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See detailPotential Role of Epithelial Endoplasmic Reticulum Stress and Anterior Gradient Protein 2 Homolog in Crohn’s Disease Fibrosis
VIEUJEAN, Sophie ULiege; Hu, Shurong; Bequet, Emeline ULiege et al

in Journal of Crohn's and Colitis (2021)

Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD). It is characterised by an accumulation of fibroblasts differentiating into myofibroblasts secreting excessive ... [more ▼]

Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD). It is characterised by an accumulation of fibroblasts differentiating into myofibroblasts secreting excessive extracellular matrix. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. Methods: We performed a pilot proteomic study comparing the proteome of surface epithelium, isolated by laser-capture microdissection, in normal and fibrotic zones of resected ileal CD strictures (13 zones collected in 5 patients). Proteins of interests were validated by immunohistochemistry (IHC) in ileal and colonic samples of stricturing CD (n=44), pure inflammatory CD (n=29) and control (n=40) subjects. The pro-fibrotic role of one selected epithelial protein was investigated through in-vitro experiments using HT-29 epithelial cells and a CCD-18Co fibroblast to myofibroblast differentiation model. Results: Proteomic study revealed an endoplasmic reticulum (ER) stress proteins increase in the epithelium of CD ileal fibrotic strictures, including Anterior gradient protein 2 homolog (AGR2) and Binding-immunoglobulin protein (BiP). This was confirmed by IHC. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2 intracellular expression and its secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin, led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. By using recombinant protein and blocking agent for AGR2, we demonstrated that the secretion of this protein by epithelial cells can play a role in the myofibroblastic differentiation. Conclusions: The development of CD fibrotic strictures could involve epithelial ER stress and particularly the secretion of AGR2. [less ▲]

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See detailPotential Role of Epithelial Protein Disulphide Isomerases in Crohn’s Disease Fibrosis
Vieujean, Sophie ULiege; Hu, Shurong; Bequet, Emeline ULiege et al

in Acta Gastro-Enterologica Belgica (2021, March 03)

Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD) characterized by an accumulation of fibroblasts differentiating into activated myofibroblasts secreting excessive ... [more ▼]

Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD) characterized by an accumulation of fibroblasts differentiating into activated myofibroblasts secreting excessive extracellular matrix. In in-vitro experiments, this myofibroblastic differentiation is elicited by a whole series of factors among which transforming growth factor β1 (TGF-β1) seems to play a key role. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. Methods: We performed a pilot proteomic study comparing the proteome of surface epithelium isolated by laser-capture microdissection in normal and fibrotic zones of resected ileal CD strictures (13 zones collected in 5 patients). The pro-fibrotic role of selected epithelial proteins was investigated through in-vitro experiments using HT-29 epithelial cells and a CCD-18Co fibroblast to myofibroblast differentiation model. Results: Proteomic study revealed an endoplasmic reticulum (ER) stress proteins increase in the epithelium of CD ileal fibrotic strictures, including Anterior gradient protein 2 homolog (AGR2), Protein disulphide isomerase A6 (PDIA6) and Endoplasmic reticulum resident protein 44 (ERP44) which are 3 protein disulphide isomerases. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2, PDIA6, ERP44 as well as TGF β1 intracellular expression and their secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin (Tm), led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. The application of blocking agents for AGR2, PDIA6, ERP44 or TGF β1 in the supernatant of these Tm pre conditioned HT-29 cells, attenuated the myofibroblastic differentiation induced by this supernatant, suggesting a pro-fibrotic role of these secreted epithelial proteins. Conclusions: The development of CD fibrotic strictures may involve ER stress in epithelial cells, releasing a whole set of proteins into their environment, including AGR2, PDIA6, ERP44 as well as TGF-β1, which could exercise a pro-fibrotic role through a paracrine action. [less ▲]

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See detailIncreased Endoplasmic Reticulum stress specific chaperones characterise CD fibrosis epithelium tissues and participates to in vitro induction of intestinal fibroblasts differentiation
Vieujean, Sophie ULiege; Hu, Shurong; Bequet, Emeline ULiege et al

Poster (2020, March 05)

Background: Intestinal fibrosis is a complication of Crohn’s disease (CD) characterized by myofibroblasts and extracellular matrix accumulation within the submucosa and smooth muscles, leading to bowel ... [more ▼]

Background: Intestinal fibrosis is a complication of Crohn’s disease (CD) characterized by myofibroblasts and extracellular matrix accumulation within the submucosa and smooth muscles, leading to bowel strictures. No medical treatment exists to treat or reverse intestinal fibrosis leading often to surgical resection. The potential role of intestinal epithelium in the fibrotic process remains poorly defined. Methods: We performed a pilot study on ileal fibrostricturing CD surgical samples (n=5), comparing the proteome of surface epithelium isolated by laser capture microdissection in normal and fibrotic zones. Confirmation of the specific protein increases was obtained by immunohistochemistry in colonic and ileal samples of CD (n=44) compared to healthy subjects (n=40), as well as in intestinal epithelial cell line under induced Endoplasmic Reticulum (ER) stress. A model of fibroblast to myofibroblast differentiation induction was also challenged using preconditioned media of intestinal epithelial cells after a pulsed ER stress. Results: Label free proteomics revealed high ER stress in the epithelium surrounding fibrotic bowel wall, involving Anterior gradient protein 2 homolog (AGR2) and 78kDA glucose regulated protein (BiP). Confirmation of both proteins increase was obtained by immunohistochemistry. ER stress induction in intestinal epithelial cells was associated with an intracellular increase of AGR2, BiP and ER stress markers as sXPB1 and CHOP. AGR2 was also detected in the culture medium of these epithelial cells and myofibroblast differentiation was obtained using this culture medium. Conclusions: The increase of ER stress proteins observed in fibrostenosing tissues together with These preliminary evidences of fibroblast to myofibrobast differentiation obtained by paracrine action of intestinal epithelial cell preconditioned to ER stress induction, suggest a role of epithelial ER stress in Crohn’s disease intestinal fibrosis. [less ▲]

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See detailIncreased Endoplasmic Reticulum stress specific chaperones characterise CD fibrosis epithelium tissues and participate to in vitro induction of intestinal fibroblasts differentiation
Vieujean, Sophie ULiege; Hu, Shurong; Bequet, Emeline ULiege et al

Poster (2020, February 14)

Background: Intestinal fibrosis is a complication of Crohn’s disease (CD) characterized by myofibroblasts and extracellular matrix accumulation within the submucosa and smooth muscles, leading to bowel ... [more ▼]

Background: Intestinal fibrosis is a complication of Crohn’s disease (CD) characterized by myofibroblasts and extracellular matrix accumulation within the submucosa and smooth muscles, leading to bowel strictures. No medical treatment exists to treat or reverse intestinal fibrosis leading often to surgical resection. The potential role of intestinal epithelium in the fibrotic process remains poorly defined. Methods: We performed a pilot study on ileal fibrostricturing CD surgical samples (n=5), comparing the proteome of surface epithelium isolated by laser capture microdissection in normal and fibrotic zones. Confirmation of the specific protein increases was obtained by immunohistochemistry in colonic and ileal samples of CD (n=44) compared to healthy subjects (n=40), as well as in intestinal epithelial cell line under induced Endoplasmic Reticulum (ER) stress. A model of fibroblast to myofibroblast differentiation induction was also challenged using preconditioned media of intestinal epithelial cells after a pulsed ER stress. Results: Label free proteomics revealed high ER stress in the epithelium surrounding fibrotic bowel wall, involving Anterior gradient protein 2 homolog (AGR2) and 78kDA glucose regulated protein (BiP). Confirmation of both proteins increase was obtained by immunohistochemistry. ER stress induction in intestinal epithelial cells was associated with an intracellular increase of AGR2, BiP and ER stress markers as sXPB1 and CHOP. AGR2 was also detected in the culture medium of these epithelial cells and myofibroblast differentiation was obtained using this culture medium. Conclusions: The increase of ER stress proteins observed in fibrostenosing tissues together with These preliminary evidences of fibroblast to myofibrobast differentiation obtained by paracrine action of intestinal epithelial cell preconditioned to ER stress induction, suggest a role of epithelial ER stress in Crohn’s disease intestinal fibrosis. [less ▲]

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See detailDiscovery of biomarker candidates associated with the risk of short-term and mid/long-term relapse after infliximab withdrawal in Crohn's patients: a proteomics-based study.
Pierre, Nicolas ULiege; Baiwir, Dominique ULiege; Huynh-Thu, Vân Anh ULiege et al

in Gut (2020)

OBJECTIVE: A subset of Crohn's disease (CD) patients experiences mid/long-term remission after infliximab withdrawal. Biomarkers are needed to identify those patients. DESIGN: New biomarkers of relapse ... [more ▼]

OBJECTIVE: A subset of Crohn's disease (CD) patients experiences mid/long-term remission after infliximab withdrawal. Biomarkers are needed to identify those patients. DESIGN: New biomarkers of relapse were searched in the baseline serum of CD patients stopping infliximab when they were under combined therapy (antimetabolite and infliximab) and stable clinical remission (diSconTinuation in CrOhn's disease patients in stable Remission on combined therapy with Immunosuppressors cohort, n=102). From shotgun proteomics experiment (discovery step), biomarker candidates were identified and further targeted by selected reaction monitoring (verification step). The dataset was stratified to search for markers of short-term (<6 months) or mid/long-term relapse (>6 months). The risk of relapse and the predicting capacity associated with biomarker candidates were evaluated using univariate Cox model and log-rank statistic, respectively. To test their complementary predicting capacity, biomarker candidates were systematically combined in pairs. RESULTS: Distinct biomarker candidates were associated with the risk (HR) of short-term (15 proteins, 2.9<HR<16.1, p<0.05) and mid/long-term (17 proteins, 2.1<HR<4.7, p<0.05) relapse, they reflect different pathophysiological processes. In stratified and non-stratified datasets, novel marker combinations exhibited a high predicting capacity as shown by their higher Z-scores (false discovery rate <0.001) than C reactive protein and faecal calprotectin (current references in predicting relapse). CONCLUSION: We identified for the first time circulating biomarker candidates associated with the risk of mid/long-term relapse in CD patients stopping infliximab. We also highlight a sequence of pathophysiological processes leading to relapse, this could help to better understand the disease progression. Our findings may pave the way for a better non-invasive evaluation of the risk of relapse when contemplating antitumour necrosis factor α withdrawal in CD patients. [less ▲]

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See detailProteomics highlights common and distinct pathophysiological processes associated with ileal and colonic ulcers in Crohn's disease.
Pierre, Nicolas ULiege; Salee, Catherine ULiege; MASSOT, Charlotte ULiege et al

in Journal of Crohn's & colitis (2019)

BACKGROUND AND AIMS: Based on genetics and natural history, Crohn's disease can be separated in two entities, an ileal and a colonic disease. Protein based-approaches are needed to elucidate whether such ... [more ▼]

BACKGROUND AND AIMS: Based on genetics and natural history, Crohn's disease can be separated in two entities, an ileal and a colonic disease. Protein based-approaches are needed to elucidate whether such subphenotypes are related to distinct pathophysiological processes. METHODS: The proteome of ulcer edge was compared to the one of paired control tissue (n=32 biopsies) by differential proteomics in the ileum and the colon of Crohn's disease patients (n=16). The results were analysed though a hypothesis-driven (based on literature) and a hypothesis-free approach (pathway enrichment analysis). To confirm one of the key pathway highlighted by proteomics, two proteins were also studied by immunochemistry in tissue biopsies. RESULTS: In the ileum and the colon, 4428 and 5204 proteins, respectively, were identified and quantified. Ileal and colonic ulcer edge differed by a distinct distribution of proteins of epithelial-mesenchymal transition, neutrophil degranulation and ribosome. Ileal and colonic ulcer edge were similarly characterised by an increase of the proteins implicated in the pathway of protein processing in endoplasmic reticulum and a decrease of mitochondrial proteins. Immunochemistry confirmed the presence of endoplasmic reticulum stress in the mucosa of ileal and colonic ulcer edge. CONCLUSION: This study provides protein-based evidences showing partly distinct pathophysiological processes associated to ileal and colonic ulcer edge in Crohn's disease patients. This could constitute a first step toward the development of gut segment-specific diagnostic markers and therapeutics. [less ▲]

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See detailSkeletal muscle ceramides do not contribute to physical inactivity-induced insulin resistance.
Appriou, Zephyra; Nay, Kevin; Pierre, Nicolas ULiege et al

in Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme (2019)

Physical inactivity increases the risk to develop type 2 diabetes, a disease characterized by a state of insulin resistance. By promoting inflammatory state, ceramides are especially recognized to alter ... [more ▼]

Physical inactivity increases the risk to develop type 2 diabetes, a disease characterized by a state of insulin resistance. By promoting inflammatory state, ceramides are especially recognized to alter insulin sensitivity in skeletal muscle. The present study was designed to analyze, in mice, whether muscle ceramides contribute to physical inactivity-induced insulin resistance. For this purpose, we used the wheel lock model to induce a sudden reduction of physical activity, in combination with myriocin treatment, an inhibitor of de novo ceramide synthesis. Mice were assigned to 3 experimental groups: voluntary wheel access group (Active), a wheel lock group (Inactive) and wheel lock group treated with myriocin (Inactive-Myr). We observed that 10 days of physical inactivity induces hyperinsulinemia and increases HOMA-IR. The muscle ceramide content was not modified by physical inactivity and myriocin. Thus, muscle ceramides do not play a role in physical inactivity-induced insulin resistance. In skeletal muscle, insulin-stimulated Akt phosphorylation and inflammatory pathway were not affected by physical inactivity whereas a reduction of GLUT4 content was observed. Based on these results, physical inactivity-induced insulin resistance seems related to a reduction in GLUT4 content rather than defects in insulin signaling. We observed in inactive mice that myriocin treatment improves glucose tolerance, insulin-stimulated Akt, AMPK activation and GLUT4 content in skeletal muscle. Such effects occur regardless of changes in muscle ceramide content. These findings open promising research perspectives to identify new mechanisms of action for myriocin on insulin sensitivity and glucose metabolism. [less ▲]

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See detailUrolithin B, a newly identified regulator of skeletal muscle mass.
Rodriguez, Julie; Pierre, Nicolas ULiege; Naslain, Damien et al

in Journal of Cachexia, Sarcopenia and Muscle (2017), 8(4), 583-597

BACKGROUND: 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 ... [more ▼]

BACKGROUND: 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. METHODS: C2C12 myotubes were treated with 15 muM of urolithin B for 24 h. For in vivo experiments, mice were implanted with mini-osmotic pumps delivering continuously 10 mug/day of urolithin B during 28 days. Muscle atrophy was studied in mice with a sciatic nerve denervation receiving urolithin B by the same way. RESULTS: Our experiments reveal that urolithin B enhances the growth and differentiation of C2C12 myotubes by increasing protein synthesis and repressing the ubiquitin-proteasome pathway. Genetic and pharmacological arguments support an implication of the androgen receptor. Signalling analyses suggest a crosstalk between the androgen receptor and the mTORC1 pathway, possibly via AMPK. In vivo experiments confirm that urolithin B induces muscle hypertrophy in mice and reduces muscle atrophy after the sciatic nerve section. CONCLUSIONS: This study highlights the potential usefulness of urolithin B for the treatment of muscle mass loss associated with various (pathological) physiological states. [less ▲]

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See detailSimulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation.
Cavey, Thibault; Pierre, Nicolas ULiege; Nay, Kevin et al

in Experimental Physiology (2017), 102(3), 291-298

NEW FINDINGS: What is the central question of this study? Although microgravity is well known to reduce circulating iron in astronauts, the underlying mechanism is still unknown. We investigated whether ... [more ▼]

NEW FINDINGS: What is the central question of this study? Although microgravity is well known to reduce circulating iron in astronauts, the underlying mechanism is still unknown. We investigated whether hepcidin, a key hormone regulating iron metabolism, could be involved in this deleterious effect. What is the main finding and its importance? We show that hindlimb suspension, a model of microgravity, stimulates the production of hepcidin in liver of rats. In agreement with the biological role of hepcidin, we found a decrease of circulating iron and an increase of spleen iron content in hindlimb-unloaded rats. Consequently, our study supports the idea that hepcidin could play a role in the alteration of iron metabolism parameters observed during spaceflight. During spaceflight, humans exposed to microgravity exhibit an increase of iron storage and a reduction of circulating iron. Such perturbations could promote oxidative stress and anaemia in astronauts. The mechanism by which microgravity modulates iron metabolism is still unknown. Herein, we hypothesized that microgravity upregulates hepcidin, a hormone produced by the liver that is the main controller of iron homeostasis. To test this hypothesis, rats were submitted to hindlimb unloading (HU), the reference model to mimic the effects of microgravity in rodents. After 7 days, the mRNA level of hepcidin was increased in the liver of HU rats (+74%, P = 0.001). In agreement with the biological role of hepcidin, we found an increase of spleen iron content (+78%, P = 0.030) and a decrease of serum iron concentration (-35%, P = 0.002) and transferrin saturation (-25%, P = 0.011) in HU rats. These findings support a role of hepcidin in microgravity-induced iron metabolism alteration. Furthermore, among the signalling pathways inducing hepcidin mRNA expression, we found that only the interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) axis was activated by HU, as shown by the increase of phospho-STAT3 (+193%, P < 0.001) and of the hepatic mRNA level of haptoglobin (+167%, P < 0.001), a STAT3-inducible gene, in HU rats. Taken together, these data support the idea that microgravity may alter iron metabolism through an inflammatory process upregulating hepcidin. [less ▲]

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See detailFrom physical inactivity to immobilization: Dissecting the role of oxidative stress in skeletal muscle insulin resistance and atrophy.
Pierre, Nicolas ULiege; Appriou, Zephyra; Gratas-Delamarche, Arlette et al

in Free Radical Biology and Medicine (2016), 98

In the literature, the terms physical inactivity and immobilization are largely used as synonyms. The present review emphasizes the need to establish a clear distinction between these two situations ... [more ▼]

In the literature, the terms physical inactivity and immobilization are largely used as synonyms. The present review emphasizes the need to establish a clear distinction between these two situations. Physical inactivity is a behavior characterized by a lack of physical activity, whereas immobilization is a deprivation of movement for medical purpose. In agreement with these definitions, appropriate models exist to study either physical inactivity or immobilization, leading thereby to distinct conclusions. In this review, we examine the involvement of oxidative stress in skeletal muscle insulin resistance and atrophy induced by, respectively, physical inactivity and immobilization. A large body of evidence demonstrates that immobilization-induced atrophy depends on the chronic overproduction of reactive oxygen and nitrogen species (RONS). On the other hand, the involvement of RONS in physical inactivity-induced insulin resistance has not been investigated. This observation outlines the need to elucidate the mechanism by which physical inactivity promotes insulin resistance. [less ▲]

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See detailEndurance Training Attenuates Catabolic Signals Induced by TNF-alpha in Muscle of Mice.
Rodriguez, Julie; Fernandez-Verdejo, Rodrigo; Pierre, Nicolas ULiege et al

in Medicine and Science in Sports and Exercise (2016), 48(2), 227-34

PURPOSE: To determine whether endurance training attenuates the negative effects induced by an acute injection of tumor necrosis factor-alpha (TNF-alpha) in skeletal muscle of mice. METHODS: Trained (6 wk ... [more ▼]

PURPOSE: To determine whether endurance training attenuates the negative effects induced by an acute injection of tumor necrosis factor-alpha (TNF-alpha) in skeletal muscle of mice. METHODS: Trained (6 wk of endurance training at 60% of maximal velocity) and untrained mice were injected with TNF-alpha 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-alpha increased the mRNA of cytokines and downregulated signals linked to protein synthesis. The phosphorylation of IKKalpha/beta and IkappaBalpha induced by TNF-alpha was blunted in trained mice, suggesting altered NF-kappaB 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-alpha. [less ▲]

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See detailIRE1alpha and TRB3 do not contribute to the disruption of proximal insulin signaling caused by palmitate in C2C12 myotubes.
Pierre, Nicolas ULiege; Fernandez-Verdejo, Rodrigo; Regnier, Pauline et al

in Cell Biology International (2016), 40(1), 91-9

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 ... [more ▼]

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 1alpha (IRE1alpha) activation and tribbles homolog 3 (TRB3) expressions. IRE1alpha 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 IRE1alpha 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 IRE1alpha. Palmitate increased TRB3 expression, activated IRE1alpha signaling, and reduced the insulin-dependent Akt phosphorylation. Knocking down TRB3 or IRE1alpha 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. [less ▲]

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See detailAging related ER stress is not responsible for anabolic resistance in mouse skeletal muscle.
Chalil, Sreeda; Pierre, Nicolas ULiege; Bakker, Astrid D. et al

in Biochemical and Biophysical Research Communications (2015), 468(4), 702-7

Anabolic resistance reflects the inability of skeletal muscle to maintain protein mass by appropriate stimulation of protein synthesis. We hypothesized that endoplasmic reticulum (ER) stress contributes ... [more ▼]

Anabolic resistance reflects the inability of skeletal muscle to maintain protein mass by appropriate stimulation of protein synthesis. We hypothesized that endoplasmic reticulum (ER) stress contributes to anabolic resistance in skeletal muscle with aging. Muscles were isolated from adult (8 mo) and old (26 mo) mice and weighed. ER stress markers in each muscle were quantified, and the anabolic response to leucine was assessed by measuring the phosphorylation state of S6K1 in soleus and EDL using an ex vivo muscle model. Aging reduced the muscle-to-body weight ratio in soleus, gastrocnemius, and plantaris, but not in EDL and tibialis anterior. Compared to adult mice, the expression of ER stress markers BiP and IRE1alpha was higher in EDL, and phospho-eIF2alpha was higher in soleus and EDL of old mice. S6K1 response to leucine was impaired in soleus, but not in EDL, suggesting that anabolic resistance contributes to soleus weight loss in old mice. Pre-incubation with ER stress inducer tunicamycin before leucine stimulation increased S6K1 phosphorylation beyond the level reached by leucine alone. Since tunicamycin did not impair leucine-induced S6K1 response, and based on the different ER stress marker regulation patterns, ER stress is probably not involved in anabolic resistance in skeletal muscle with aging. [less ▲]

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See detailPomegranate and green tea extracts protect against ER stress induced by a high-fat diet in skeletal muscle of mice.
Rodriguez, Julie; Gilson, Helene; Jamart, Cecile et al

in European Journal of Nutrition (2015), 54(3), 377-89

PURPOSE: We tested the hypothesis that polyphenol-rich extracts can reduce endoplasmic reticulum (ER) stress induced by a high-fat diet (HFD) in skeletal muscle of mice. METHODS: Mice were randomly ... [more ▼]

PURPOSE: We tested the hypothesis that polyphenol-rich extracts can reduce endoplasmic reticulum (ER) stress induced by a high-fat diet (HFD) in skeletal muscle of mice. METHODS: Mice were randomly assigned to four groups receiving during 20 weeks either a standard chow control (CTRL), or a HFD supplemented, or not, with pomegranate (HFD + P) or green tea (HFD + GT) extracts. After the nutritional intervention, mice were killed and gastrocnemius muscles were taken. Proteins and mRNA were measured by Western blot and RT-qPCR, respectively. RESULTS: Body weight gain and visceral fat were higher in HFD, HFD + P and HFD + GT than in CTRL. The markers of the unfolded protein response BiP, XBP1u, XBP1s and ATF4 were higher only in HFD. In HFD + P and HFD + GT, this increase was not observed except for CHOP, which was elevated in all HFD groups. HFD increased also markers of ubiquitin-proteasome pathway, autophagy and oxidative stress, which were kept low in HFD + P and HFD + GT groups. CONCLUSION: Our data provide evidence for a protective effect of pomegranate and green tea extracts against ER stress, oxidative stress and protein degradation induced by HFD in skeletal muscle. They give arguments for a usefulness of these natural nutritional compounds to fight against cellular dysfunctions related to fat excess. [less ▲]

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See detailActivation of ER stress by hydrogen peroxide in C2C12 myotubes.
Pierre, Nicolas ULiege; Barbe, Caroline; Gilson, Helene et al

in Biochemical and Biophysical Research Communications (2014), 450(1), 459-63

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 muM) and ... [more ▼]

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 muM) 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 2alpha (eIF2alpha, 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 1alpha (IRE1alpha). 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. [less ▲]

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See detailToll-like receptor 4 knockout mice are protected against endoplasmic reticulum stress induced by a high-fat diet.
Pierre, Nicolas ULiege; Deldicque, Louise; Barbe, Caroline et al

in PLoS ONE (2013), 8(5), 65061

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 ... [more ▼]

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-eIF2alpha. TLR4(-/-) mice were protected against HFD-induced ER stress. Then, we investigated the main signaling downstream of TLR4 namely the NF-kappaB pathway, expecting to identify the mechanism by which TLR4 is able to activate ER stress. The mRNA levels of cytokines regulated by NF-kappaB namely TNFalpha, IL-1beta and IL-6, were not changed after HFD and phospho-IkappaB-alpha (ser 32) was not changed. Our results indicate that TLR4 is essential for the development of ER stress related to HFD. Nevertheless, the NFkappa-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. [less ▲]

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See detailTLR2 and TLR4 activation induces p38 MAPK-dependent phosphorylation of S6 kinase 1 in C2C12 myotubes.
Zbinden-Foncea, Hermann; Deldicque, Louise; Pierre, Nicolas ULiege et al

in Cell Biology International (2012), 36(12), 1107-13

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 ... [more ▼]

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. [less ▲]

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