Jean-Marc Raymackers

The unfolded protein response is activated in skeletal muscle by high-fat feeding: potential role in the downregulation of protein synthesis

High-fat diets are known to decrease muscle protein synthesis, the adaptation to overload, and insulin sensitivity. Conditions that disrupt endoplasmic reticulum (ER) homeostasis lead to the activation of the unfolded protein response (UPR) that is associated with decreases in protein synthesis, chronic inflammation, and insulin resistance. The purpose of the present study was to establish whether ER stress is induced by a high-fat diet in skeletal muscle and whether ER stress can decrease mTORC1 activity and protein synthesis in muscle cells. Two independent protocols of high-fat feeding activated the UPR in mice. In the first study, mice consuming a high-fat diet containing 70% fat and <1% carbohydrates for 6 wk showed higher markers of the UPR (BiP, IRE1α, and MBTPS2) in the soleus and in the tibialis anterior muscles and ATF4 in the tibialis anterior (P < 0.05). In the second study, a 20-wk high-fat diet containing 46% fat and 36% carbohydrates also increased BiP, IRE1α, and phospho-PERK protein and the expression of ATF4, CHOP, and both the spliced and unspliced forms of XBP1 in the plantar flexors (P < 0.05). In C(2)C(12) muscle cells, tunicamycin, thapsigargin, and palmitic acid all increased UPR markers and decreased phosphorylation of S6K1 (P < 0.05). Collectively, these data show that a high-fat diet activates the UPR in mouse skeletal muscle in vivo. In addition, in vitro studies indicate that palmitic acid, and other well-known ER stress inducers, triggered the UPR in myogenic cells and led to a decrease in protein synthesis and mTORC1 activity.

Resolution of early diffusion-weighted and FLAIR MRI abnormalities in a patient with TIA

Article abstract We report a patient with a clinical history and neurologic examination consistent with acute stroke. Diffusion-weighted and fast fluid-attenuated inversion recovery MRI obtained 4 hours after stroke onset detected focal abnormalities suggestive of acute ischemic brain damage. The neurologic deficit and the imaging abnormalities both resolved completely at follow-up. This patient illustrates complete resolution of early changes observed with diffusion-weighted MRI at the hyperacute phase in a TIA.

Endoplasmic reticulum stress markers and ubiquitin–proteasome pathway activity in response to a 200-km run.

PURPOSE This study investigated whether a 200-km run modulates signaling pathways implicated in cellular stress in skeletal muscle, with special attention paid to the endoplasmic reticulum (ER) stress and to the activation of the ubiquitin-proteasome pathway. METHODS Eight men ran 200 km (28 h 03 min ± 2 h 01 min). Two muscle biopsies were obtained from the vastus lateralis muscle 2 wk before and 3 h after the race. Mitogen-activated protein kinase, ubiquitin-proteasome pathway, ER stress, inflammation, and oxidative stress markers were assayed by Western blot analysis or by quantitative real-time polymerase chain reaction. Chymotrypsin-like activity of the proteasome was measured by a fluorimetric assay. RESULTS Phosphorylation states of extracellular signal-related kinase 1/2 (+401% ± 173.8%, P = 0.027) and c-Jun N-terminal (+149% ± 61.9%, P = 0.023) increased after the race, whereas p38 phosphorylation remained unchanged. Increases in BiP (+235% ± 94.7%, P = 0.021) and in the messenger RNA level of total (+138% ± 31.2%, P = 0.002) and spliced X-box binding protein 1 (+241% ± 53.3%, P = 0.001) indicated the presence of ER stress. Transcripts of inflammatory markers interleukin-6 (+403% ± 96.1%, P = 0.002) and tumor necrosis factor-α (+233% ± 58.4%, P = 0.003) as well as oxidative stress markers metallothionein 1F (+519% ± 258.3%, P = 0.042), metallothionein 1H (+666% ± 157.5%, P = 0.002), and nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) (+162% ± 60.5%, P = 0.016) were increased. The messenger RNA level of the ubiquitin ligases muscle-specific RING finger 1 (+583% ± 244.3%, P = 0.024) and muscle atrophy F-box (+249% ± 83.8%, P = 0.011) and the C2 proteasome subunit (+116% ± 40.6%, P = 0.012) also increased. Surprisingly, the amount of ubiquitin-conjugated proteins and the chymotrypsin-like activity of the proteasome were decreased by 20% ± 8.3% (P = 0.025) and 21% ± 4.4% (P = 0.001), respectively. The expression of ubiquitin-specific protease 28 deubiquitinase was increased (+81% ± 37.9%, P = 0.034). CONCLUSIONS In the skeletal muscle, a 200-km run activates the expression of ubiquitin ligases muscle-specific RING finger 1 and muscle atrophy F-box as well as various cellular stresses, among which are ER stress, oxidative stress, and inflammation. Meanwhile, compensatory mechanisms seem also triggered: the unfolded protein response is up-regulated, and the chymotrypsin-like activity of the proteasome is repressed.

Enhancing translation: guidelines for standard pre-clinical experiments in mdx mice

Duchenne Muscular Dystrophy is an X-linked disorder that affects boys and leads to muscle wasting and death due to cardiac involvement and respiratory complications. The cause is the absence of dystrophin, a large structural protein indispensable for muscle cell function and viability. The mdx mouse has become the standard animal model for pre-clinical evaluation of potential therapeutic treatments. Recent years have seen a rapid increase in the number of experimental compounds being evaluated in the mdx mouse. There is, however, much variability in the design of these pre-clinical experimental studies. This has made it difficult to interpret and compare published data from different laboratories and to evaluate the potential of a treatment for application to patients. The authors therefore propose the introduction of a standard study design for the mdx mouse model. Several aspects, including animal care, sampling times and choice of tissues, as well as recommended endpoints and methodologies are addressed and, for each aspect, a standard procedure is proposed. Testing of all new molecules/drugs using a widely accepted and agreed upon standard experimental protocol would greatly improve the power of pre-clinical experimentations and help identifying promising therapies for the translation into clinical trials for boys with Duchenne Muscular Dystrophy.

Sprint Interval Training in Hypoxia Stimulates Glycolytic Enzyme Activity

PURPOSE In this study, we compared the effect of sprint interval training (SIT) in normoxia versus hypoxia on muscle glycolytic and oxidative capacity, monocarboxylate transporter content, and endurance exercise performance. METHODS Healthy male volunteers (18-30 yr) performed 6 wk of SIT on a cycling ergometer (30-s sprints vs 4.5-min rest intervals; 3 d · wk(-1)) in either normobaric hypoxia (HYP, FiO2 = 14.4%, n = 10) or normoxia (NOR, FiO2 = 20.9%, n = 9). The control group did not train (CON, n = 10). Training load was increased from four sprints per session in week 1 to nine sprints in week 6. Before and after SIT, subjects performed a maximal incremental exercise test plus a 10-min simulated time trial on a cycle ergometer in both normoxia (MAX nor and TT nor) and hypoxia (MAX hyp and TT hyp). A needle biopsy was taken from musculus vastus lateralis at rest 5-6 d after the last exercise session. RESULTS SIT increased muscle phosphofructokinase activity more in HYP (+59%, P < 0.05) than that in NOR (+17%), whereas citrate synthase activity was similar between groups. Compared with the pretest, power outputs corresponding to 4 mmol blood lactate in HYP during MAX nor (+7%) and MAX hyp (+9%) were slightly increased (P < 0.05), whereas values were constant in NOR. V·O 2max in MAX nor and TT performance in TT nor and TT hyp were increased by ≈ 6%-8% (P < 0.05) in either group. The training elevated monocarboxylate transporter 1 protein content by ≈ 70% (P < 0.05). In CON, all measurements were constant throughout the study. CONCLUSION SIT in hypoxia up-regulated muscle phosphofructokinase activity and the anaerobic threshold more than SIT in normoxia but did not enhance endurance exercise performance.

TLR2 and TLR4 Activate p38 MAPK and JNK during Endurance Exercise in Skeletal Muscle

PURPOSE Toll-like receptors 2 and 4 (TLR2, TLR4) are found in the membrane of skeletal muscle cells. A variety of molecular components can activate TLR2 and TLR4, among others, long-chain fatty acids. The subsequent downstream signaling triggers the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways. Therefore, the purpose of this study was to test whether an elevation of extracellular nonesterified fatty acids (NEFA) observed during endurance exercise may activate the MAPK and NF-κB pathways via TLR2 and TLR4. METHODS tlr2 and tlr4 mice and wild-type C57BL/6J animals (WT) were submitted to a standardized endurance exercise. RESULTS Immediately after exercise, the phosphorylation state of p38 MAPK, c-Jun NH2-terminal kinase (JNK), and c-Jun was increased in the tibialis anterior (TA) and soleus (SOL) muscles of WT (P < 0.05). The phosphorylation state of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and IκB kinase α/β and the DNA-binding of NF-κB remained unchanged. The activation of p38 MAPK, JNK, and c-Jun was completely blunted in TA of tlr2 and tlr4 mice, whereas in SOL, it represented only 25% of the increase observed in WT mice. The causal relationship between NEFA concentration and MAPK activation was evaluated by injecting mice with heparin. A similar increase in plasma NEFA was observed after heparin injection than after endurance exercise. JNK and p38 MAPK were activated under heparin in TA and SOL of WT (P < 0.05) but not in muscles of tlr2 and tlr4 mice. CONCLUSIONS The present study supports the idea that during endurance exercise, TLR2 and TLR4 mediate a signal linking the elevated plasma NEFA concentration to the activation of p38 MAPK and JNK.

Prevention of muscle disuse atrophy by MG132 proteasome inhibitor.

Introduction: Our goal was to determine whether in vivo administration of the proteasome inhibitor MG132 can prevent muscle atrophy caused by hindlimb unloading (HU). Methods: Twenty‐seven NMRI mice were assigned to a weight‐bearing control, a 6‐day HU, or a HU+MG132 (1 mg/kg/48 h) treatment group. Results: Gastrocnemius wasting was significantly less in HU+MG132 mice (−6.7 ± 2.0%) compared with HU animals (−12.6 ± 1.1%, P = 0.011). HU was also associated with an increased expression of MuRF‐1 (P = 0.006), MAFbx (P = 0.001), and USP28 (P = 0.027) mRNA, whereas Nedd4, E3α, USP19, and UBP45 mRNA did not change significantly. Increases in MuRF‐1, MAFbx, and USP28 mRNA were largely repressed after MG132 administration. β5 proteasome activity tended to increase in HU (+16.7 ± 6.1%, P = 0.086). Neither β1 and β2 proteasome activities nor ubiquitin‐conjugated proteins were changed by HU. Conclusions: Our results indicate that in vivo administration of MG132 partially prevents muscle atrophy associated with disuse and highlight an unexpected regulation of MG132 proteasome inhibitor on ubiquitin‐ligases. Muscle Nerve, 2011

Glycine antagonist and NO synthase inhibitor protect the developing mouse brain against neonatal excitotoxic lesions.

The prevention of cerebral palsy and neuroprotection of the immature brain continue to be health care priorities. The pathophysiology of perinatal brain lesions associated with cerebral palsy seems to be multifactorial and includes pre- and perinatal factors such as preconceptional events, hormone and growth factors deficiencies, maternal infections with production of cytokines, and hypoxic/ischemic perfusion failures. Excitotoxic cascade could represent a common pathway that leads to neural cell death and subsequent brain damage. Brain injuries induced by ibotenate, a glutamatergic analog, which are essentially mediated through the N-methyl-D-aspartate receptor, mimic some aspects of the white matter cysts and transcortical necrosis observed in human perinatal brain damage. The purpose of the present study was to assess the protective role of several pharmacological agents, administered in conjunction with ibotenate, against induced excitotoxic lesions. We injected ibotenate in the developing mouse brain 5 d postnatally, after the full settlement of neuronal layers. Co-treatment with kynurenic acid, and antagonist of the facilitating glycine site of the N-methyl-D-asparate receptor, or with NG-nitro-L-arginine, an inhibitor of nitric oxide synthesis, induced a dose-dependent neuroprotective effect. Conversely, zinc gluconate, a blocking agent of the channel linked to the N-methyl-D-aspartate receptor, and a free radical scavenger (U74389F), were unable to protect the developing brain against excitotoxic attack. These data help to clarify some molecular mechanisms involved in excitotoxic lesions of the developing mouse brain and permit us to envision new strategies in the prevention of cerebral palsy.

Effect of creatine supplementation on skeletal muscle of mdx mice

Dystrophic mice (mdx) and their controls (C57/Bl10) were fed for 1 month with a diet with or without creatine (Cr) enrichment. Cr supplementation reduced mass (by 19%, P < 0.01) and mean fiber surface (by 25%, P < 0.05) of fast‐twitch mdx muscles. In both strains, tetanic tension increased slightly (9.2%) without reaching statistical significance (P = 0.08), and relaxation time increased by 16% (P < 0.001). However, Cr had no protective effect on the other hallmarks of dystrophy such as susceptibility to eccentric contractions; large numbers of centrally nucleated fibers in tibialis anterior; and elevated total calcium content, which increased by 85% (P = 0.008) in gastrocnemius mdx muscles. In conclusion, Cr may be a positive intervention for improving function of dystrophic muscle. Muscle Nerve 29: 687–692, 2004

Activation of ER stress by hydrogen peroxide in C2C12 myotubes.

The purpose of this study was to examine the link between oxidative stress and endoplasmic reticulum (ER) stress in myogenic cells. C2C12 myotubes were incubated with hydrogen peroxide (H2O2, 200 μM) and harvested 4h or 17 h after the induction of this oxidative stress. A massive upregulation of binding immunoglobulin protein (BiP) was found, indicating the presence of ER stress. Nevertheless, the three branches of the unfolded protein response (UPR) were not activated to the same extent. The double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK) branch was the most activated as shown by the increase of phospho-eukaryotic translation-initiation factor 2α (eIF2α, Ser51) and the mRNA levels of activating transcription factor 4 (ATF4), C/EBP homologous (CHOP) and tribbles homolog 3 (TRB3). The slight increase in the spliced form of X-box binding protein 1 (XBP1s) together with the decrease of the unspliced form (XBP1u) indicated a higher endoribonuclease activity of inositol-requiring 1α (IRE1α). The transcriptional activity of activating transcription factor 6 (ATF6) remained unchanged after incubation with H2O2. The mechanisms by which the three branches of UPR can be specifically regulated by oxidative stress are currently unresolved and need further investigations.