Exercise Reduces the Protein Abundance of TXNIP and Its Interacting Partner REDD1 in Skeletal Muscle: Potential Role for a PKA-Mediated Mechanism

Thioredoxin-interacting protein (TXNIP) negatively effects the redox state and growth signaling via its interactions with thioredoxin (TRX) and regulated in development and DNA damage response 1 (REDD1), respectively. TXNIP expression is downregulated by pathways activated during aerobic exercise (AE), via posttranslational modifications (PTMs; serine phosphorylation and ubiquitination). The purpose of this investigation was to determine the effects of acute AE on TXNIP expression, posttranslational modifications, and its interacting partners, REDD1 and TRX. Fifteen healthy adults performed 30 minutes of aerobic exercise (80% VO2max) with muscle biopsies taken before, immediately following, and three hours following the exercise bout. To explore potential mechanisms underlying our in vivo findings, primary human myotubes were exposed to two models of exercise, electrical pulse stimulation (EPS) and palmitate-forskolin-ionomycin (PFI). Immediately following exercise, TXNIP protein decreased, but returned to pre-exercise levels three hours post exercise. These results were replicated in our PFI exercise model only. Although not statistically significant, there was a trending main effect in serine-phosphorylation status of TXNIP (p=0.07) immediately following exercise. REDD1 protein decreased three hours after exercise. AE had no effect on TRX protein expression, gene expression or the activity of its reducing enzyme, thioredoxin reductase. Consequently, AE had no effect on the TRX: TXNIP interaction. Our results indicate that AE leads to acute reductions in TXNIP and REDD1 protein expression. However, these changes did not result in alterations in the TRX: TXNIP interaction and could not be entirely explained by alterations in TXNIP PTMs or changes in TRX expression or activity.

Mitochondrial And Myogenic Gene Expression Pathways Are Influenced By The Exercise Mimetic Formoterol In Human Myotubes

Background: Exercise is an effective treatment for establishing and maintaining skeletal muscle (SKM) health. The interconnected cascade of gene expression pathways related to myogenesis, mitochondrial homeostasis, and thyroid hormone metabolism are critical to SKM health. This in vitro study was conducted to investigate the effects of exercise mimetic (formoterol) stimulation on human SKM cell signaling during myogenesis, and to provide insight on potential targets for future studies exploring therapies for SKM atrophy.Methods: Human myoblasts were cultured and differentiated to evaluate the effects of exercise mimetic stimulation on gene expression during mid and late myogenesis. We characterized the expression of 24 genes related to myogenesis, mitochondrial biogenesis, thyroid hormone metabolism, and cellular homeostasis.Results: Formoterol stimulated the gene expression for SKM pathways related to mitochondrial biogenesis, thyroid metabolism, and cellular homeostasis. Additionally, formoterol resulted in a myogenic program that appears to favor prolonged myoblast proliferation and delayed myotube maturation.Conclusion: Robust, yet differential effects of exercise mimetic stimulation on gene expression during mid-myogenesis and at terminal differentiation were found. The results of our study support the groundwork for establishing further experiments utilizing exercise signaling as a therapeutic treatment in models targeting dysfunctional SKM cell growth.

Muscle-Derived Cytokines Reduce Growth, Viability and Migratory Activity of Pancreatic Cancer Cells

The evidence that regular physical exercise reduces the risk of developing cancer is well described. However, the interaction between physical exercise and cancer is not fully clarified yet. Several myokines released by skeletal muscle appear to have a direct anti-tumour function. There are few data on myokine secretion after exercise in patients with advanced tumours. Pancreatic cancer (PC) is a very aggressive and usually fatal cancer. To investigate the effects of exercise in PC, the blood of advanced-stage PC patients was analysed after 12 weeks of resistance training using whole-body electromyostimulation. After the 12-week training period, the patient serum inhibited the proliferation and the motility of PC cells and enhanced PC cell apoptosis. The impact of exercise training was also investigated in an exercise-mimicking in vitro model using electric pulse stimulation of human myotubes and revealed similar anti-tumour effects on PC cells, clearly indicating direct cancer-protective properties of activated skeletal muscle. Protein and gene expression analyses in plasma from exercise-trained patients and in myotube cultures after in vitro exercise showed that interleukin 10 (IL10), C-X-C motif ligand 1 (CXCL1) and C-C motif chemokine ligand 4 (CCL4) are myokines released from activated skeletal muscle. In accordance with the effects of serum from exercise-trained patients, the supplementation with recombinant IL10, CXCL1 and CCL4 impaired growth and migration of PC cells. Treatment of PC cells with these myokines upregulated caspase 3/7 expression and the cleavage of poly(ADP-ribose) polymerase, leading to enhanced PC cell death. The identification of myokines with anti-tumour properties in advanced-stage PC patients after exercise opens a new perspective in supportive therapy with sports and exercise for cancer patients.

Branched-chain amino acid metabolism is regulated by ERRα in primary human myotubes and is further impaired by glucose loading in type 2 diabetes

Aims/hypothesis Increased levels of branched-chain amino acids (BCAAs) are associated with type 2 diabetes pathogenesis. However, most metabolomic studies are limited to an analysis of plasma metabolites under fasting conditions, rather than the dynamic shift in response to a metabolic challenge. Moreover, metabolomic profiles of peripheral tissues involved in glucose homeostasis are scarce and the transcriptomic regulation of genes involved in BCAA catabolism is partially unknown. This study aimed to identify differences in circulating and skeletal muscle BCAA levels in response to an OGTT in individuals with normal glucose tolerance (NGT) or type 2 diabetes. Additionally, transcription factors involved in the regulation of the BCAA gene set were identified. Methods Plasma and vastus lateralis muscle biopsies were obtained from individuals with NGT or type 2 diabetes before and after an OGTT. Plasma and quadriceps muscles were harvested from skeletal muscle-specific Ppargc1a knockout and transgenic mice. BCAA-related metabolites and genes were assessed by LC-MS/MS and quantitative RT-PCR, respectively. Small interfering RNA and adenovirus-mediated overexpression techniques were used in primary human skeletal muscle cells to study the role of PPARGC1A and ESRRA in the expression of the BCAA gene set. Radiolabelled leucine was used to analyse the impact of oestrogen-related receptor α (ERRα) knockdown on leucine oxidation. Results Impairments in BCAA catabolism in people with type 2 diabetes under fasting conditions were exacerbated after a glucose load. Branched-chain keto acids were reduced 37–56% after an OGTT in the NGT group, whereas no changes were detected in individuals with type 2 diabetes. These changes were concomitant with a stronger correlation with glucose homeostasis biomarkers and downregulated expression of branched-chain amino acid transaminase 2, branched-chain keto acid dehydrogenase complex subunits and 69% of downstream BCAA-related genes in skeletal muscle. In primary human myotubes overexpressing peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α, encoded by PPARGC1A), 61% of the analysed BCAA genes were upregulated, while 67% were downregulated in the quadriceps of skeletal muscle-specific Ppargc1a knockout mice. ESRRA (encoding ERRα) silencing completely abrogated the PGC-1α-induced upregulation of BCAA-related genes in primary human myotubes. Conclusions/interpretation Metabolic inflexibility in type 2 diabetes impacts BCAA homeostasis and attenuates the decrease in circulating and skeletal muscle BCAA-related metabolites after a glucose challenge. Transcriptional regulation of BCAA genes in primary human myotubes via PGC-1α is ERRα-dependent. Graphical abstract

A unified model of amino acid homeostasis in mammalian cells

Homeostasis is one of the fundamental concepts in physiology. Despite remarkable progress in our molecular understanding of amino acid transport, metabolism and signalling, it remains unclear by what mechanisms cytosolic amino acid concentrations are maintained. We propose that amino acid transporters are the primary determinants of intracellular amino acid levels. We show that a cell’s endowment with amino acid transporters can be deconvoluted by a logical series of experiments. This was used to computationally simulate amino acid translocation across the plasma membrane. For two different cancer cell lines and human myotubes, transport simulation generates cytosolic amino acid concentrations that are close to those observed in vitro. Perturbations of the system were replicated in silico and could be applied to systems where only transcriptomic data are available. The methodology developed in this study is widely applicable to other transport processes and explain amino acid homeostasis at the systems-level.

Effects of Physiological Doses of Resveratrol and Quercetin on Glucose Metabolism in Primary Myotubes

Phenolic compounds have emerged in recent years as an option to face insulin resistance and diabetes. The central aim of this study was: (1) to demonstrate that physiological doses of resveratrol (RSV) or quercetin (Q) can influence glucose metabolism in human myotubes, (2) to establish whether AMP-activated protein kinase (AMPK) and protein kinase B –PKB- (Akt) pathways are involved in this effect. In addition, the effects of these polyphenols on mitochondrial biogenesis and fatty acid oxidation were analysed. Myotubes from healthy donors were cultured for 24 h with either 0.1 μM of RSV or with 10 μM of Q. Glucose metabolism, such as glycogen synthesis, glucose oxidation, and lactate production, were measured with D[U-14C]glucose. β-oxidation using [1–14C]palmitate as well as the expression of key metabolic genes and proteins by Real Time PCR and Western blot were also assessed. Although RSV and Q increased pgc1α expression, they did not significantly change either glucose oxidation or β-oxidation. Q increased AMPK, insulin receptor substrate 1 (IRS-1), and AS160 phosphorylation in basal conditions and glycogen synthase kinase 3 (GSK3β) in insulin-stimulated conditions. RSV tended to increase the phosphorylation rates of AMPK and GSK3β. Both of the polyphenols increased insulin-stimulated glycogen synthesis and reduced lactate production in human myotubes. Thus, physiological doses of RSV or Q may exhibit anti-diabetic actions in human myotubes.

A Novel in vitro Model for Mature Toxoplasma gondii Bradyzoites Reveals their Metabolome and a Diminished Role of the Mitochondrial Tricarboxylic Acid Cycle

SummaryThe apicomplexan parasite Toxoplasma gondii causes chronic and drug-tolerant infections, yet current models do not permit metabolomic characterization of these persisting tissue cysts. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts that enabled direct measurements of their metabolome. The cysts are functionally mature and tolerate exposure to a range of antibiotics and to extended temperature stresses and are orally infectious to mice. Metabolomic characterization of purified cysts reveals global changes that comprise systematically increased levels of amino acids and decrease abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. Consequently, pharmacological modulation of the TCA cycle in T. gondii bradyzoites reveals that this pathway is rendered dispensable during parasite stage conversion. Direct access to persisting parasite stages will be essential for the dissection of functionally important host-parasite interactions and drug evasion mechanisms and also help to identify new strategies for therapeutic intervention.Highlights–Toxoplasma gondii forms mature tissue cysts in immortalized human myotubes–In vitro cysts of T. gondii develop drug tolerance and temperature stress resistance–Untargeted metabolomic characterization of tissue cysts reveals a distinct metabolome–The mitochondrial tricarboxylic acid cycle is dispensable in T. gondii bradyzoites

Substrate oxidation in primary human skeletal muscle cells is influenced by donor age

Primary human myotubes represent an alternative system to intact skeletal muscle for the study of human diseases related to changes in muscle energy metabolism. This work aimed to study if fatty acid and glucose metabolism in human myotubes in vitro were related to muscle of origin, donor gender, age, or body mass index (BMI). Myotubes from a total of 82 donors were established from three different skeletal muscles, i.e., musculus vastus lateralis, musculus obliquus internus abdominis, and musculi interspinales, and cellular energy metabolism was evaluated. Multiple linear regression analyses showed that donor age had a significant effect on glucose and oleic acid oxidation after correcting for gender, BMI, and muscle of origin. Donor BMI was the only significant contributor to cellular oleic acid uptake, whereas cellular glucose uptake did not rely on any of the variables examined. Despite the effect of age on substrate oxidation, cellular mRNA expression of pyruvate dehydrogenase kinase 4 (PDK4) and peroxisome proliferator–activated receptor gamma coactivator 1 alpha (PPARGC1A) did not correlate with donor age. In conclusion, donor age significantly impacts substrate oxidation in cultured human myotubes, whereas donor BMI affects cellular oleic acid uptake.