loss of muscle cross sectional area (CSA) impairs force production. In addition, muscle fiber-type shifts from slow to fast-twitch fibers. Unloading also results in a downregulation of heat shock proteins (e.g., HSP70) and anabolic signaling, which further exacerbate these morphological changes. Our lab recently showed reactive oxygen species (ROS) are causal in unloading-induced alterations in Akt and FoxO3a phosphorylation, muscle fiber atrophy, and fiber-type shift. Nutritional supplements such as fish oil and curcumin enhance anabolic signaling, glutathione levels, and heat shock proteins. We hypothesized that fish oil, rich in omega-3-fatty acids, combined with the polyphenol curcumin would enhance stress protective proteins and anabolic signaling in the rat soleus muscle, concomitant with synergistic protection of morphology. C57BL/6 mice were assigned to 3 groups (n = 6/group): ambulatory controls (CON), hindlimb unloading (HU), and hindlimb unloading with 5% fish oil, 1% curcumin in diet (FOC). FOC treatments began 10 days prior to HU and tissues were harvested following 7 days of HU. FOC mitigated the unloading induced decrease in CSA. FOC also enhanced abundance of HSP70 and anabolic signaling (Akt phosphorylation, p70S6K phosphorylation), while reducing Nox2, a source of oxidative stress. Therefore, we concluded that the combination of fish oil and curcumin prevents skeletal muscle atrophy due to a boost of heat shock proteins and anabolic signaling in an unloaded state.

The effects of hindlimb unloading versus dietary cholesterol and resistance training on rat skeletal muscle responses.

BACKGROUND: The loss of muscle mass and concomitantly strength, poses a serious risk to the elderly and to astronauts. Dietary cholesterol (CL), in conjunction with resistance training (RT), has been strongly associated with improvements in lean mass. The purpose of this study was to examine the effects of two opposing environments on rat skeletal muscle: (1) hindlimb unloading and (2) CL and RT. METHODS: In protocol 1, 13 male Sprague-Dawley rats were unloaded for 28 days (HU; n = 6) or served as cage controls (CC; n = 7). In protocol 2, 42 rats were assigned to 1 of 6 groups: CC (n = 7), CC + CL (n = 4), RT controls (RTC; n = 7), RTC + CL (n = 8), RT (n = 8) and RT + CL (n = 8). RT/RTC consisted of squat-like exercise. RT had weights added progressively from 80 to 410 g over 5 weeks. CL was supplemented in the chow with either 180 ppm (controls) or 1800 ppm (CL). Lower limb muscles were harvested at the end of both protocols and analyzed by Western Blotting for sterol regulatory element-binding protein-2 (SREBP-2) and low-density lipoprotein-receptor (LDL-R) and protein synthesis. RESULTS: Gastrocnemius and plantaris masses and their body mass ratios were significantly lower in the HU rats than control rats. The RT rats gained significantly less body and lean mass than the RTC groups, but the plantar flexor muscles did not show any significant differences among groups. Moreover, RT groups had significantly higher plantaris mixed muscle fractional synthesis rate (FSR) than the RTC and CC animals, with the CL groups showing greater FSR than control rats. No significant differences among groups in SREBP-2 or LDL-R were observed in either protocol. CONCLUSIONS: These studies provide evidence for a relationship between skeletal muscle and cholesterol metabolism, but the exact nature of that association remains unclear.

Responses of skeletal muscle size and anabolism are reproducible with multiple periods of unloading/reloading.

Mechanical unloading has long been understood to contribute to rapid and substantial adaptations within skeletal muscle, most notably, muscle atrophy. Studies have often demonstrated that many of the alterations resulting from disuse are reversed with a reintroduction of load and have supported the concept of muscle plasticity. We hypothesized that adaptations during disuse and recovery were a repeatable/reproducible phenomenon, which we tested with repeated changes in mechanical load. Rats were assigned to one of the following five groups: animals undergoing one or two bouts of hindlimb unloading (28 days), with or without recovery (56 day), or control. Following the completion of their final time point, posterior crural muscles were studied. Muscle sizes were lower following 28 days of disuse but fully recovered with a 56-day reloading period, regardless of the number of disuse/recovery cycles. Mixed protein fractional synthesis rates consistently reflected mass and loading conditions (supported by anabolic signaling), whereas the myofibrillar protein synthesis response varied among muscles. Amino acid concentrations were assessed in the gastrocnemius free pool and did not correlate with muscle atrophy associated with mechanical unloading. Muscle collagen concentrations were higher following the second unloading period and remained elevated following 56 days of recovery. Anabolic responses to alterations in load are preserved throughout multiple perturbations, but repeated periods of unloading may cause additive strain to muscle structure (collagen). This study suggests that whereas mass and anabolism are reproducibly reflective of the loading environment, repeated exposure to unloading and/or reloading may impact the overall structural integrity of muscle. NEW & NOTEWORTHY Repeatability should be considered a component of skeletal muscle plasticity during atrophy and recovery. Muscle anabolism is equally affected during a first or second disuse bout and returns equally with adequate recovery. Elevated muscle collagen concentrations observed after the second unloading period suggest altered structural integrity with repeated disuse.

Effect of Eukarion-134 on Akt-mTOR signalling in the rat soleus during 7 days of mechanical unloading.

NEW FINDINGS: What is the central question of this study? Translocation of nNOSµ initiates catabolic signalling via FoxO3a and skeletal muscle atrophy during mechanical unloading. Recent evidence suggests that unloading-induced muscle atrophy and FoxO3a activation are redox sensitive. Will a mimetic of superoxide dismutase and catalase (i.e. Eukarion-134) also mitigate suppression of the Akt-mTOR pathway? What is the main finding and its importance? Eukarion-134 rescued Akt-mTOR signalling and sarcolemmal nNOSµ, which were linked to protection against the unloading phenotype, muscle fibre atrophy and partial fibre-type shift from slow to fast twitch. The loss of nNOSµ from the sarcolemma appears crucial to Akt phosphorylation and is redox sensitive, although the mechanisms remain unresolved. ABSTRACT: Mechanical unloading stimulates rapid changes in skeletal muscle morphology, characterized by atrophy of muscle fibre cross-sectional area and a partial fibre-type shift from slow to fast twitch. Recent studies revealed that oxidative stress contributes to activation of forkhead box O3a (FoxO3a), proteolytic signalling and unloading-induced muscle atrophy via translocation of the µ-splice variant of neuronal nitric oxide synthase (nNOSµ) and activation of FoxO3a. There is limited understanding of the role of reactive oxygen species in the Akt-mammalian target of rapamycin (mTOR) pathway signalling during unloading. We hypothesized that Eukarion-134 (EUK-134), a mimetic of the antioxidant enzymes superoxide dismutase and catalase, would protect Akt-mTOR signalling in the unloaded rat soleus. Male Fischer 344 rats were separated into the following three study groups: ambulatory control (n = 11); 7 days of hindlimb unloading + saline injections (HU, n = 11); or 7 days of HU + EUK-134; (HU + EUK-134, n = 9). EUK-134 mitigated unloading-induced dephosphorylation of Akt, as well as FoxO3a, in the soleus. Phosphorylation of mTOR in the EUK-treated HU rats was not different from that in control animals. However, EUK-134 did not significantly rescue p70S6K phosphorylation. EUK-134 attenuated translocation of nNOSµ from the membrane to the cytosol, reduced nitration of tyrosine residues and suppressed upregulation of caveolin-3 and dysferlin. EUK-134 ameliorated HU-induced remodelling, atrophy of muscle fibres and the 12% increase in type II myosin heavy chain-positive fibres. Attenuation of the unloaded muscle phenotype was associated with decreased reactive oxygen species, as assessed by ethidium-positive nuclei. We conclude that oxidative stress affects Akt-mTOR signalling in unloaded skeletal muscle. Direct linkage of abrogation of nNOSµ translocation with Akt-mTOR signalling during unloading is the subject of future investigation.

The influence of chronic IL-6 exposure, in vivo, on rat Achilles tendon extracellular matrix.

When compared to placebo, acetaminophen (APAP) reduces tendon stiffness and collagen cross-linking. APAP also enhances the exercise-induced increase in peritendinous levels of IL-6. Elevated levels of IL-6 are associated with tendinopathy, thus we hypothesized that chronic, elevated peritendinous IL-6 would alter tendon extracellular matrix (ECM). IL-6 (∼3000pgml-1) was injected (3dwk-1 for 8-wks) into the Achilles peritendinous region of male Wistar rats (n=16) with the opposite leg serving as a sham. Fractional synthesis rates (FSR) were determined using deuterium oxide. Collagen (hydroxyproline) and hydroxylysl pyridinoline (HP) cross-linking were analyzed by HPLC. ECM and IL-6 related genes were evaluated using qRT-PCR. Relative to sham, collagen (Col) 1a1 but not Col3a1 expression was suppressed (47%) in tendons exposed to IL-6 (p<0.05). Lysyl oxidase (LOX) and MMP-1 expression were also reduced (37%) in IL-6 treated tendons (p<0.05). Relative to sham the expression of MMP-2, -3, -9, and TIMP-1 were not altered by IL-6 treatment (p>0.05). Interleukin-6 receptor subunit beta precursor (IL6st) was lower (16%) in IL-6 treated tendons when compared to sham (p<0.05). Suppressor of cytokine signaling 3 (Socs3), signal transducer and activator of transcription 3 (STAT3), and protein inhibitor of activated STAT 1 (Pias1) were not altered by IL-6 exposure (p>0.05). Neither collagen nor cross-linking content were altered by IL-6 (p>0.05). Additionally, IL-6 treatment did not alter tendon FSR. Chronic treatment with physiologically relevant levels of IL-6 suppresses expression of Col1a1 and LOX while also altering expression of select MMPs but does not alter Achilles tendon collagen synthesis.

Hyperglycemia- and hyperinsulinemia-induced insulin resistance causes alterations in cellular bioenergetics and activation of inflammatory signaling in lymphatic muscle.

Insulin resistance is a well-known risk factor for obesity, metabolic syndrome (MetSyn) and associated cardiovascular diseases, but its mechanisms are undefined in the lymphatics. Mesenteric lymphatic vessels from MetSyn or LPS-injected rats exhibited impaired intrinsic contractile activity and associated inflammatory changes. Hence, we hypothesized that insulin resistance in lymphatic muscle cells (LMCs) affects cell bioenergetics and signaling pathways that consequently alter contractility. LMCs were treated with different concentrations of insulin or glucose or both at various time points to determine insulin resistance. Onset of insulin resistance significantly impaired glucose uptake, mitochondrial function, oxygen consumption rates, glycolysis, lactic acid, and ATP production in LMCs. Hyperglycemia and hyperinsulinemia also impaired the PI3K/Akt while enhancing the ERK/p38MAPK/JNK pathways in LMCs. Increased NF-κB nuclear translocation and macrophage chemoattractant protein-1 and VCAM-1 levels in insulin-resistant LMCs indicated activation of inflammatory mechanisms. In addition, increased phosphorylation of myosin light chain-20, a key regulator of lymphatic muscle contraction, was observed in insulin-resistant LMCs. Therefore, our data elucidate the mechanisms of insulin resistance in LMCs and provide the first evidence that hyperglycemia and hyperinsulinemia promote insulin resistance and impair lymphatic contractile status by reducing glucose uptake, altering cellular metabolic pathways, and activating inflammatory signaling cascades.-Lee, Y., Fluckey, J. D., Chakraborty, S., Muthuchamy, M. Hyperglycemia- and hyperinsulinemia-induced insulin resistance causes alterations in cellular bioenergetics and activation of inflammatory signaling in lymphatic muscle.

Cumulative Muscle Protein Synthesis and Protein Intake Requirements.

Muscle protein synthesis (MPS) fluctuates widely over the course of a day and is influenced by many factors. The time course of MPS responses to exercise and the influence of training and nutrition can only be pieced together from several different investigations and methods, many of which create unnatural experimental conditions. Measurements of cumulative MPS, the sum synthesis over an extended period, using deuterium oxide have been shown to accurately reflect muscle responses and may allow investigations of the response to exercise, total protein intake requirements, and interaction with protein timing in free-living experimental conditions; these factors have yet to be carefully integrated. Such studies could include clinical and athletic populations to integrate nutritional and exercise recommendations and help guide their revisions to optimize the skeletal muscle function that is so important to overall health.

Acute and chronic safety and efficacy of dose dependent creatine nitrate supplementation and exercise performance.

BACKGROUND: Creatine monohydrate (CrM) and nitrate are popular supplements for improving exercise performance; yet have not been investigated in combination. We performed two studies to determine the safety and exercise performance-characteristics of creatine nitrate (CrN) supplementation. METHODS: Study 1 participants (N = 13) ingested 1.5 g CrN (CrN-Low), 3 g CrN (CrN-High), 5 g CrM or a placebo in a randomized, crossover study (7d washout) to determine supplement safety (hepatorenal and muscle enzymes, heart rate, blood pressure and side effects) measured at time-0 (unsupplemented), 30-min, and then hourly for 5-h post-ingestion. Study 2 participants (N = 48) received the same CrN treatments vs. 3 g CrM in a randomized, double-blind, 28d trial inclusive of a 7-d interim testing period and loading sequence (4 servings/d). Day-7 and d-28 measured Tendo™ bench press performance, Wingate testing and a 6x6-s bicycle ergometer sprint. Data were analyzed using a GLM and results are reported as mean ± SD or mean change ± 95 % CI. RESULTS: In both studies we observed several significant, yet stochastic changes in blood markers that were not indicative of potential harm or consistent for any treatment group. Equally, all treatment groups reported a similar number of minimal side effects. In Study 2, there was a significant increase in plasma nitrates for both CrN groups by d-7, subsequently abating by d-28. Muscle creatine increased significantly by d-7 in the CrM and CrN-High groups, but then decreased by d-28 for CrN-High. By d-28, there were significant increases in bench press lifting volume (kg) for all groups (PLA, 126.6, 95 % CI 26.3, 226.8; CrM, 194.1, 95 % CI 89.0, 299.2; CrN-Low, 118.3, 95 % CI 26.1, 210.5; CrN-High, 267.2, 95 % CI 175.0, 359.4, kg). Only the CrN-High group was significantly greater than PLA (p < 0.05). Similar findings were observed for bench press peak power (PLA, 59.0, 95 % CI 4.5, 113.4; CrM, 68.6, 95 % CI 11.4, 125.8; CrN-Low, 40.9, 95 % CI -9.2, 91.0; CrN-High, 60.9, 95 % CI 10.8, 111.1, W) and average power. CONCLUSIONS: Creatine nitrate delivered at 3 g was well-tolerated, demonstrated similar performance benefits to 3 g CrM, in addition, within the confines of this study, there were no safety concerns.

microRNA-16 Is Downregulated During Insulin Resistance and Controls Skeletal Muscle Protein Accretion.

Insulin resistant diabetes, currently at epidemic levels in developed countries, begins in the skeletal muscle and is linked to altered protein turnover. microRNAs downregulate targeted mRNA translation decreasing the amount of translated protein, thereby regulating many cellular processes. Regulation of miRNAs and their function in skeletal muscle insulin resistance is largely unexplored. The purpose of this study was to identify the effects of insulin resistance on contents of skeletal muscle miRNAs with potential functions in protein turnover. We examined miRs -1, -16, -23, -27, -133a, -133b, and -206 in muscles of Zucker rats. miR-1 was 5- to 10-fold greater in obesity, whereas miRs-16 and -133b were repressed ∼50% in obese compared to lean rats, with no other alterations in miRNA contents. miR-16 correlated to protein synthesis in lean, but not obese rats. miR-16 reduction by lipid overload was verified in-vivo by diet-induced obesity and in-vitro using a diacylglycerol analog. A role for miR-16 in protein turnover of skeletal myocytes was established using transient overexpression and anti-miR inhibition. miR-16 overexpression resulted in lower protein synthesis (puromycin incorporation, ∼25-50%), mTOR (∼25%), and p70S6K1 (∼40%) in starved and insulin stimulated myoblasts. Conversely, anti-miR-16 increased basal protein synthesis (puromycin incorporation, ∼75%), mTOR (∼100%), and p70S6K1 (∼100%). Autophagy was enhanced by miR-16 overexpression (∼50% less BCL-2, ∼100% greater LC3II/I, ∼50% less p62) and impaired with miR-16 inhibition (∼45% greater BCL-2, ∼25% less total LC3, ∼50% greater p62). This study demonstrates reduced miR-16 during insulin resistance and establishes miR-16 control of protein accretion in skeletal muscle. J. Cell. Biochem. 117: 1775-1787, 2016. © 2015 Wiley Periodicals, Inc.