Age‐related skeletal muscle wasting is associated with elevated 5′‐AMP‐Activated Protein Kinase (AMPK) activity, which inhibits overload‐induced skeletal muscle protein synthesis (MPS) and growth. Glycogen, an inhibitor of AMPK, is reduced in aged skeletal muscle. We recently found that glycogen enhancement augments 21‐day overload‐induced MPS and growth in aged rat skeletal muscle. Unexpectedly, these findings were largely independent of AMPK and other signaling regulating MPS. However, the effects of glycogen enhancement on the skeletal muscle response to other anabolic stimuli, such as leucine, are largely unknown. Therefore, the purpose of this investigation was to examine the effects of enhanced glycogen on AMPK phosphorylation status and activity, MPS, and MPS‐related signaling in response to leucine in C2C12 myotubes. We hypothesized that glycogen enhancement in myotubes would enhance the anabolic effect of leucine by inhibiting AMPK and increasing anabolic signaling and MPS, and that this effect would be reduced by rapamycin‐induced mechanistic target of rapamycin (mTOR) blockade. Four days after the onset of differentiation, myotubes were transfected (via Lipofectamine 3000) for 48 hrs with a mutant glycogen synthase plasmid (mutant‐GS; designed to enhance skeletal muscle glycogen content) or an empty‐vector plasmid. At six days post‐differentiation, transfected myotubes were serum‐starved for 4 hrs prior to an amino acid starvation for 1 hr. Myotubes were then treated with either a vehicle control (CT), 10 mM leucine (LEU), 100 nM rapamycin (RAP), or leucine/rapamycin co‐treatment (LEU+RAP) for 30 min. All treatment conditions contained 100 μM puromycin for measurement of MPS rate. All values were normalized to the empty‐vector CT condition. As expected, mutant‐GS expression and glycogen content were significantly (p ≤ 0.05) higher in all four mutant‐GS myotube conditions vs. corresponding empty‐vector conditions. Within both the mutant‐GS conditions and the empty‐vector conditions, MPS was stimulated with LEU treatment and suppressed with RAP treatment as expected; however, MPS was significantly (p ≤ 0.05) higher in all four mutant‐GS conditions vs. corresponding empty‐vector conditions. LEU treatment increased the phosphorylation status of mTOR (Ser2448), p70S6K (Thr389), and 4EBP1 (Thr37/46), and this effect was blocked with RAP treatment within both the mutant‐GS myotubes or the empty‐vector myotubes. However, there were no significant (p > 0.05) differences between mutant‐GS or empty‐vector myotubes in this signaling. Additionally, markers of AMPK phosphorylation status (Thr172) and activity (ACC at Ser79) and other MPS signaling intermediates (TSC2 Ser1387 & Thr1462) were not significantly different between mutant‐GS vs. empty‐vector conditions and were unaffected by LEU or RAP treatments. Although the stimulation of MPS by leucine is mTOR‐dependent regardless of glycogen enhancement in myotubes, glycogen enhancement augments MPS independent of changes in AMPK, mTOR, or other MPS‐related signaling markers assessed. Even though the signaling mechanism(s) remain to be identified, these findings along with our recent in vivo data support the translatable potential to enhance human skeletal muscle glycogen content to augment leucine‐stimulated MPS or augment MPS and growth under conditions of chronic overload (such as resistance exercise training).Support or Funding InformationThis research was partially supported by an ACSM Foundation Doctoral Student Research Grant from the American College of Sports Medicine Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Age-related skeletal muscle (SkM) wasting is associated with elevated 5’-AMP-Activated Protein Kinase (AMPK) activity, which inhibits overload-induced (OI) SkM protein synthesis (MPS) and growth. Glycogen, an inhibitor of AMPK, is reduced in aged SkM. PURPOSE: To examine the effects of manipulating glycogen on AMPK, MPS and related signaling, and OI-growth in aged SkM. METHODS: Mutant glycogen synthase (GS; designed to enhance SkM glycogen content [GC]) or empty-vector plasmids were electrotransferred into fast-twitch plantaris muscles prior to 21-day synergist ablation-induced unilateral overload in young adult (8 mo.; empty vector; YE, n=9) and old (33 mo.; empty vector, OE, n=11; or mutant GS, OM, n=13) male FBN rats. Contralateral limbs underwent SHAM ablations with no plasmid. RESULTS: As expected, mutant GS expression and GC were significantly higher in OM overloaded muscles (the only muscles receiving the mutant GS plasmid) vs SHAM OM muscles or vs both SHAM or overloaded YE and OE muscles. There were significant increases in OI-(all vs SHAM) MPS and hypertrophy in all groups and OM was greater than OE. Markers of AMPK activity and other signaling intermediates affecting MPS were largely unaltered by glycogen enhancement. However, there was a strong and significant effect of enhancing GC (via mutant GS vs empty vector plasmid) on myogenic regulatory factors MyoD and myogenin, embryonic myosin heavy chain-positive fibers, and total fiber number in aged muscle under conditions of overload. CONCLUSIONS: Thus, enhancing GC may lead to enhanced MPS and OI growth in aged SkM. This effect may be due, in part, to an enhanced myogenesis.

•Training causes differential responses of proenkephalin Peptide F and epinephrine.•Using both high intensity resistance and aerobic training may result in overtraining.•Adrenal medulla release mechanisms may reflect the type of exercise training.•Adaptations may impact immune and analgesic roles of proenkephalin peptides.

Objective: Prior research has indicated that the loss of skeletal muscle mass and bone mineral density observed with aging is related to the prominent age-related decline in the concentration of serum growth hormone (GH). However, there is limited data on the effects of aging on GH responses to acute bouts of heavy resistance exercise (HRE) and aerobic exercise (AE).Design: The present investigation examined the effects of a HRE protocol and an AE protocol on immunoreactive GH (IGH) and bioactive GH (BGH) in active young and old women.Results: Older women had a diminished serum IGH response to both the HRE and AE protocols compared to the younger women, however a similar response was not observed in serum BGH. Additionally, the HRE protocol elicited a greater BGH response than the AE protocol exclusively in the younger group.Conclusions: Regardless of exercise mode, aging induces an increase in growth hormone polymerization that specifically results in a loss of serum growth hormone immunoreactivity without a concurrent loss of serum growth hormone bioactivity. The greater BGH response to the HRE protocol found in the younger group can be attributed to an unknown serum factor of molecular weight between 30 and 55 kD that either potentiated growth hormone bioactivity in response to HRE or inhibited growth hormone bioactivity in response to AE. (C) 2014 Elsevier Ltd. All rights reserved.

Proenkephalin Peptide F [107-140] is an enkephalin-containing peptide found predominantly within the adrenal medulla, co-packaged with epinephrine within the chromaffin granules. In vivo studies indicate that Peptide F has classic opioid analgesia effects: in vitro studies suggest potential immune cell interactions. In this investigation we examined patterns of Peptide F concentrations in different bio-compartmyents of the blood at rest and following sub-maximal cycle exercise to determine if Peptide F interacts with the white blood cell (WBC) bio-compartment during aerobic exercise. Eight physically active men (n = 8) performed sub-maximal (80-85% (V) over dotO(2) (peak)) cycle ergometer exercise for 30 min. Plasma Peptide F and WBC Peptide F immunoreactivity were examined pre-exercise, mid-exercise and immediately post-, 5-min post-, 15-min post-, 30-min post- and 60-min post-exercise and at similar time-points during a control condition (30 min rest). Peptide F concentrations significantly (p < 0.05) increased at 5 and 60 min post-exercise, compared to pre-exercise concentrations. No significant increases in Peptide F concentrations in the WBC fraction were observed during or after exercise. However, a significant decrease was observed at 30 min post-exercise. An ultradian pattern of Peptide F distribution was apparent during rest. Furthermore, concentrations of T cells, B cells, NK cells, and total WBCs demonstrated significant changes in response to aerobic exercise. Data indicated that Peptide F was bound in significant molar concentrations in the WBC fraction and that this biocompartment may be one of the tissue targets for binding interactions. These data indicate that Peptide F is involved with immune cell modulation in the white blood circulatory biocompartment of blood. (C) 2013 Published by Elsevier Inc.

Immunosenescence involves changes on the cellular and molecular levels of the immune system, and is associated with aging. Higher incidence of autoimmune disorders and other inflammatory conditions, as well as increased rates of infectious disease, cancer, and mortality have been linked to this condition. Proinflammatory interleukins (IL‐6) have been shown to increase with age, while anti‐inflammatory interleukins (IL‐4, IL‐10) decrease. Additionally, a low‐fat diet has been correlated with lower IL‐6 levels in older adults. The purpose of this study was to examine the effects of fat intake on IL‐4 and IL‐10 levels in older adults.IL‐4 and IL‐10 concentrations were analyzed from venous samples of 10 older adults (46–85 yrs) and compared to venous samples of 9 younger adults (18–45 yrs). Five older adults were counseled to consume a high‐carbohydrate, low‐fat diet for three days prior. All other subjects were instructed to consume their normal diet. Contrary to our hypothesis, a low‐fat diet did not result in increased levels of IL‐4 or IL‐10 levels, and no reversal of immunosenescence. Further investigation is warranted to investigate contributing factors to changes in immunosenescence. This research was supported by the East Carolina University Division of Research and Graduate Studies.

Introduction: The hypertrophic response to overload in fast-twitch skeletal muscle is impaired in aged humans and rats, and impaired protein synthesis pathway activation is hypothesized to be a contributing factor. Muscle growth occurs when protein synthesis exceeds protein degradation. Dietary supplementation of the essential amino acid leucine has been shown to enhance protein synthesis in both young and aged skeletal muscle. Leucine acts in part by activating mammalian target of rapamycin (mTOR; a key upstream regulator of protein synthesis pathways) as well as by attenuating the activation of 5'-AMP-activated protein kinase (AMPK; a negative regulator of mTOR and protein synthesis). During the aging process, AMPK Thr172 phosphorylation (and thus its activation) is increased, purportedly inhibiting gains in muscle mass and strength. Although dietary leucine supplementation has been shown to enhance strength gains in response to resistance training in young humans, the potential for leucine supplementation to enhance overload-induced muscle hypertrophy in aged humans or animal models has not been examined. Thus, the aim of this study was to determine whether dietary leucine supplementation can enhance markers of protein synthesis and rescue hypertrophy in overloaded fast-twitch skeletal muscles of aged rats to levels comparable to their younger counterparts. It was hypothesized that dietary leucine supplementation during 7 days of fast-twitch plantaris muscle overload would enhance plantaris muscle hypertrophy in aged rats to levels observed in young adult rats not receiving leucine. It was also hypothesized that dietary leucine supplementation during the overload period would suppress AMPK phosphorylation and enhance markers of protein synthesis [70 kDa ribosomal protein S6 kinase (p70S6k), ribosomal protein S6 (rpS6), and eukaryotic elongation factor 2 (eEF2)] in the overloaded fast-twitch plantaris muscles of the aged rats to levels observed in young adult rats not receiving leucine.

We examined the relationship between glycogen content, AMP‐activated protein kinase (AMPK), and eukaryotic elongation factor 2 (eEF2) in response to resistance exercise in young and old skeletal muscle. Seven young (21.7 ± 2.1 yrs) and 11 old (67.0 ± 8.6 yrs) men and women performed an acute bout of leg extension exercise. Muscle biopsies were obtained pre‐ (PRE), immediately post‐ (0P), 1‐hr post‐ (1P), and 2‐hr post‐exercise (2P). Glycogen content was measured in muscle samples, as were the phosphorylations (via western blot) of AMPK, acetyl‐CoA carboxylase (ACC; a marker of AMPK activity), and eEF2. AMPK phosphorylation was significantly increased vs. PRE in old, but not young, subjects at 0P. However, no age‐related differences were observed in AMPK activity, which was significantly elevated at 0P and 1P in both age groups. Similarly, inhibitory eEF2 phosphorylation (elevated at 0P and decreased at 1P and 2P vs. PRE in both age groups) was also unaffected by age. Regardless of age, higher muscle glycogen content was associated with lower AMPK activity at 0P and 1P, and this lower AMPK activity was associated with lower inhibitory phosphorylation of eEF2 at those same timepoints. Thus, higher muscle glycogen content may result in lower AMPK activation and consequently lower inhibitory eEF2 phosphorylation in response to resistance exercise in the muscles of both younger and older individuals.

Immunosenescence involves changes on the cellular and molecular levels in the immune system, and is associated with aging. Higher incidence of autoimmune disorders and other inflammatory conditions, as well as increased rates of infectious disease, cancer, and mortality have been linked to these conditions. Proinflammatory interleukins (IL‐6) have been shown to increase while anti‐inflammatory interleukins (IL‐4, IL‐10) decrease with age. Energy restriction has been associated with altered cytokine production; thus, the purpose of this study is to examine the effects of a prescribed diet on immunosenescence in older adults.IL‐6 concentrations were analyzed from venous samples of 10 older adults (46–85 yrs) and compared to 9 younger adults (18–45 yrs). Five older adults consumed a high‐carbohydrate diet for three days. All other subjects consumed a normal diet. Older adults who consumed less than the Acceptable Macronutrient Distribution Range for fat (20–35% of total calories) had lower IL‐6 levels comparable to younger subjects. Contrary to our hypothesis, a high‐carbohydrate diet did not lower IL‐6 levels however fat intake may contribute to a reversal of immunosenescence. Further investigation is warranted to investigate anti‐inflammatory factors contributing to changes in immunosenescence.This research was supported by the East Carolina University Division of Research and Graduate Studies.

In addition to suppressing protein synthesis, 5'-AMP-activated protein kinase (AMPK) stimulates muscle protein degradation through forkhead box transcription factor 3A (FOXO3A), known to promote transcription of mRNAs encoding degradation pathway proteins. Phosphorylation of FOXO3A at Ser318/321 by Akt normally prevents FOXO3A action by preventing its translocation to the nucleus. However, AMPK inhibits FOXO3A Ser318/321 phosphorylation by Akt, thus allowing its translocation and consequently transcription and upregulation of degradation pathways. AMPK phosphorylation as well as degradation pathways are more greatly elevated in old vs. young rats and humans in response to muscle loading. Moreover, older individuals exhibit lower muscle glycogen content, a condition known to accentuate AMPK activity at rest and during aerobic exercise. PURPOSE: To examine the relationship between muscle glycogen content, AMPK activation, and FOXO3A phosphorylation in response to resistance exercise in young and old individuals. METHODS: Seven young (21.7 ± 2.1 yrs) and 11 old (67.0 ± 8.6 yrs) men and women performed an acute bout of leg extension resistance exercise. Muscle biopsies were obtained pre-exercise (PRE), immediately post-exercise (0P), 1-hour post-exercise (1P), and 2-hours post-exercise (2P). Glycogen content was measured in muscle samples, as were the phosphorylations (via western blot) of AMPK, acetyl-CoA carboxylase (ACC; as a marker of AMPK activity), and FOXO3A. RESULTS: AMPK phosphorylation was significantly increased in old, but not young, subjects immediately post-exercise; however, no differences were observed in the responses of AMPK activity or FOXO3A phosphorylation between age groups. Nevertheless, amongst all subjects regardless of age, higher glycogen content was associated with lower phospho/total ACC ratio and higher FOXO3A phosphorylation at the PRE, 0P, and 1P time points. Additionally, the FOXO3A phosphorylation response to resistance exercise was significantly greater in subjects selected for high vs. low pre-exercise muscle glycogen content. CONCLUSION: These findings suggest that higher muscle glycogen content may enhance the FOXO3A Ser318/321 phosphorylation response, and thus potentially lessen the muscle protein degradation response, to resistance exercise regardless of age. However, the observed relationship between AMPK activity and FOXO3A Ser318/321 phosphorylation was not strong, suggesting that another glycogen-mediated factor may play a role in influencing the protein degradation pathway following resistance exercise in younger and older subjects.

One characteristic of ageing skeletal muscle is a decline in mitochondrial function. Activation of AMP‐activated protein kinase (AMPK) occurs in response to an increased AMP/ATP ratio, which is one potential result of mitochondrial dysfunction. We have previously observed higher AMPK activity in old (O; 30 months) vs young adult (YA; 8 months) fast‐twitch muscle in response to chronic overload. Here we tested the hypothesis that AMPK would also be hyperactivated in O vs YA fast‐twitch extensor digitorum longus muscles from Fischer344× Brown Norway (FBN) rats (n= 8 per group) in response to high‐frequency electrical stimulation of the sciatic nerve (HFES) or injection of AICAR, an activator of AMPK. Muscles were harvested immediately after HFES (10 sets of six 3‐s contractions, 10 s rest between contractions, 1 min rest between sets) or 1 h after AICAR injection (1 mg (g body weight)−1 subcutaneously). The phosphorylations of AMPKα and acetyl‐CoA carboxylase (ACC2; a downstream AMPK target) were both greatly increased (P≤ 0.05) in response to HFES in O muscles, but were either unresponsive (AMPK α) or much less responsive (ACC) in YA muscles. AMPK α2 activity was also greatly elevated in response to HFES in O muscles (but not YA muscles) despite a lower total AMPK α2 protein content in O vs YA muscles. In contrast, AMPK α2 activity was equally responsive to AICAR treatment in both age groups. Since mitochondrial content and/or efficiency could potentially underlie AMPK hyperactivation, we measured levels of mitochondrial proteins as well as citrate synthase (CS) activity. While CS activity was increased by 25% in O vs YA muscles, uncoupling protein‐3 (UCP‐3) protein level was upregulated with age by 353%. Thus, AMPK hyperactivation in response to contractile activity in aged fast‐twitch muscle may be the result of compromised cellular energetics and not necessarily due to an inherent defect in responsiveness of the AMPK molecule per se.

Regulation of protein translation through Akt and the downstream mammalian target of rapamycin (mTOR) pathway is an important component of the cellular response to hypertrophic stimuli. It has been proposed that 5'-AMP-activated protein kinase (AMPK) activation during muscle contraction may limit the hypertrophic response to resistance-type exercise by inhibiting translational signaling. However, experimental manipulation of AMPK activity during such a stimulus has not been attempted. Therefore, we investigated whether AMPK activation can attenuate the downstream signaling response of the Akt/mTOR pathway to electrically stimulated lengthening muscle contractions. Extensor digitorum longus muscles (n = 8/group) were subjected to a 22-min bout of lengthening contractions by high-frequency sciatic nerve electrical stimulation (STIM) in young adult (8 mo) Fischer 344 x Brown Norway male rats. Forty minutes before electrical stimulation, rats were subcutaneously injected with saline or 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR; 1 mg/g body wt), an AMPK activator. Stimulated and contralateral resting muscles were removed at 0, 20, and 40 min post-STIM, and AMPK, acetyl CoA carboxylase (ACC), Akt, eukaryotic initiation factor 4E-binding protein (4E-BP1), 70-kDa ribosomal protein S6 kinase (S6K1), and eukaryotic elongation factor 2 (eEF2) phosphorylations were assessed by Western blot. AICAR treatment increased (P < or = 0.05) post-STIM AMPK (Thr172) and ACC phosphorylation (Ser79/221), inhibited post-STIM S6K1 (Thr389) and 4E-BP1 (gel shift) phosphorylation, and elevated post-STIM eEF2 phosphorylation (Thr56). These findings suggest that translational signaling downstream of Akt/mTOR can be inhibited after lengthening contractions when preceded by AMPK activation and that energetic stress may be antagonistic to the hypertrophic translational signaling response to loaded muscle contractions.

The activity of 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), a negative regulator of cell size, is up-regulated with age in resting and overloaded fast-twitch skeletal muscle but not slow-twitch muscle. Here, we provide evidence to support the hypothesis that elevated AMPK activity plays a potentially important integrative role in the age-related atrophy and diminished capacity for growth specific to fast-twitch skeletal muscle.

Objective: This is a study estimating diagnostic accuracy of CSF asialotransferrin to transferrin ratio measurement in eIF2B related disorders by using clinical evaluation and EIF2B mutation analysis as the reference standard. eIF2B-related disorder is a relatively common leukodystrophy with broad phenotypic variation that is caused by mutations in any of the five EIF2B genes. There is a need for a simple and clinically valid screening tool for physicians evaluating patients with an unclassified leukodystrophy.Methods: CSF two-dimensional gel (2DG) electrophoresis analyses to measure asialotransferrin to transferrin ratios were performed in 60 subjects including 6 patients with documented EIF2B gene mutations, patients with other types of leukodystrophy, and patients with no leukodystrophy.Results: All six patients with mutation proven eIF2B-related disease showed low to nearly undetectable amounts of asialotransferrin in their CSF when compared to 54 unaffected controls by CSF 2DG analyses in this study. eIF2B-like patients, with clinically similar presentations but no mutations in EIF2B1-5, were distinguished from patients with mutations in EIF2B1-5 by this biomarker. Patients with mutations in EIF2B1-5 had asialotransferrin/transferrin ratio levels significantly different from the group as a whole (p < 0.001). Using 8% asialotransferrin/transferrin ratio as a cutoff, this biomarker has a 100% sensitivity (95% CI = 52-100%) and 94% specificity (95% CI = 84-99%).Conclusion: Decreased asialotransferrin/ transferrin ratio in the CSF of patients with eIF2-Brelated disorder is highly sensitive and specific. This rapid (< 48 hours) and inexpensive diagnostic tool for eIF2B-related disorders has the potential to identify patients with likely eIF2B-related disorder for mutation analysis.

The purpose of this study was to examine the effect of blocking nitric oxide synthase (NOS) activity via NG-nitro-L-arginine methyl ester (L-NAME) on myonuclear addition in skeletal muscle under basal and overloaded conditions. Female Sprague-Dawley rats (approx. 220 g) were placed into 1 of the following 4 groups (n = 7-9/group): 7-day skeletal muscle overload (O), sham operation (S), skeletal muscle overload with L-NAME treatment (OLN), and sham operation with L-NAME treatment (SLN). Plantaris muscles were overloaded via bilateral surgical ablation of the gastrocnemius muscles and L-NAME (0.75 mg/mL) was administered in the animals' daily drinking water starting 2 days prior to surgery and continued until sacrifice. Myonuclear addition was assessed as subsarcolemmal incorporation of nuclei labeled with 5-bromo-2'-deoxyuridine (approx. 25 mg.(kg body mass)-1.day-1) delivered via osmotic pump during the overload period. As expected, muscle wet mass, total protein content, fiber cross-sectional area, and myonuclear addition were significantly higher (p <or= 0.05) in O vs. S; however, only the increase in wet mass and total protein content (per body mass) were attenuated by L-NAME administration. Interestingly, L-NAME significantly reduced myonuclear addition by 75% in non overloaded muscles (SLN vs. S). Muscle hepatocyte growth factor protein content increased with overload, but was unaffected by L-NAME in either loading state. These data indicate that NOS inhibition in rat plantaris muscle attenuates myonuclear addition under basal, but not overloaded, conditions.