Skeletal Muscle Protein Content Research Articles

Assessment of Body Mass Index, Body Composition, Physical Activity, and Dietary Preferences in University Students: A Pilot Study

(1) Background: Body composition analysis, particularly the assessment of the amount and distribution of body fat and muscle mass in young people, is of considerable clinical importance for the detection of nutritional disorders. (2) Methods: University students aged 19–25 years had their body composition measured using a bioimpedance (BIA) device InBody 370S. Furthermore, a questionnaire survey was performed using the questionnaires: factors that influence your choice of food (FCQ); food preference questionnaire for adolescents and adults (FPQ); the international physical activity questionnaire (IPAQ). (3) Results: Body mass index (BMI) values were within a normal range in 89.5% of men and 77.9% of women, while statistically significant differences between the sexes were confirmed for all body parameters (p < 0.001; p = 0.025). The waist-to-hip ratio (WHR) and percentage body fat (PBF) were abnormally high in 50.4% and 44.3% of women, respectively. High values of skeletal muscle mass (SMM), protein, minerals, and bone mineral content (BMC) were identified in 36.8% of men. A total of 88 students (66.7% of men and women) had a higher level of physical activity, i.e., achieved metabolic equivalent (MET) values of more than 3000 per week. (4) Conclusions: BMI does not always have explanatory power for assessing body weight, as it does not consider the percentage distribution of fat and non-fat body mass in the total body weight. Physical activity and a varied diet have a positive effect on achieving optimal body weight and are effective in preventing nutritional disorders (such as obesity and malnutrition) and associated health problems.

Impact of short-term, pharmacological CARM1 inhibition on skeletal muscle mass, function, and atrophy in mice.

Coactivator-associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of arginine residues on target proteins critical for health and disease. The purpose of this study was to characterize the effects of short-term, pharmacological CARM1 inhibition on skeletal muscle size, function, and atrophy. Adult mice (n = 10 or 11/sex) were treated with either a CARM1 inhibitor (150 mg/kg EZM2302; EZM) or vehicle (Veh) via oral gavage for 11-13 days and muscle mass, function, and exercise capacity were assessed. In addition, we investigated the effect of CARM1 suppression on unilateral hindlimb denervation (DEN)-induced muscle atrophy (n = 8/sex). We report that CARM1 inhibition caused significant reductions in the asymmetric dimethylation of known CARM1 substrates but no change in CARM1 protein or mRNA content in skeletal muscle. Reduced CARM1 activity did not affect body or muscle mass, however, we observed a decrease in exercise capacity and muscular endurance in male mice. CARM1 methyltransferase activity increased in the muscle of Veh-treated mice following 7 days of DEN, and this response was blunted in EZM-dosed mice. Skeletal muscle mass and myofiber cross-sectional area were significantly reduced in DEN compared with contralateral, non-DEN limbs to a similar degree in both treatment groups. Furthermore, skeletal muscle atrophy and autophagy gene expression programs were elevated in response to DEN independent of CARM1 suppression. Collectively, these results suggest that short-term, pharmacological CARM1 inhibition in adult animals affects muscle performance in a sex-specific manner but does not impact the maintenance and remodeling of skeletal muscle mass during conditions of neurogenic muscle atrophy.NEW & NOTEWORTHY Short-term pharmacological inhibition of coactivator-associated arginine methyltransferase 1 (CARM1) was effective at significantly reducing CARM1 methyltransferase function in skeletal muscle. CARM1 inhibition did not impact muscle mass, but exercise capacity was impaired, particularly in male mice, whereas morphological and molecular signatures of denervation-induced muscle atrophy were largely maintained in animals administered the inhibitor. Altogether, the role of CARM1 in neuromuscular biology remains complex and requires further investigation of its therapeutic potential in muscle-wasting conditions.

Targeting NAT10 protects against sepsis-induced skeletal muscle atrophy by inhibiting ROS/NLRP3

AimsTo identify N-acetyltransferase 10 (NAT10) and its downstream signaling pathways in myocytes and skeletal muscle, and to investigate its role in inflammation-induced muscle atrophy. Materials and methodsCecal ligation and puncture models were used to induce sepsis in C57BL/6 mice, which were treated with either a NAT10 inhibitor or a control agent. The therapeutic effect of NAT10 inhibitor was investigated by evaluating the mass, morphology, and molecular characteristics of mouse skeletal muscle. C2C12 cells were stimulated with LPS, and the expression of the NAT10 gene, downstream protein content, and atrophy phenotype were analyzed using a NAT10 inhibitor, to further explore the atrophic effect of NAT10 on C2C12 differentiated myotubes. ResultsGene set enrichment analysis revealed that NAT10 expression was elevated in the Lateral femoris muscle of patients with ICUAW. In vitro and in vivo experiments showed that sepsis or LPS induced the upregulation of NAT10 expression in skeletal muscles and C2C12 myotubes. Skeletal muscle mass, tissue morphology, gene expression, and protein content were associated with atrophic response in sepsis models. Remodelin ameliorated the LPS-induced skeletal muscle weight loss, as well as muscular atrophy, and improved survival. Remodelin reversed the atrophy program that was induced by inflammation through the downregulation of the ROS/NLRP3 pathway, along with the inhibition of the expression of MuRF1 and Atrogin-1. ConclusionNAT10 is closely related to skeletal muscle atrophy during sepsis. Remodelin improves the survival rate of mice by improving the systemic inflammatory response and skeletal muscle atrophy by downregulating the ROS/NLRP3 signaling pathway.

Acute resistance exercise training does not augment mitochondrial remodelling in master athletes or untrained older adults.

Background: Ageing is associated with alterations to skeletal muscle oxidative metabolism that may be influenced by physical activity status, although the mechanisms underlying these changes have not been unraveled. Similarly, the effect of resistance exercise training (RET) on skeletal muscle mitochondrial regulation is unclear. Methods: Seven endurance-trained masters athletes ([MA], 74 ± 3years) and seven untrained older adults ([OC]. 69 ± 6years) completed a single session of knee extension RET (6 x 12 repetitions, 75% 1-RM, 120-s intra-set recovery). Vastus lateralis muscle biopsies were collected pre-RET, 1h post-RET, and 48h post-RET. Skeletal muscle biopsies were analyzed for citrate synthase (CS) enzyme activity, mitochondrial content, and markers of mitochondrial quality control via immunoblotting. Results: Pre-RET CS activity and protein content were ∼45% (p < .001) and ∼74% greater in MA compared with OC (p = .006). There was a significant reduction (∼18%) in CS activity 48h post-RET (p < .05) in OC, but not MA. Pre-RET abundance of individual and combined mitochondrial electron transport chain (ETC) complexes I-V were significantly greater in MA compared with OC, as were markers of mitochondrial fission and fusion dynamics (p-DRP-1Ser616, p-MFFSer146, OPA-1 & FIS-1, p < .05 for all). Moreover, MA displayed greater expression of p-AMPKThr172, PGC1α, TFAM, and SIRT-3 (p < .05 for all). Notably, RET did not alter the expression of any marker of mitochondrial content, biogenesis, or quality control in both OC and MA. Conclusion: The present data suggest that long-term aerobic exercise training supports superior skeletal muscle mitochondrial density and protein content into later life, which may be regulated by greater mitochondrial quality control mechanisms and supported via superior fission-fusion dynamics. However, a single session of RET is unable to induce mitochondrial remodelling in the acute (1h post-RET) and delayed (48h post-RET) recovery period in OC and MA.

Decreased methylglyoxal-mediated protein glycation in the healthy aging mouse model of ectopic expression of UCP1 in skeletal muscle

Mice with ectopic expression of uncoupling protein-1 (UCP1) in skeletal muscle exhibit a healthy aging phenotype with increased longevity and resistance to impaired metabolic health. This may be achieved by decreasing protein glycation by the reactive metabolite, methylglyoxal (MG). We investigated protein glycation and oxidative damage in skeletal muscle of mice with UCP1 expression under control of the human skeletal actin promoter (HSA-mUCP1) at age 12 weeks (young) and 70 weeks (aged). We found both young and aged HSA-mUCP1 mice had decreased advanced glycation endproducts (AGEs) formed from MG, lysine-derived Nε(1-carboxyethyl)lysine (CEL) and arginine-derived hydroimidazolone, MG-H1, whereas protein glycation by glucose forming Nε-fructosyl-lysine (FL) was increased ca. 2-fold, compared to wildtype controls. There were related increases in FL-linked AGEs, Nε-carboxymethyl-lysine (CML) and 3-deoxylglucosone-derived hydroimidazolone 3DG-H, and minor changes in protein oxidative and nitration adducts. In aged HSA-mUCP1 mice, urinary MG-derived AGEs/FL ratio was decreased ca. 60% whereas there was no change in CML/FL ratio – a marker of oxidative damage. This suggests that, normalized for glycemic status, aged HSA-mUCP1 mice had a lower flux of whole body MG-derived AGE exposure compared to wildtype controls. Proteomics analysis of skeletal muscle revealed a shift to increased heat shock proteins and mechanoprotection and repair in HSA-mUCP1 mice. Decreased MG-derived AGE protein content in skeletal muscle of aged HSA-mUCP1 mice is therefore likely produced by increased proteolysis of MG-modified proteins and increased proteostasis surveillance of the skeletal muscle proteome. From this and previous transcriptomic studies, signaling involved in enhanced removal of MG-modified protein is likely increased HSPB1-directed HUWE1 ubiquitination through eIF2α-mediated, ATF5-induced increased expression of HSPB1. Decreased whole body exposure to MG-derived AGEs may be linked to increased weight specific physical activity of HSA-mUCP1 mice. Decreased formation and increased clearance of MG-derived AGEs may be associated with healthy aging in the HSA-mUCP1 mouse.

Glucose 6-P Dehydrogenase-An Antioxidant Enzyme with Regulatory Functions in Skeletal Muscle during Exercise.

Hypomorphic Glucose 6-P dehydrogenase (G6PD) alleles, which cause G6PD deficiency, affect around one in twenty people worldwide. The high incidence of G6PD deficiency may reflect an evolutionary adaptation to the widespread prevalence of malaria, as G6PD-deficient red blood cells (RBCs) are hostile to the malaria parasites that infect humans. Although medical interest in this enzyme deficiency has been mainly focused on RBCs, more recent evidence suggests that there are broader implications for G6PD deficiency in health, including in skeletal muscle diseases. G6PD catalyzes the rate-limiting step in the pentose phosphate pathway (PPP), which provides the precursors of nucleotide synthesis for DNA replication as well as reduced nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is involved in the detoxification of cellular reactive oxygen species (ROS) and de novo lipid synthesis. An association between increased PPP activity and the stimulation of cell growth has been reported in different tissues including the skeletal muscle, liver, and kidney. PPP activity is increased in skeletal muscle during embryogenesis, denervation, ischemia, mechanical overload, the injection of myonecrotic agents, and physical exercise. In fact, the highest relative increase in the activity of skeletal muscle enzymes after one bout of exhaustive exercise is that of G6PD, suggesting that the activation of the PPP occurs in skeletal muscle to provide substrates for muscle repair. The age-associated loss in muscle mass and strength leads to a decrease in G6PD activity and protein content in skeletal muscle. G6PD overexpression in Drosophila Melanogaster and mice protects against metabolic stress, oxidative damage, and age-associated functional decline, and results in an extended median lifespan. This review discusses whether the well-known positive effects of exercise training in skeletal muscle are mediated through an increase in G6PD.

Phosphoproteomic mapping reveals distinct signaling actions and activation of muscle protein synthesis by Isthmin-1.

The secreted protein isthmin-1 (Ism1) mitigates diabetes by increasing adipocyte and skeletal muscle glucose uptake by activating the PI3K-Akt pathway. However, while both Ism1 and insulin converge on these common targets, Ism1 has distinct cellular actions suggesting divergence in downstream intracellular signaling pathways. To understand the biological complexity of Ism1 signaling, we performed phosphoproteomic analysis after acute exposure, revealing overlapping and distinct pathways of Ism1 and insulin. We identify a 53% overlap between Ism1 and insulin signaling and Ism1-mediated phosphoproteome-wide alterations in ~450 proteins that are not shared with insulin. Interestingly, we find several unknown phosphorylation sites on proteins related to protein translation, mTOR pathway, and, unexpectedly, muscle function in the Ism1 signaling network. Physiologically, <i>Ism1</i> ablation in mice results in altered proteostasis, including lower muscle protein levels under fed and fasted conditions, reduced amino acid incorporation into proteins, and reduced phosphorylation of the key protein synthesis effectors Akt and downstream mTORC1 targets. As metabolic disorders such as diabetes are associated with accelerated loss of skeletal muscle protein content, these studies define a non-canonical mechanism by which this antidiabetic circulating protein controls muscle biology.

Human adipose and skeletal muscle tissue DNA, RNA, and protein content.

The purpose of this project was to provide a profile of DNA, RNA, and protein content in adipose tissue, which is relatively understudied in humans, to gain more insight into the amount of tissue that may be required for various analyses. Skeletal muscle tissue was also investigated to provide a direct comparison into potential differences between these two highly metabolically active tissues. Basal adipose and skeletal muscle tissue samples were obtained from 10 (7 M, 3 W) recreationally active participants [25 ± 1 yr; 84 ± 3 kg, maximal oxygen consumption (V̇o2max): 3.5 ± 0.2 L/min, body fat: 29 ± 2%]. DNA, RNA, and protein were extracted and subsequently analyzed for quantity and quality. DNA content of adipose and skeletal muscle tissue was 52 ± 14 and 189 ± 44 ng DNA·mg tissue-1, respectively (P < 0.05). RNA content of adipose and skeletal muscle tissue was 46 ± 14 and 537 ± 72 ng RNA·mg tissue-1, respectively (P < 0.05). Protein content of adipose and skeletal muscle tissue was 4 ± 1 and 177 ± 10 µg protein·mg tissue-1, respectively (P < 0.05). In summary, human adipose had 28% of the DNA, 9% of the RNA, and 2% of the protein found in skeletal muscle per mg of tissue. This information should be useful across a wide range of human clinical investigation designs and various laboratory analyses.NEW & NOTEWORTHY This investigation studied DNA, RNA, and protein contents of adipose and skeletal muscle tissues from young active individuals. A series of optimization steps were investigated to aid in determining the optimal approach to extract high-yield and high-quality biomolecules. These findings contribute to the knowledge gap in adipose tissue requirements for molecular biology assays, which is of increasing importance due to the growing interest in adipose tissue research involving human exercise physiology research.

Reduced mitochondrial DNA and OXPHOS protein content in skeletal muscle of children with cerebral palsy.

To provide a detailed gene and protein expression analysis related to mitochondrial biogenesis and assess mitochondrial content in skeletal muscle of children with cerebral palsy (CP). Biceps brachii muscle samples were collected from 19 children with CP (mean [SD] age 15y 4mo [2y 6mo], range 9-18y, 16 males, three females) and 10 typically developing comparison children (mean [SD] age 15y [4y], range 7-21y, eight males, two females). Gene expression (quantitative reverse transcription polymerase chain reaction [PCR]), mitochondrial DNA (mtDNA) to genomic DNA ratio (quantitative PCR), and protein abundance (western blotting) were analyzed. Microarray data sets (CP/aging/bed rest) were analyzed with a focused query investigating metabolism- and mitochondria-related gene networks. The mtDNA to genomic DNA ratio was lower in the children with CP compared to the typically developing group (-23%, p=0.002). Out of five investigated complexes in the mitochondrial respiratory chain, we observed lower protein levels of all complexes (I, III, IV, V, -20% to -37%; p<0.05) except complex II. Total peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) messenger RNA (p<0.004), isoforms PGC1α1 (p=0.05), and PGC1α4 (p<0.001) were reduced in CP. Transcriptional similarities were observed between CP, aging, and 90 days' bed rest. Mitochondrial biogenesis, mtDNA, and oxidative phosphorylation protein content are reduced in CP muscle compared with typically developing muscle. Transcriptional pathways shared between aging and long-term unloading suggests metabolic dysregulation in CP, which may guide therapeutic strategies for combatting CP muscle pathology. What this paper adds Cerebral palsy (CP) muscle contains fewer energy-generating organelles than typically developing muscle. Gene expression in CP muscle is similar to aging and long-term bed rest.

Protein Abundance of NOR‐1 is Diminished During Aging and Skeletal Muscle Disuse

INTRODUCTION Skeletal muscle mass and function are crucial for metabolic health and quality of life. Mitochondrial defects and loss of proteostasis driven by inactivity and aging predispose skeletal muscle to apoptosis and greater degradative signaling. Nuclear orphan receptor NOR-1 has been shown to be responsive to exercise and promotes an oxidative phenotype. NOR-1 is also an important regulator of glucose uptake, fatty acid oxidation, and mitochondrial biogenesis. A gap in our knowledge is that we do not know if NOR-1 is altered by aging or muscle disuse. PURPOSE Exercise is beneficial for skeletal muscle and overall health. However, exercise participation is low among the elderly, which represent a clinically relevant population prone to inactivity and bed rest. Disuse due to prolonged immobility can result in significant atrophy and metabolic impairments to the immobilized limb. Identifying genes that are dysregulated during aging or disuse is crucial for discovering therapeutic targets that might be modulated to delay or mitigate detriments to skeletal muscle mass and function. Although NOR-1 is responsive to exercise and is associated with improved metabolic and mitochondrial function, the role of NOR-1 in disuse and aging-related muscle atrophy has yet to be investigated. Therefore, the purpose of this study was to determine if aging and limb immobilization impact skeletal muscle protein content of NOR-1. We hypothesized that both disuse and aging reduce skeletal muscle protein abundance of NOR-1 in mice. METHODS Aged (33 months) and adult (6-10 months) C57/BL6 mice were used in this study. To investigate the effect of age, the tibialis anterior (TA) of aged and adult control mice were harvested and analyzed for skeletal muscle protein content of NOR-1. In a separate set of experiments, adult mice were subjected to 14 days of unilateral cast immobilization to induce disuse associated atrophy in the hindlimb plantarflexors. After immobilization, the gastrocnemius and soleus muscles of both the immobilized and contralateral limb were harvested, weighed, and analyzed for NOR-1 protein content. Skeletal muscle abundance of NOR-1 protein was measured by western blot. Paired t-test were used to determine differences between immobilized and contralateral limbs. To compare aged and adult animals, data were analyzed via unpaired t-test. RESULTS Protein abundance of NOR-1 was reduced (72%; p<0.05) in aged mice when compared to adult control mice. Additionally, hindlimb immobilization in adult mice resulted in marked (35% and 18%; p<0.05) decrease in the wet weight of soleus and gastrocnemius muscles respectively and reduced NOR-1 protein abundance (51% and 42%; p<0.05) in soleus and gastrocnemius muscles respectively, as compared to the contralateral control muscles. Taken together, these data suggest muscle protein content of NOR-1 is reduced during disuse and aging.

Exercise training improves mitochondrial respiration and is associated with an altered intramuscular phospholipid signature in women with obesity

Aims/hypothesisWe sought to determine putative relationships among improved mitochondrial respiration, insulin sensitivity and altered skeletal muscle lipids and metabolite signature in response to combined aerobic and resistance training in women with obesity.MethodsThis study reports a secondary analysis of a randomised controlled trial including additional measures of mitochondrial respiration, skeletal muscle lipidomics, metabolomics and protein content. Women with obesity were randomised into 12 weeks of combined aerobic and resistance exercise training (n = 20) or control (n = 15) groups. Pre- and post-intervention testing included peak oxygen consumption, whole-body insulin sensitivity (intravenous glucose tolerance test), skeletal muscle mitochondrial respiration (high-resolution respirometry), lipidomics and metabolomics (mass spectrometry) and lipid content (magnetic resonance imaging and spectroscopy). Proteins involved in glucose transport (i.e. GLUT4) and lipid turnover (i.e. sphingomyelin synthase 1 and 2) were assessed by western blotting.ResultsThe original randomised controlled trial showed that exercise training increased insulin sensitivity (median [IQR]; 3.4 [2.0–4.6] to 3.6 [2.4–6.2] x10−5 pmol l−1 min−1), peak oxygen consumption (mean ± SD; 24.9 ± 2.4 to 27.6 ± 3.4 ml kg−1 min−1), and decreased body weight (84.1 ± 8.7 to 83.3 ± 9.7 kg), with an increase in weight (pre intervention, 87.8± 10.9 to post intervention 88.8 ± 11.0 kg) in the control group (interaction p < 0.05). The current study shows an increase in mitochondrial respiration and content in response to exercise training (interaction p < 0.05). The metabolite and lipid signature at baseline were significantly associated with mitochondrial respiratory capacity (p < 0.05) but were not associated with whole-body insulin sensitivity or GLUT4 protein content. Exercise training significantly altered the skeletal muscle lipid profile, increasing specific diacylglycerol(32:2) and ceramide(d18:1/24:0) levels, without changes in other intermediates or total content of diacylglycerol and ceramide. The total content of cardiolipin, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) increased with exercise training with a decrease in the PC:PE ratios containing 22:5 and 20:4 fatty acids. These changes were associated with content-driven increases in mitochondrial respiration (p < 0.05), but not with the increase in whole-body insulin sensitivity or GLUT4 protein content. Exercise training increased sphingomyelin synthase 1 (p < 0.05), with no change in plasma-membrane-located sphingomyelin synthase 2.Conclusions/interpretationThe major findings of our study were that exercise training altered specific intramuscular lipid intermediates, associated with content-driven increases in mitochondrial respiration but not whole-body insulin sensitivity. This highlights the benefits of exercise training and presents putative target pathways for preventing lipotoxicity in skeletal muscle, which is typically associated with the development of type 2 diabetes.Graphical abstract

Resting skeletal muscle PNPLA2 (ATGL) and CPT1B are associated with peak fat oxidation rates in men and women but do not explain observed sex differences

What is the central question of this study? What is the relationship between proteins in skeletal muscle and adipose tissue determined at rest and at peak rates of fat oxidation in men and women? What is the main finding and its importance? The resting contents of proteins in skeletal muscle involved in triglyceride hydrolysis and mitochondrial lipid transport were more strongly associated with peak fat oxidation rates than proteins related to lipid transport or hydrolysis in adipose tissue. Although females displayed higher relative rates of fat oxidation than males, this was not explained by the proteins measured in this study, suggesting that other factors determine sex differences in fat metabolism. We explored key proteins involved in fat metabolism that might be associated with peak fat oxidation (PFO) and account for sexual dimorphism in fuel metabolism during exercise. Thirty-six healthy adults [15 women; 40±11years of age; peak oxygen consumption 42.5±9.5ml(kg body mass)-1 min-1 ; mean±SD] completed two exercise tests to determine PFO via indirect calorimetry. Resting adipose tissue and/or skeletal muscle biopsies were obtained to determine the adipose tissue protein content of PLIN1, ABHD5 (CGI-58), LIPE (HSL), PNPLA2 (ATGL), ACSL1, CPT1B and oestrogen receptorα (ERα) and the skeletal muscle protein content of FABP 3 (FABPpm), PNPLA2 (ATGL), ACSL1, CTP1B and ESR1 (ERα). Moderate strength correlations were found between PFO [in milligrams per kilogram of fat-free mass (FFM) per minute] and the protein content of PNPLA2 (ATGL) [rs =0.41 (0.03-0.68), P<0.05] and CPT1B [rs =0.45 (0.09-0.71), P<0.05] in skeletal muscle. No other statistically significant bivariate correlations were found consistently. Females had a greater relative PFO than males [7.1±1.9 vs. 4.5±1.3 and 7.3±1.7 vs. 4.8±1.2mg(kg FFM)-1 min-1 in the adipose tissue (n=14) and skeletal muscle (n=12) subgroups, respectively (P<0.05)]. No statistically significant sex differences were found in the content of these proteins. The regulation of PFO might involve processes relating to intramyocellular triglyceride hydrolysis and mitochondrial fatty acid transport, and adipose tissue is likely to play a more minor role than muscle. Sex differences in fat metabolism are likely to be attributable to factors other than the resting content of proteins in skeletal muscle and adipose tissue relating to triglyceride hydrolysis and fatty acid transport.

Abstract 15747: The Sarcoplasmic Reticulum Calcium ATPase Pump Modulates Whole-body Glucose Homeostasis Through a Protein Disulfide Isomerase-dependent Pathway

Introduction: The heart is a very active metabolic organ which exerts endocrine effects by secreting cardiokines. However, its role in regulating whole-body metabolism has received scarce attention. Hypothesis: We hypothesized that cardiac-specific overexpression of the sarcoplasmic reticulum calcium ATPase (SERCA) pump, which tightly regulates cytosolic calcium, modulates whole-body glucose homeostasis by secreting cardiokines during healthy and diabetic states. Methods: Diabetes was induced by streptozotocin in wild type (WT) and transgenic (TG) mice overexpressing the SERCA pump in the heart (n=6-10/group). Translocation of glucose transporters (GLUTs) to the cell surface was measured via a photolabeling biotinylation assay. Glucose and palmitate oxidation rates, cardiac work and ATP production were quantified in isolated working hearts. Results: Blood glucose levels were lower in diabetic TG mice compared to diabetic WT mice (P=0.023). Diabetes induced a downregulation of cell surface GLUT4 protein content in cardiac and skeletal muscle (by 56% and 79%, respectively, P&lt;0.05), as well as in white and brown adipose tissue of WT mice (by 68% and 74%, respectively, P&lt;0.05), which was rescued in TG diabetic mice. WT diabetic hearts had decreased cardiac glucose oxidation rates and cardiac efficiency, and increased palmitate oxidation rates (P&lt;0.05), which were restored in TG diabetic hearts. Using quantitative mass spectrometry, we found protein disulfide isomerase (PDI) upregulated in the heart of TG mice (by 1822%, P&lt;0.0001), which was confirmed in the serum of TG mice via an ELISA assay (by 243%, P=0.03). Inoculation with exogenous PDI reduced blood glucose in WT healthy mice and partially rescued hyperglycemia in WT diabetic mice compared to untreated counterparts (P=0.009 and P=0.044, respectively). Conclusions: These data suggest that cardiac-specific SERCA overexpression rescues hyperglycemia by improving glucose transport in striated muscle and adipose tissue of diabetic TG mice. Identification of the pathways by which the SERCA pump and cardiac secreted proteins (i.e. PDI) are involved in the regulation of whole-body glucose homeostasis could lead to the identification of novel pharmacological targets in diabetic patients.

Differences in intrinsic aerobic capacity alters sensitivity to ischemia-reperfusion injury but not cardioprotective capacity by ischemic preconditioning in rats.

IntroductionAerobic capacity is a strong predictor of cardiovascular mortality. Whether aerobic capacity influences myocardial ischemia and reperfusion (IR) injury is unknown.PurposeTo investigate the impact of intrinsic differences in aerobic capacity and the cardioprotective potential on IR injury.MethodsWe studied hearts from rats developed by selective breeding for high (HCR) or low (LCR) capacity for treadmill running. The rats were randomized to: (1) control, (2) local ischemic preconditioning (IPC) or (3) remote ischemic preconditioning (RIC) followed by 30 minutes of ischemia and 120 minutes of reperfusion in an isolated perfused heart model. The primary endpoint was infarct size. Secondary endpoints included uptake of labelled glucose, content of selected mitochondrial proteins in skeletal and cardiac muscle, and activation of AMP-activated kinase (AMPK).ResultsAt baseline, running distance was 203±7 m in LCR vs 1905±51 m in HCR rats (p<0.01). Infarct size was significantly lower in LCR than in HCR controls (49±5% vs 68±5%, p = 0.04). IPC reduced infarct size by 47% in LCR (p<0.01) and by 31% in HCR rats (p = 0.01). RIC did not modulate infarct size (LCR: 52±5, p>0.99; HCR: 69±6%, p>0.99, respectively). Phosphorylaion of AMPK did not differ between LCR and HCR controls. IPC did not modulate cardiac phosphorylation of AMPK. Glucose uptake during reperfusion was similar in LCR and HCR rats. IPC increased glucose uptake during reperfusion in LCR animals (p = 0.02). Mitochondrial protein content in skeletal muscle was lower in LCR than in HCR (0.77±0.10 arbitrary units (AU) vs 1.09±0.07 AU, p = 0.02), but not in cardiac muscle.ConclusionAerobic capacity is associated with altered myocardial sensitivity to IR injury, but the cardioprotective effect of IPC is not. Glucose uptake, AMPK activation immediately prior to ischemia and basal mitochondrial protein content in the heart seem to be of minor importance as underlying mechanisms for the cardioprotective effects.

Regulation of Proteins in Human Skeletal Muscle: The Role of Transcription

Regular low intensity aerobic exercise (aerobic training) provides effective protection against various metabolic disorders. Here, the roles played by transient transcriptome responses to acute exercise and by changes in baseline gene expression during up-regulation of protein content in human skeletal muscle were investigated after 2 months of aerobic training. Seven untrained males were involved in a 2 month aerobic cycling training program. Mass-spectrometry and RNA sequencing were used to evaluate proteome and transcriptome responses to training and acute exercise. We found that proteins with different functions are regulated differently at the transcriptional level; for example, a training-induced increase in the content of extracellular matrix-related proteins is regulated at the transcriptional level, while an increase in the content of mitochondrial proteins is not. An increase in the skeletal muscle content of several proteins (including mitochondrial proteins) was associated with increased protein stability, which is related to a chaperone-dependent mechanism and/or reduced regulation by proteolysis. These findings increase our understanding of the molecular mechanisms underlying regulation of protein expression in human skeletal muscle subjected to repeated stress (long term aerobic training) and may provide an opportunity to control the expression of specific proteins (e.g., extracellular matrix-related proteins, mitochondrial proteins) through physiological and/or pharmacological approaches.

Lipid Metabolism Links Nutrient-Exercise Timing to Insulin Sensitivity in Men Classified as Overweight or Obese.

ContextPre-exercise nutrient availability alters acute metabolic responses to exercise, which could modulate training responsiveness.ObjectiveTo assess acute and chronic effects of exercise performed before versus after nutrient ingestion on whole-body and intramuscular lipid utilization and postprandial glucose metabolism.Design(1) Acute, randomized, crossover design (Acute Study); (2) 6-week, randomized, controlled design (Training Study).SettingGeneral community.ParticipantsMen with overweight/obesity (mean ± standard deviation, body mass index: 30.2 ± 3.5 kg⋅m-2 for Acute Study, 30.9 ± 4.5 kg⋅m-2 for Training Study).InterventionsModerate-intensity cycling performed before versus after mixed-macronutrient breakfast (Acute Study) or carbohydrate (Training Study) ingestion.ResultsAcute Study—exercise before versus after breakfast consumption increased net intramuscular lipid utilization in type I (net change: –3.44 ± 2.63% versus 1.44 ± 4.18% area lipid staining, P < 0.01) and type II fibers (–1.89 ± 2.48% versus 1.83 ± 1.92% area lipid staining, P < 0.05). Training Study—postprandial glycemia was not differentially affected by 6 weeks of exercise training performed before versus after carbohydrate intake (P > 0.05). However, postprandial insulinemia was reduced with exercise training performed before but not after carbohydrate ingestion (P = 0.03). This resulted in increased oral glucose insulin sensitivity (25 ± 38 vs –21 ± 32 mL⋅min-1⋅m-2; P = 0.01), associated with increased lipid utilization during exercise (r = 0.50, P = 0.02). Regular exercise before nutrient provision also augmented remodeling of skeletal muscle phospholipids and protein content of the glucose transport protein GLUT4 (P < 0.05).ConclusionsExperiments investigating exercise training and metabolic health should consider nutrient-exercise timing, and exercise performed before versus after nutrient intake (ie, in the fasted state) may exert beneficial effects on lipid utilization and reduce postprandial insulinemia.

Oral glutamine supplementation attenuates inflammation and oxidative stress-mediated skeletal muscle protein content degradation in immobilized rats: Role of 70 kDa heat shock protein

Skeletal muscle disuse results in myofibrillar atrophy and protein degradation, via inflammatory and oxidative stress-mediated NF-kB signaling pathway activation. Nutritional interventions, such as l-glutamine (GLN) supplementation have shown antioxidant properties and cytoprotective effects through the modulation on the 70-kDa heat shock protein (HSP70) expression. However, these GLN-mediated effects on cell signaling pathways and biochemical mechanisms that control the myofibrillar protein content degradation in muscle disuse situations are poorly known yet. This study investigated the effects of oral GLN plus l-alanine (ALA; GLN ​+ ​ALA-solution) supplementation, either in their free or dipeptide (L-alanyl-l-glutamine-DIP) form, on GLN-glutathione (GSH) axis and cytoprotection mediated by HSP70 protein expression in the slow-twitch soleus and fast-twitch gastrocnemius skeletal muscle of rats submitted to 14-days of hindlimb immobilization-induced disuse muscle atrophy. Forty-eight Wistar rats were distributed into 6 groups: hindlimb immobilized (IMOB group) and hindlimb immobilized orally supplemented with either GLN (1 g kg−1) plus ALA (0.61 g kg−1) ​(GLN ​+ ​ALA-IMOB group) or 1.49 ​g ​kg−1 of DIP (DIP-IMOB group) and; no-immobilized (CTRL) and no-immobilized supplemented GLN ​+ ​ALA and DIP baselines groups. All animals, including CTRL and IMOB rats (water), were supplemented via intragastric gavage for 14 days, concomitantly to immobilization period. Plasma and muscle GLN levels, lipid (thiobarbituric acid reactive substances-TBARS) and protein (carbonyl) peroxidation, erythrocyte concentration of reduced GSH and GSH disulfide (GSSG), plasma and muscle pro-inflammatory TNF-α levels, muscle IKKα/β-NF-kB signaling pathway and, the myofibrillar protein content (MPC) were measured. The MPC was significantly lower in IMOB rats, compared to CTRL, GLN ​+ ​ALA, and DIP animals (p ​< ​0.05). This finding was associated with reduced plasma and muscle GLN concentration, equally in IMOB animals. Conversely, both GLN ​+ ​ALA and DIP supplementation restored plasma and muscle GLN levels, which equilibrated GSH and intracellular redox status (GSSG/GSH ratio) in erythrocytes and skeletal muscle even as, increased muscle HSP70 protein expression; attenuating oxidative stress and TNF-α-mediated NF-kB pathway activation, fact that reverberated on reduction of MPC degradation in GLN ​+ ​ALA-IMOB and DIP-IMOB animals (p ​< ​0.05). In conclusion, the findings shown herein support the oral GLN ​+ ​ALA and DIP supplementations as a therapeutic and effective nutritional alternative to attenuate the deleterious effects of the skeletal muscle protein degradation induced by muscle disuse.

Serum amyloid A1 mediates myotube atrophy via Toll-like receptors.

BackgroundCritically ill patients frequently develop muscle atrophy and weakness in the intensive‐care‐unit setting [intensive care unit‐acquired weakness (ICUAW)]. Sepsis, systemic inflammation, and acute‐phase response are major risk factors. We reported earlier that the acute‐phase protein serum amyloid A1 (SAA1) is increased and accumulates in muscle of ICUAW patients, but its relevance was unknown. Our objectives were to identify SAA1 receptors and their downstream signalling pathways in myocytes and skeletal muscle and to investigate the role of SAA1 in inflammation‐induced muscle atrophy.MethodsWe performed cell‐based in vitro and animal in vivo experiments. The atrophic effect of SAA1 on differentiated C2C12 myotubes was investigated by analysing gene expression, protein content, and the atrophy phenotype. We used the cecal ligation and puncture model to induce polymicrobial sepsis in wild type mice, which were treated with the IкB kinase inhibitor Bristol‐Myers Squibb (BMS)‐345541 or vehicle. Morphological and molecular analyses were used to investigate the phenotype of inflammation‐induced muscle atrophy and the effects of BMS‐345541 treatment.ResultsThe SAA1 receptors Tlr2, Tlr4, Cd36, P2rx7, Vimp, and Scarb1 were all expressed in myocytes and skeletal muscle. Treatment of differentiated C2C12 myotubes with recombinant SAA1 caused myotube atrophy and increased interleukin 6 (Il6) gene expression. These effects were mediated by Toll‐like receptors (TLR) 2 and 4. SAA1 increased the phosphorylation and activity of the transcription factor nuclear factor ‘kappa‐light‐chain‐enhancer' of activated B‐cells (NF‐κB) p65 via TLR2 and TLR4 leading to an increased binding of NF‐κB to NF‐κB response elements in the promoter region of its target genes resulting in an increased expression of NF‐κB target genes. In polymicrobial sepsis, skeletal muscle mass, tissue morphology, gene expression, and protein content were associated with the atrophy response. Inhibition of NF‐κB signalling by BMS‐345541 increased survival (28.6% vs. 91.7%, P < 0.01). BMS‐345541 diminished inflammation‐induced atrophy as shown by a reduced weight loss of the gastrocnemius/plantaris (vehicle: −21.2% and BMS‐345541: −10.4%; P < 0.05), tibialis anterior (vehicle: −22.7% and BMS‐345541: −17.1%; P < 0.05) and soleus (vehicle: −21.1% and BMS‐345541: −11.3%; P < 0.05) in septic mice. Analysis of the fiber type specific myocyte cross‐sectional area showed that BMS‐345541 reduced inflammation‐induced atrophy of slow/type I and fast/type II myofibers compared with vehicle‐treated septic mice. BMS‐345541 reversed the inflammation‐induced atrophy program as indicated by a reduced expression of the atrogenes Trim63/MuRF1, Fbxo32/Atrogin1, and Fbxo30/MuSA1.ConclusionsSAA1 activates the TLR2/TLR4//NF‐κB p65 signalling pathway to cause myocyte atrophy. Systemic inhibition of the NF‐κB pathway reduced muscle atrophy and increased survival of septic mice. The SAA1/TLR2/TLR4//NF‐κB p65 atrophy pathway could have utility in combatting ICUAW.

Myotube Protein Content Associates with Intracellular L-Glutamine Levels.

Skeletal mass loss is reported in several catabolic conditions and it has been associated with a reduced intracellular L-glutamine content. We investigated the association of intracellular L-glutamine concentration with the protein content in skeletal muscle cells. We cultivated C2C12 myotubes in the absence or presence of 2 (reference condition), 8 or 16 mM L-glutamine for 48 hours, and the variations in the contents of amino acids and proteins measured. We used an inhibitor of L-glutamine synthesis (L-methionine sulfoximine - MSO) to promote a further reduction in intracellular L-glutamine levels. Amino acids contents in cells and media were measured using LC-MS/MS. We measured changes in phosphorylated Akt, RP-S6, and 4E-BP1contents in the absence or presence of insulin by western blotting. Reduced intracellular L-glutamine concentration was associated with decreased protein content and increased protein breakdown. Low intracellular glutamine levels were also associated with decreased p-Akt contents in the presence of insulin. A further decrease in intracellular L-glutamine caused by glutamine synthetase inhibitor reduced protein content and levels of amino acids generated from glutamine metabolism and increased bAib still further. Cells exposed to high medium glutamine levels did not have any change in protein content but exhibited increased contents of the amino acids derived from L-glutamine metabolism. Intracellular L-glutamine levels per se play a role in the control of protein content in skeletal muscle myotubes.

Reduced delta-6 desaturase activity partially protects against high-fat diet-induced impairment in whole-body glucose tolerance

Delta-6 desaturase (D6D), which is encoded by the fatty acid desaturase (Fads2) gene, is the rate-limiting enzyme for the endogenous production of n-3 long-chain polyunsaturated fatty acids. The absence of D6D activity in Fads2−/− knockout mice results in the inability to produce eicosapentaenoic acid and docosahexaenoic acid, and has previously been associated with altered glucose and lipid metabolism. Skeletal muscle is a major site for insulin-stimulated glucose disposal; however, the consequences of reduced D6D activity on skeletal muscle metabolism are unknown. The objective of this study was to examine the role of a partial reduction in D6D activity on whole-body glucose tolerance, skeletal muscle fatty acid profiles and protein content of key markers of carbohydrate and fat signaling pathways in the context of both low- and high-fat diets. Male C57BL/6J heterozygous (Fads2+/−) and wild-type (WT) mice were fed either a low-fat (16% kcal from fat) or high-fat (HFD; 45% kcal from fat) diet for 21 weeks. Fads2+/− mice were protected from the HFD-induced impairment in glucose tolerance. Unexpectedly, HFD-fed Fads2+/− mice had reduced GLUT4 skeletal muscle protein content compared to their WT counterparts. No changes were detected in total protein content of key markers of fatty acid uptake, glycogen formation or substrate oxidation. This study shows that reduced D6D activity is partially protective against HFD-induced impairments in whole-body glucose tolerance but does not appear to be due to increased muscle GLUT4 content or total content of proteins regulating substrate utilization.