Temporal regulation of transgene expression controlled by amino acid availability in human T cells.

T cell therapy strategies, from allogeneic stem cell transplantation toward genetically-modified T cells infusion, develop powerful anti-tumor effects but are often accompanied by side effects and their efficacy remains sometimes to be improved. It therefore appears important to provide a flexible and easily reversible gene expression regulation system to control T cells activity. We developed a gene expression regulation technology that exploits the physiological GCN2-ATF4 pathway's ability to induce gene expression in T cells in response to one essential amino acid deficiency. We first demonstrated the functionality of NUTRIREG in human T cells by transient expression of reporter genes. We then validated that NUTRIREG can be used in human T cells to transiently express a therapeutic gene such as IL-10. Overall, our results represent a solid basis for the promising use of NUTRIREG to regulate transgene expression in human T cells in a reversible way, and more generally for numerous preventive or curative therapeutic possibilities in cellular immunotherapy strategies.

Bovine extracellular matrix proteins and potential role in meat quality: First in silico Bos taurus compendium.

The extracellular matrix (ECM) is a three-dimensional set of proteins that binds tissues and defines their biomechanical properties. Among the ECM components known to be involved in beef sensory qualities, authors have often studied fibrillar collagens but also, although less often, proteoglycans and some glycoproteins. The ECM contains many other proteins. Our hypothesis is that to deepen the role of ECM proteins in beef qualities and to identify new ones among the vast amount of data generated by high throughput methods, it is necessary to have a list of proteins of this matrix to refer to for the bovine species. We have therefore defined the Bos taurus matrisome known as the set of genes encoding ECM (core matrisome proteins and matrisome-associated proteins). We have used orthology, as a reference method, and a bioinformatic approach based on a computational pipeline previously published for Homo sapiens, Mus musculus and Danio rerio for the definition of their respective matrisome. We have reported here that the Bos taurus matrisome is composed of 1022 genes that we have classified according to the different matrisome categories. This list is the only matrisome of a livestock species to be defined to date. SIGNIFICANCE: In this study, we provide the first definition of matrisome of a livestock species, the Bos taurus. We believe that the Bos taurus matrisome will be of great interest for several reasons. It comes as a complement to the matrisomes of several other species such as Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster and Caenorabditis elegans previously defined by other authors. It could be used to identify matrisome molecules among the vast amount of data generated by the high throughput methods. It thus can be used in addition to the other matrisomes as a model by the scientific community to study cell behavior and mechanotransduction and could lead to the identification of novel biomarkers for several diseases and cancers in which ECM is involved. Moreover, in the domain of studies on livestock, the dataset that we have provided here can be used in the context of product quality studies, especially meat quality, but also, for example, for lactation studies.

Activation of the eIF2α-ATF4 Pathway by Chronic Paracetamol Treatment Is Prevented by Dietary Supplementation with Cysteine.

Chronic treatment with acetaminophen (APAP) induces cysteine (Cys) and glutathione (GSH) deficiency which leads to adverse metabolic effects including muscle atrophy. Mammalian cells respond to essential amino acid deprivation through the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). Phosphorylated eIF2α leads to the recruitment of activating transcription factor 4 (ATF4) to specific CCAAT/enhancer-binding protein-ATF response element (CARE) located in the promoters of target genes. Our purpose was to study the activation of the eIF2α-ATF4 pathway in response to APAP-induced Cys deficiency, as well as the potential contribution of the eIF2α kinase GCN2 and the effect of dietary supplementation with Cys. Our results showed that chronic treatment with APAP activated both GCN2 and PERK eIF2α kinases and downstream target genes in the liver. Activation of the eIF2α-ATF4 pathway in skeletal muscle was accompanied by muscle atrophy even in the absence of GCN2. The dietary supplementation with cysteine reversed APAP-induced decreases in plasma-free Cys, liver GSH, muscle mass, and muscle GSH. Our new findings demonstrate that dietary Cys supplementation also reversed the APAP-induced activation of GCN2 and PERK and downstream ATF4-target genes in the liver.

Dataset on transcriptome signature of skeletal muscle of young, adult and aged mice.

This data article reports the level of expression of messenger RNA (mRNA) obtained from a set of 18 skeletal muscle samples using Affymetrix Genechips Exon arrays. Data were obtained from Gastrocnemius muscle of C57BL6 male mice at 3 distinct age groups, 2, 11 and 25 months old representing young, mature adult and aged groups. The data submitted to GEO constitute a large dataset of 15,300 mRNA levels. The data include eighteen .CEL files obtained after scanning mouse exon arrays and one .xls file obtained after processing with Genespring GX 14.9. Three distinct files containing affymetrix data processed using Genespring and analyzed for differences between stages 2 per 2 are provided as supplementary data.

When idiopathic male infertility is rooted in maternal malnutrition during the perinatal period in mice†.

Infertility represents a growing burden worldwide, with one in seven couples presenting difficulties conceiving. Among these, 10-15% of the men have idiopathic infertility that does not correlate with any defect in the classical sperm parameters measured. In the present study, we used a mouse model to investigate the effects of maternal undernutrition on fertility in male progeny. Our results indicate that mothers fed on a low-protein diet during gestation and lactation produce male offspring with normal sperm morphology, concentration, and motility but exhibiting an overall decrease of fertility when they reach adulthood. Particularly, in contrast to control, sperm from these offspring show a remarkable lower capacity to fertilize oocytes when copulation occurs early in the estrus cycle relative to ovulation, due to an altered sperm capacitation. Our data demonstrate for the first time that maternal nutritional stress can have long-term consequences on the reproductive health of male progeny by affecting sperm physiology, especially capacitation, with no observable impact on spermatogenesis and classical quantitative and qualitative sperm parameters. Moreover, our experimental model could be of major interest to study, explain, and ultimately treat certain categories of infertilities.

Induction of ATF4-Regulated Atrogenes Is Uncoupled from Muscle Atrophy during Disuse in Halofuginone-Treated Mice and in Hibernating Brown Bears.

Activating transcription factor 4 (ATF4) is involved in muscle atrophy through the overexpression of some atrogenes. However, it also controls the transcription of genes involved in muscle homeostasis maintenance. Here, we explored the effect of ATF4 activation by the pharmacological molecule halofuginone during hindlimb suspension (HS)-induced muscle atrophy. Firstly, we reported that periodic activation of ATF4-regulated atrogenes (Gadd45a, Cdkn1a, and Eif4ebp1) by halofuginone was not associated with muscle atrophy in healthy mice. Secondly, halofuginone-treated mice even showed reduced atrophy during HS, although the induction of the ATF4 pathway was identical to that in untreated HS mice. We further showed that halofuginone inhibited transforming growth factor-ß (TGF-ß) signalling, while promoting bone morphogenetic protein (BMP) signalling in healthy mice and slightly preserved protein synthesis during HS. Finally, ATF4-regulated atrogenes were also induced in the atrophy-resistant muscles of hibernating brown bears, in which we previously also reported concurrent TGF-ß inhibition and BMP activation. Overall, we show that ATF4-induced atrogenes can be uncoupled from muscle atrophy. In addition, our data also indicate that halofuginone can control the TGF-ß/BMP balance towards muscle mass maintenance. Whether halofuginone-induced BMP signalling can counteract the effect of ATF4-induced atrogenes needs to be further investigated and may open a new avenue to fight muscle atrophy. Finally, our study opens the way for further studies to identify well-tolerated chemical compounds in humans that are able to fine-tune the TGF-ß/BMP balance and could be used to preserve muscle mass during catabolic situations.

Transgenerational supplementation with eicosapentaenoic acid reduced the metabolic consequences on the whole body and skeletal muscle in mice receiving an obesogenic diet.

PURPOSE: The effect of manipulating the fatty acid profile of the diet over generations could affect the susceptibility to develop obesity and metabolic disorders. Although some acute effects were described, the impact of transgenerational continuous supplementation with omega 3 fatty acids on metabolic homeostasis and skeletal muscle metabolic flexibility during a nutritional stress is unknown. METHODS: We analyzed the effect of an obesogenic diet in mice after transgenerational supplementation with an omega-3 rich oil (mainly EPA) or a control oil. Young F3 animals received a high fat and high sucrose diet for 4 months. Whole-body biometric data were recorded and lipidomic/transcriptomic adaptations were explored in the skeletal muscle. RESULTS: F3 mice from the lineage supplemented with EPA gained less weight, fat mass, and exhibited better metabolic parameters after the obesogenic diet compared to mice from the control lineage. Transcriptomic exploration of skeletal muscle showed differential regulation of biological processes such as fibrosis, fatty acid catabolism, and inflammation between lineages. These adaptations were associated to subtle lipid remodeling of cellular membranes with an enrichment in phospholipids with omega 3 fatty acid in mice from the EPA lineage. CONCLUSION: Transgenerational and continuous intake of EPA could help to reduce cardiovascular and metabolic risks related to an unbalanced diet by the modulation of insulin sensitivity, fatty acid metabolism, and fibrosis in skeletal muscle.

"Do my qPCR calculation", a web tool.

In order to automatically process qPCR raw data, we present the tool "Do my qPCR calculation". We offer a website to automatically calculate the data normalization and represent the different samples graphically in an Excel file. This tool is also available on Github for installation and local use with or without web interface.

Protein restricted diet during gestation and/or lactation in mice affects 15N natural isotopic abundance of organs in the offspring: Effect of diet 15N content and growth.

OBJECTIVES AND STUDY: This study aimed at measuring the effect in normal to restricted protein diets with specific 15N natural isotopic abundance (NIA) given during gestation and/or lactation on the 15N NIA of fur, liver and muscle in dams and their offspring from birth to adulthood. The secondary aim was to study the effect of growth on the same parameters. METHODS: Female Balb/c mice were fed normal protein diet containing 22% protein or isocaloric low protein diet containing 10% protein throughout gestation. Dam's diets were either maintained or switched to the other diet until weaning at 30 days. All animals were fed standard chow thereafter. Offspring were sacrificed at 1, 11, 30, 60, 480 days and a group of dams at d1. Growth was modeled as an exponential function on the group followed up until 480 days. Fur, liver and muscle were sampled at sacrifice and analyzed for bulk 15N NIA. Fixed effects and interactions between fixed effects and random elements were tested by three-way ANOVA. RESULTS: Higher 15N NIA in the diet resulted in higher organ 15N NIA. Switching from one diet to another changed 15N NIA in each organ. Although dam and offspring shared the same isotopic environment during gestation, 15N NIA at day 1 was higher in dams. Growth rate did not differ between groups after 10 days and decreased between 1 and 5 months. 15N NIA differed between organs and was affected by growth and gestation/lactation. CONCLUSION: Dietary 15N NIA is a major determinant of the 15N NIA of organs. 15N NIA depended on organ and age (i.e. growth) suggesting an effect of metabolism and/or dilution space. Post-natal normal-protein diet of lactating dams could reverse the effect of a protein-restricted diet during gestation on the offspring growth. Measuring 15N NIA in various matrices may open a field of application particularly useful in studying the pre- and post-natal origins of health and disease.

Decreased ATF4 expression as a mechanism of acquired resistance to long-term amino acid limitation in cancer cells.

The uncontrolled growth of tumor can lead to the formation of area deprived in nutrients. Due to their high genetic instability, tumor cells can adapt and develop resistance to this pro-apoptotic environment. Among the resistance mechanisms, those involved in the resistance to long-term amino acid restriction are not elucidated. A long-term amino acid restriction is particularly deleterious since nine of them cannot be synthetized by the cells. In order to determine how cancer cells face a long-term amino acid deprivation, we developed a cell model selected for its capacity to resist a long-term amino acid limitation. We exerted a selection pressure on mouse embryonic fibroblast to isolate clones able to survive with low amino acid concentration. The study of several clones revealed an alteration of the eiF2α/ATF4 pathway. Compared to the parental cells, the clones exhibited a decreased expression of the transcription factor ATF4 and its target genes. Likewise, the knock-down of ATF4 in parental cells renders them resistant to amino acid deprivation. Moreover, this association between a low level of ATF4 protein and the resistance to amino acid deprivation was also observed in the cancer cell line BxPC-3. This resistance was abolished when ATF4 was overexpressed. Therefore, decreasing ATF4 expression may be one important mechanism for cancer cells to survive under prolonged amino acid deprivation.

Age-Dependent Control of Energy Homeostasis by Brown Adipose Tissue in Progeny Subjected to Maternal Diet-Induced Fetal Programming.

Epidemiological and animal studies show that deleterious maternal environments predispose aging offspring to metabolic disorders and type 2 diabetes. Young progenies in a rat model of maternal low-protein (LP) diet are normoglycemic despite collapsed insulin secretion. However, without further worsening of the insulin secretion defect, glucose homeostasis deteriorates in aging LP descendants. Here we report that normoglycemic and insulinopenic 3-month-old LP progeny shows increased body temperature and energy dissipation in association with enhanced brown adipose tissue (BAT) activity. In addition, it is protected against a cold challenge and high-fat diet (HFD)-induced obesity with associated insulin resistance and hyperglycemia. Surgical BAT ablation in 3-month-old LP offspring normalizes body temperature and causes postprandial hyperglycemia. At 10 months, BAT activity declines in LP progeny with the appearance of reduced protection to HFD-induced obesity; at 18 months, LP progeny displays a BAT activity comparable to control offspring and insulin resistance and hyperglycemia occur. Together our findings identify BAT as a decisive physiological determinant of the onset of metabolic dysregulation in offspring predisposed to altered ß-cell function and hyperglycemia and place it as a critical regulator of fetal programming of adult metabolic disease.

GCN2 contributes to mTORC1 inhibition by leucine deprivation through an ATF4 independent mechanism.

It is well known that the GCN2 and mTORC1 signaling pathways are regulated by amino acids and share common functions, in particular the control of translation. The regulation of GCN2 activity by amino acid availability relies on the capacity of GCN2 to sense the increased levels of uncharged tRNAs upon amino acid scarcity. In contrast, despite recent progress in the understanding of the regulation of mTORC1 by amino acids, key aspects of this process remain unsolved. In particular, while leucine is well known to be a potent regulator of mTORC1, the mechanisms by which this amino acid is sensed and control mTORC1 activity are not well defined. Our data establish that GCN2 is involved in the inhibition of mTORC1 upon leucine or arginine deprivation. However, the activation of GCN2 alone is not sufficient to inhibit mTORC1 activity, indicating that leucine and arginine exert regulation via additional mechanisms. While the mechanism by which GCN2 contributes to the initial step of mTORC1 inhibition involves the phosphorylation of eIF2α, we show that it is independent of the downstream transcription factor ATF4. These data point to a novel role for GCN2 and phosphorylation of eIF2α in the control of mTORC1 by certain amino acids.

Regulating the expression of therapeutic transgenes by controlled intake of dietary essential amino acids.

Widespread application of gene therapy will depend on the development of simple methods to regulate the expression of therapeutic genes. Here we harness an endogenous signaling pathway to regulate therapeutic gene expression through diet. The GCN2-eIF2α signaling pathway is specifically activated by deficiencies in any essential amino acid (EAA); EAA deficiency leads to rapid expression of genes regulated by ATF4-binding cis elements. We found that therapeutic genes under the control of optimized amino acid response elements (AAREs) had low basal expression and high induced expression. We applied our system to regulate the expression of TNFSF10 (TRAIL) in the context of glioma therapy and found that intermittent activation of this gene by EEA-deficient meals retained its therapeutic efficacy while abrogating its toxic effects on normal tissue. The GCN2-eIF2α pathway is expressed in many tissues, including the brain, and is highly specific to EAA deficiency. Our system may be particularly well suited for intermittent regulation of therapeutic transgenes over short or long time periods.

Method for collecting mouse milk without exogenous oxytocin stimulation.

It has been reported that breast-feeding more than 6 months strongly decreases the risk of allergy, diabetes, obesity, and hypertension in humans. In order to understand the mechanisms responsible for this benefit, it is important to evaluate precisely the composition of maternal milk, especially in response to environmental cues. Mouse models offer a unique opportunity to study the impact of maternal milk composition on the development and health of offspring. Oxytocin injection of the dam is usually used to stimulate milk ejection; however, exogenous oxytocin might have deleterious effects under some experimental conditions by modifying milk content as well as the physiology and behavior of the dam. Taking advantage of the natural stimulation of the mammary gland that occurs after the reunion of a dam that has been separated from her pups, we developed a new procedure to collect mouse milk without the injection of oxytocin. This method is easy to use, low-cost ,and non-invasive. Moreover, it provides a sufficient amount of milk for use in a wide range of biological analyses.

In vivo imaging of the spatiotemporal activity of the eIF2α-ATF4 signaling pathway: Insights into stress and related disorders.

The eIF2α-ATF4 pathway is involved in cellular adaptation to stress and is dysregulated in numerous diseases. Activation of this pathway leads to phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and the recruitment of the transcription factor ATF4 (activating transcription factor 4) to specific CCAAT/enhancer binding protein (C/EBP)-ATF response elements (CAREs) located in the promoters of target genes. To monitor the spatiotemporal modulation of this pathway in living animals, we generated a novel CARE-driven luciferase mouse model (CARE-LUC). These transgenic mice enable the investigation of the eIF2α-ATF4 pathway activity in the whole organism and at the tissue and cellular levels by combining imaging, luciferase assays, and immunochemistry. Using this mouse line, we showed the tissue-specific activation pattern of this pathway in response to amino acid deficiency or endoplasmic reticulum stress and the hepatic induction of this pathway in a stress-related pathology model of liver fibrosis. The CARE-LUC mouse model represents an innovative tool to investigate the eIF2α-ATF4 axis and to develop drugs targeting this important pathway in the remediation of related pathologies.

[Adaptation to the availability of essential amino-acids: role of GCN2/eIF2α/ATF4 pathway]. / Mécanismes d'adaptation à la disponibilité en acides aminés indispensables: rôles de la voie GCN2/eIF2α/ATF4.

In mammals, metabolic adaptations are required to overcome nutritional deprivation in amino-acids/proteins as well as episodes of malnutrition. GCN2 protein kinase, which phosphorylates the α subunit of the translation initiation factor eIF2, is a sensor of amino-acid(s) deficiencies. On one hand, this review briefly describes the main features of amino-acid metabolism. On the other hand, it describes the role of GCN2 in regulating numerous physiological functions.

Methyl donor deficiency in H9c2 cardiomyoblasts induces ER stress as an important part of the proteome response.

Deficiency of methyl donors (MDs, folate, vitamin B12, and choline) causes increased plasma level of Hcy, a risk factor for cardiovascular diseases. Previously, we showed that maternal MD deprivation altered the cardiac proteome of rat pups. To better understand its impact on cardiac cells, we exposed rat H9c2 cardiomyoblasts to selectively a synthetic folate- or MD-deficient (FD or MDD) medium. We found that a 4-day exposure to the FD medium, unlike the MDD one, did not cause an abnormal extracellular release of Hcy relatively to similar exposure to the control complete (C) medium. Comparative analyses of the proteomes of FD, MDD, and C cells identified 7 and 6 proteins up- or downregulated by either deficiency, respectively. Most proteins were found interrelated in a single network dealing with "post-translational modification, protein folding and cell death/survival" (FD cells) or "DNA replication/recombination/repair and cell morphology/compromise" (MDD cells). Both deficiencies altered the protein and mRNA levels of the chaperones α-crystallin B, protein disulfide-isomerase A4, and prohibitin. This was concurrent with rapid induction of several key genes of the ER stress response, notably gadd153/chop, and increased expression of the E3 ubiquitin ligases, Hrd1, and MAFbx. In conclusion, the effects of folate and MD deficiencies on the cardiomyoblast proteome display some dissimilarities possibly related to different cellular production of Hcy. In both cases activation of the ER stress could occur in response to accumulation of ubiquitinated misfolded proteins.

Perinatal protein malnutrition affects mitochondrial function in adult and results in a resistance to high fat diet-induced obesity.

Epidemiological findings indicate that transient environmental influences during perinatal life, especially nutrition, may have deleterious heritable health effects lasting for the entire life. Indeed, the fetal organism develops specific adaptations that permanently change its physiology/metabolism and that persist even in the absence of the stimulus that initiated them. This process is termed "nutritional programming". We previously demonstrated that mothers fed a Low-Protein-Diet (LPD) during gestation and lactation give birth to F1-LPD animals presenting metabolic consequences that are different from those observed when the nutritional stress is applied during gestation only. Compared to control mice, adult F1-LPD animals have a lower body weight and exhibit a higher food intake suggesting that maternal protein under-nutrition during gestation and lactation affects the energy metabolism of F1-LPD offspring. In this study, we investigated the origin of this apparent energy wasting process in F1-LPD and demonstrated that minimal energy expenditure is increased, due to both an increased mitochondrial function in skeletal muscle and an increased mitochondrial density in White Adipose Tissue. Importantly, F1-LPD mice are protected against high-fat-diet-induced obesity. Clearly, different paradigms of exposure to malnutrition may be associated with differences in energy expenditure, food intake, weight and different susceptibilities to various symptoms associated with metabolic syndrome. Taken together these results demonstrate that intra-uterine environment is a major contributor to the future of individuals and disturbance at a critical period of development may compromise their health. Consequently, understanding the molecular mechanisms may give access to useful knowledge regarding the onset of metabolic diseases.