Nicolas Pierre

Toll-Like Receptor 4 Knockout Mice Are Protected against Endoplasmic Reticulum Stress Induced by a High-Fat Diet

The purpose of this study was to investigate whether toll-like receptor 4 (TLR4) is implicated in the development of endoplasmic reticulum stress (ER stress) observed after a high-fat diet (HFD) in liver, skeletal muscle and adipose tissue. TLR4−/− and C57BL/6J wild-type mice (WT) were fed with chow or HFD (45% calories from fat) during 18 weeks. An oral glucose tolerance-test was performed. The animals were sacrificed in a fasted state and the tissues were removed. TLR4 deletion protected from body weight gain and glucose intolerance induced by HFD whereas energy intake was higher in transgenic mice suggesting larger energy expenditure. HFD induced an ER stress in skeletal muscle, liver and adipose tissue of WT mice as assessed by BiP, CHOP, spliced and unspliced XBP1 and phospho-eIF2α. TLR4−/− mice were protected against HFD-induced ER stress. Then, we investigated the main signaling downstream of TLR4 namely the NF-κB pathway, expecting to identify the mechanism by which TLR4 is able to activate ER stress. The mRNA levels of cytokines regulated by NF-κB namely TNFα, IL-1β and IL-6, were not changed after HFD and phospho-IκB-α (ser 32) was not changed. Our results indicate that TLR4 is essential for the development of ER stress related to HFD. Nevertheless, the NFκ-B pathway does not seem to be directly implicated. The reduced fat storage in TLR4−/− mice could explain the absence of an ER stress after HFD.

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.

TLR2 and TLR4 activation induces p38 MAPK-dependent phosphorylation of S6 kinase 1 in C2C12 myotubes.

Toll‐like receptors 2 (TLR2) and 4 (TLR4) are present in the plasma membrane of skeletal muscle cells where their functions remain incompletely resolved. They can bind various extracellular ligands, such as FSL‐1, lipopolysaccharide (LPS) and/or palmitic acid (PA). We have investigated the link between PA, TLR2/4 and ribosomal S6 kinase 1 (S6K1) in C2C12 myotubes. Incubation with agonists of either TLR2 or TLR4, and with a high concentration of PA, increased S6K1 phosphorylation. Canonical upstream kinases of S6K1, protein kinase B (PKB) and mammalian target of rapamycin complex 1 (mTORC1), were regulated in the opposite way by PA, indicating that these kinases were probably not involved. By using the SB202190 inhibitor, p38 MAPK (mitogen‐activated protein kinase) was found to be a key mediator of PA‐induced phosphorylation of S6K1. Downregulation of either tlr2 or tlr4 gene expression by small interfering RNAs prevented the activation of both p38 MAPK and S6K1 by FSL‐1, LPS or PA. Thus TLR2 and TLR4 agonists can increase the level of S6K1 phosphorylation in a p38 MAPK‐dependent way in C2C12 myotubes. As PA induced the same intracellular signalling, a novel atypical pathway for PA is induced at the cellular membrane level and results in a higher phosphorylation state of S6K1.

Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence

Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment.

Endurance Training Attenuates Catabolic Signals Induced by TNF-α in Muscle of Mice.

PURPOSE To determine whether endurance training attenuates the negative effects induced by an acute injection of tumor necrosis factor-α (TNF-α) in skeletal muscle of mice. METHODS Trained (6 wk of endurance training at 60% of maximal velocity) and untrained mice were injected with TNF-α or vehicle and killed 6 h after. Tibialis anterior muscles were analyzed using Western blot and qRT-PCR for markers of inflammation and protein synthesis/degradation. RESULTS Independently of training, TNF-α increased the mRNA of cytokines and downregulated signals linked to protein synthesis. The phosphorylation of IKKα/β and IκBα induced by TNF-α was blunted in trained mice, suggesting altered NF-κB activation. This was associated with lower induction of several markers of protein degradation (FoxO1, MURF1, MAFbx, myostatin, Gabarapl1, and LC3BII/LC3BI ratio). CONCLUSIONS Endurance training protects skeletal muscle against the activation of protein degradation signaling pathways induced by TNF-α.

IRE1α and TRB3 do not contribute to the disruption of proximal insulin signaling caused by palmitate in C2C12 myotubes.

Endoplasmic reticulum (ER) stress is a central actor in the physiopathology of insulin resistance (IR) in various tissues. The subsequent unfolded protein response (UPR) interacts with insulin signaling through inositol‐requiring 1α (IRE1α) activation and tribbles homolog 3 (TRB3) expressions. IRE1α impairs insulin actions through the activation of c‐Jun N‐terminal kinase (JNK), and TRB3 is a pseudokinase inhibiting Akt. In muscle cells, the link between ER stress and IR has only been demonstrated by using chemical ER stress inducers or overexpression techniques. However, the involvement of ER stress in lipid‐induced muscle IR remains controversial. The aim of the study is to test whether palmitate‐induced IRE1α signaling and TRB3 expression disturb insulin signaling in myogenic cells. C2C12 myotubes were exposed to palmitate and then stimulated with insulin. siRNA transfection was used to downregulate TRB3 and IRE1α. Palmitate increased TRB3 expression, activated IRE1α signaling, and reduced the insulin‐dependent Akt phosphorylation. Knocking down TRB3 or IRE1α did not prevent the inhibitory effect of palmitate on Akt phosphorylation. Our results support the idea that ER stress is not responsible for lipid‐induced IR in C2C12 myotubes.

Urolithin B, a newly identified regulator of skeletal muscle mass

The control of muscle size is an essential feature of health. Indeed, skeletal muscle atrophy leads to reduced strength, poor quality of life, and metabolic disturbances. Consequently, strategies aiming to attenuate muscle wasting and to promote muscle growth during various (pathological) physiological states like sarcopenia, immobilization, malnutrition, or cachexia are needed to address this extensive health issue. In this study, we tested the effects of urolithin B, an ellagitannin‐derived metabolite, on skeletal muscle growth.

ER Stress Induced By Hydrogen Peroxide In C2C12 Cells

Oxidative stress and endoplasmic reticulum stress (ER stress) are both involved in a number of diseases amongst which insulin resistance, Parkinson syndrome and atherosclerosis. Although, those stresses are often regarded in a separately way, it is known that they can be interrelated. Oxidative stress could lead to ER stress and vice versa as demonstrated by studies focused on the potential cross talk between the signaling pathways triggered by each of them in various cell types including mouse fibroblast cells, Chinese hamster ovary cells and mesencephalic cells. Skeletal muscle is a great provider of reactive oxygen species (ROS) and ER stress has been found to be present in skeletal muscle in certain conditions such as high-fat feeding and extreme endurance exercise. In agreement with those observations we have investigated the consequences of oxidative stress on ER stress in C2C12 myotubes. PURPOSE: To establish if oxidative stress, induced by hydrogen peroxide (H2O2), can lead to ER stress in C2C12 myotubes. METHODS: Myogenic C2C12 cells were grown until confluence and further differentiated for 4 days. Preliminary experiments were carried out to determine the optimal H2O2 dose and incubation period for inducing ER stress. A dose of 200μM and incubation periods of 4h and 17h were the most potent conditions for triggering ER stress and were used subsequently. Several downstream targets of the IRE1α and PERK pathways were analyzed at the protein level by western blot and at the mRNA level by qPCR. Three independent experiments were carried out. Unpaired Student t-test was used for the statistical analysis. RESULTS: After 4h H2O2 incubation, P-eIF2 α increased 7.7 +/- 1.3 fold (p 0.05). At the mRNA level, bip and atf4 increased 1.9 +/- 0.2 and 2.4 +/- 0.4 fold, respectively (p<0.05), whereas spliced xbp1 (0.90 +/- 0.2 fold) and unspliced xbp1 (0.8 +/- 0.2 fold) were not altered (ns). Finally chop and trb3 mRNA levels augmented drastically with H2O2 (17.9 +/- 4.8 and 34.4 +/- 6.3 fold, respectively, p<0.05). CONCLUSION: Hydrogen peroxide induces ER stress in C2C12 myotubes through at least the PERK pathway.