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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., (© 2023 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

Purpose: Protein synthesis and proteolysis are known to be controlled through mammalian target of rapamycin, AMP-activated kinase (AMPK) and general control non-derepressible 2 (GCN2) pathways, depending on the nutritional condition. This study aimed at investigating the contribution of liver AMPK and GCN2 on the adaptation to high variations in protein intake., Methods: To evaluate the answer of protein pathways to high- or low-protein diet, male wild-type mice and genetically modified mice from C57BL/6 background with liver-specific AMPK- or GCN2-knockout were fed from day 25 diets differing in their protein level as energy: LP (5%), NP (14%) and HP (54%). Two hours after a 1 g test meal, protein synthesis rate was measured after a 13 C valine flooding dose. The gene expression of key enzymes involved in proteolysis and GNC2 signaling pathway were quantified., Results: The HP diet but not the LP diet was associated with a decrease in fractional synthesis rate by 29% in the liver compared to NP diet. The expression of mRNA encoding ubiquitin and Cathepsin D was not sensitive to the protein content. The deletion of AMPK or GCN2 in the liver did not affect nor protein synthesis rates and neither proteolysis markers in the liver or in the muscle, whatever the protein intake. In the postprandial state, protein level alters protein synthesis in the liver but not in the muscle., Conclusions: Taken together, these results suggest that liver AMPK and GCN2 are not involved in this adaptation to high- and low-protein diet observed in the postprandial period., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

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-β (TGF-β) 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-β 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-β/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-β/BMP balance and could be used to preserve muscle mass during catabolic situations.

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.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cyclosporin A (CsA) and tacrolimus (FK506) are valuable immunosuppressants for a range of clinical settings, including (but not limited to) organ transplantation and the treatment of autoimmune diseases. They function by inhibiting the activity of the Ca 2+ /calmodulin-dependent phosphatase calcineurin toward nuclear factor of activated T-cells (NF-AT) in T-lymphocytes. However, use of CsA is associated with more serious side effects and worse clinical outcomes than FK506. Here we show that CsA, but not FK506, causes activation of the integrated stress response (ISR), an event which is normally an acute reaction to various types of intracellular insults, such as nutrient deficiency or endoplasmic reticulum stress. These effects of CsA involve at least two of the stress-activated protein kinases (GCN2 and PERK) that act on the translational machinery to slow down protein synthesis via phosphorylation of the eukaryotic initiation factor (eIF) 2α and thereby induce the ISR. These actions of CsA likely contribute to the adverse effects associated with its clinical application., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Fedele et al.)

Background: mTOR signaling is an essential nutrient and energetic sensing pathway. Here we describe AIMTOR, a sensitive genetically encoded BRET (Bioluminescent Resonance Energy Transfer) biosensor to study mTOR activity in living cells., Results: As a proof of principle, we show in both cell lines and primary cell cultures that AIMTOR BRET intensities are modified by mTOR activity changes induced by specific inhibitors and activators of mTORC1 including amino acids and insulin. We further engineered several versions of AIMTOR enabling subcellular-specific assessment of mTOR activities. We then used AIMTOR to decipher mTOR signaling in physio-pathological conditions. First, we show that mTORC1 activity increases during muscle cell differentiation and in response to leucine stimulation in different subcellular compartments such as the cytosol and at the surface of the lysosome, the nucleus, and near the mitochondria. Second, in hippocampal neurons, we found that the enhancement of neuronal activity increases mTOR signaling. AIMTOR further reveals mTOR-signaling dysfunctions in neurons from mouse models of autism spectrum disorder., Conclusions: Altogether, our results demonstrate that AIMTOR is a sensitive and specific tool to investigate mTOR-signaling dynamics in living cells and phenotype mTORopathies.

General control nonderepressible 2 (GCN2) is a kinase that detects amino acid deficiency and is involved in the control of protein synthesis and energy metabolism. However, the role of hepatic GCN2 in the metabolic adaptations in response to the modulation of dietary protein has been seldom studied. Wild-type (WT) and liver GCN2-deficient (KO) mice were fed either a normo-protein diet, a low-protein diet, or a high-protein diet for 3 wk. During this period, body weight, food intake, and metabolic parameters were followed. In mice fed normo- and high-protein diets, GCN2 pathway in the liver is not activated in WT mice, leading to a similar metabolic profile with the one of KO mice. On the contrary, a low-protein diet activates GCN2 in WT mice, inducing FGF21 secretion. In turn, FGF21 maintains a high level of lipid oxidation, leading to a different postprandial oxidation profile compared with KO mice. Hepatic GCN2 controls FGF21 secretion under a low-protein diet and modulates a whole body postprandial oxidation profile.

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.

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.

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.

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.

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., (Copyright © 2015, American Association for the Advancement of Science.)

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., (© Société de Biologie, 2016.)

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and metabolism. It controls many cell functions by integrating nutrient availability and growth factor signals. Amino acids, and in particular leucine, are among the main positive regulators of mTORC1 signaling. The current model for the regulation of mTORC1 by amino acids involves the movement of mTOR to the lysosome mediated by the Rag-GTPases. Here, we have examined the control of mTORC1 signaling and mTOR localization by amino acids and leucine in serum-fed cells, because both serum growth factors (or, e.g., insulin) and amino acids are required for full activation of mTORC1 signaling. We demonstrate that mTORC1 activity does not closely correlate with the lysosomal localization of mTOR. In particular, leucine controls mTORC1 activity without any detectable modification of the lysosomal localization of mTOR, indicating that the signal(s) exerted by leucine is likely distinct from those exerted by other amino acids. In addition, knock-down of the Rag-GTPases attenuated the inhibitory effect of amino acid- or leucine-starvation on the phosphorylation of mTORC1 targets. Furthermore, data from cells where Rag expression has been knocked down revealed that leucine can promote mTORC1 signaling independently of the lysosomal localization of mTOR. Our data complement existing models for the regulation of mTORC1 by amino acids and provide new insights into this important topic., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

CHOP encodes a ubiquitous transcription factor that is one of the most important components in the network of stress-inducible transcription. In particular, this factor is known to mediate cell death in response to stress. The focus of this work is to study its pivotal role in the control of cell viability according to the duration of a stress like amino acid starvation. We show that during the first 6h of starvation, CHOP upregulates a number of autophagy genes but is not involved in the first steps of the autophagic process. By contrast, when the amino acid starvation is prolonged (16-48h), we demonstrated that CHOP has a dual role in both inducing apoptosis and limiting autophagy through the transcriptional control of specific target genes. Overall, this study reveals a novel regulatory role for CHOP in the crosstalk between autophagy and apoptosis in response to stress., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ribosome biogenesis is a key process for maintaining protein synthetic capacity in dividing or growing cells, and requires coordinated production of ribosomal proteins and ribosomal RNA (rRNA), including the processing of the latter. Signalling through mammalian target of rapamycin complex 1 (mTORC1) activates all these processes. Here, we show that, in human cells, impaired rRNA processing, caused by expressing an interfering mutant of BOP1 or by knocking down components of the PeBoW complex elicits activation of mTORC1 signalling. This leads to enhanced phosphorylation of its substrates S6K1 and 4E-BP1, and stimulation of proteins involved in translation initiation and elongation. In particular, we observe both inactivation and downregulation of the eukaryotic elongation factor 2 kinase, which normally inhibits translation elongation. The latter effect involves decreased expression of the eEF2K mRNA. The mRNAs for ribosomal proteins, whose translation is positively regulated by mTORC1 signalling, also remain associated with ribosomes. Therefore, our data demonstrate that disrupting rRNA production activates mTORC1 signalling to enhance the efficiency of the translational machinery, likely to help compensate for impaired ribosome production.

The reversible phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) is a highly conserved signal implicated in the cellular adaptation to numerous stresses such as the one caused by amino acid limitation. In response to dietary amino acid deficiency, the brain-specific activation of the eIF2α kinase GCN2 leads to food intake inhibition. We report here that GCN2 is rapidly activated in the mediobasal hypothalamus (MBH) after consumption of a leucine-deficient diet. Furthermore, knockdown of GCN2 in this particular area shows that MBH GCN2 activity controls the onset of the aversive response. Importantly, pharmacological experiments demonstrate that the sole phosphorylation of eIF2α in the MBH is sufficient to regulate food intake. eIF2α signaling being at the crossroad of stress pathways activated in several pathological states, our study indicates that hypothalamic eIF2α phosphorylation could play a critical role in the onset of anorexia associated with certain diseases., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

In response to different environmental stresses, eIF2α phosphorylation represses global translation coincident with preferential translation of ATF4, a master regulator controlling the transcription of key genes essential for adaptative functions. Here, we establish that the eIF2α/ATF4 pathway directs an autophagy gene transcriptional program in response to amino acid starvation or endoplasmic reticulum stress. The eIF2α-kinases GCN2 and PERK and the transcription factors ATF4 and CHOP are also required to increase the transcription of a set of genes implicated in the formation, elongation and function of the autophagosome. We also identify three classes of autophagy genes according to their dependence on ATF4 and CHOP and the binding of these factors to specific promoter cis elements. Furthermore, different combinations of CHOP and ATF4 bindings to target promoters allow the trigger of a differential transcriptional response according to the stress intensity. Overall, this study reveals a novel regulatory role of the eIF2α-ATF4 pathway in the fine-tuning of the autophagy gene transcription program in response to stresses.

Purpose: Selection of a balanced diet has a determinant impact on human health. Individual food preferences involve socio-cultural as well as physiological factors and evolve during aging. In mammals, physiological mechanisms governing food choices appear to require the sensing of nutrient concentrations in diet. This is particularly the case for dietary amino acids that are sensed by the protein kinase GCN2. It has been reported that GCN2 is involved in the adaptive response to amino acid imbalanced diets at the level of food intake and lipid metabolism. Here, we hypothesized that GCN2 may play a role in macronutrient selection and its age-related changes., Methods: Two groups of wild-type and GCN2 knock-out mice were subjected to a food self-selection protocol at ages 6, 12, 18 and 24 months. During each test, mice were allowed to create their own diets by selecting between three separate food sources, each containing either protein, fat or carbohydrates., Results: Our results show that the absence of GCN2 had two main age-related effects. First, it exacerbated fat preference at the expense of carbohydrate consumption. Second, it prevented the increase in protein intake., Conclusion: These findings indicate that, in omnivores, the GCN2 ancient pathway participates in the control of food preference.

Constitution of oxidative defense systems and, correspondingly, oxidative stress prevention are highly dependent on amino acid supply. In vitro, experiments have demonstrated that amino acid availability participates to the homeostasis of reactive oxygen species. However the molecular mechanisms involved in the maintenance of redox homeostasis responsive to circulating amino acid levels remain unclear. As GCN2 is a protein kinase considered to be an important sensor for amino acids availability and a potential regulator of redox homeostasis, we hypothesized that this kinase can modulate redox homeostasis in vivo, in response to an amino acid-imbalanced diet. We investigated the response of GCN2+/+ and GCN2-/- mice to a long-term (24 weeks) leucine-imbalanced diet (EDΔLeu). In order to evaluate the oxidation level in each group of mice, we determined the degree of protein oxidation in the liver. Interestingly, GCN2-/- mice exhibited an increase in protein carbonylation, a marker of oxidative stress, in response to the EDΔLeu diet. These data correlate with a decrease in hepatic GPX1 expression, a major antioxidant enzyme, and a decrease in total GPX activity in the liver. Our results suggest that GCN2 and its downstream signaling pathway have an important role in the protection against oxidative injuries induced by an amino acid-imbalanced diet, and that it can play a critical role in the prevention of oxidative damage., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Transient environmental influences, such as perinatal nutritional stress, may induce deleterious metabolic symptoms that last for the entire life of individuals, implying that epigenetic modifications play an important role in this process. We have investigated, in mice, the consequences of maternal undernutrition during gestation and lactation on DNA methylation and expression of the leptin gene, which plays a major regulatory role in coordinating nutritional state with many aspects of mammalian biology. We show that animals born to mothers fed a low-protein-diet (F1-LPD group) have a lower body weight/adiposity and exhibit a higher food intake than animals born to mothers fed a control diet (F1-CD group). These modifications persisted throughout life and were associated with lower levels of leptin mRNA and protein in starved F1-LPD mice, emphasizing that maternal protein-undernutrition affects the balance between food intake and energy expenditure in adults. Moreover, this nutritional stress resulted in the removal of methyls at CpGs located in the promoter of leptin, causing a permanent specific modification in the dynamics of the expression of leptin, which exhibits a stronger induction in the F1-LPD than in F1-CD mice in response to a meal. This study is an example of a molecular rationale linking transient environmental influences to permanent phenotypic consequences.

In mammals, plasma amino acid concentrations are markedly affected by dietary or pathological conditions. It has been well established that amino acids are involved in the control of gene expression. Up to now, all the information concerning the molecular mechanisms involved in the regulation of gene transcription by amino acid availability has been obtained in cultured cell lines. The present study aims to investigate the mechanisms involved in transcriptional activation of the TRB3 gene following amino acid limitation in mice liver. The results show that TRB3 is up-regulated in the liver of mice fed a leucine-deficient diet and that this induction is quickly reversible. Using transient transfection and chromatin immunoprecipitation approaches in hepatoma cells, we report the characterization of a functional Amino Acid Response Element (AARE) in the TRB3 promoter and the binding of ATF4, ATF2 and C/EBPβ to this AARE sequence. We also provide evidence that only the binding of ATF4 to the AARE plays a crucial role in the amino acid-regulated transcription of TRB3. In mouse liver, we demonstrate that the GCN2/eIF2α/ATF4 pathway is essential for the induction of the TRB3 gene transcription in response to a leucine-deficient diet. Therefore, this work establishes for the first time that the molecular mechanisms involved in the regulation of gene transcription by amino acid availability are functional in mouse liver.

In mammals, metabolic adaptations are required to cope with episodes of protein deprivation and malnutrition. Consequently, mammals have to adjust physiological functions involved in the adaptation to amino acid availability. Part of this regulation involves the modulation of the expression of numerous genes. In particular, it has been shown that amino acids by themselves can modify the expression of target genes. This review describes the regulation of amino acids homeostasis and the their role as signal molecules. The recent advances in the understanding of the molecular mechanisms involved in the control of mammalian gene expression in response to amino acid limitation will be described., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

Aging is characterized by a decline in muscle mass that could be explained by a defect in the regulation of postprandial muscle protein metabolism. This study was undertaken to examine a possible link between the development of low-grade inflammation (LGI) in elderly and the resistance of muscle protein synthesis and degradation pathways to food intake. Fifty-five 20-month-old-rats were studied for 5 months; blood was withdrawn once a month to assess plasma fibrinogen and alpha2-macroglobulin. Animals were then separated into two groups at 25 months old according to their inflammation status: a control non-inflamed (NI, n=24) and a low-grade inflamed group (LGI, n=23). The day of the experiment, rats received no food or a meal. Muscle protein synthesis was assessed in vivo using the flooding dose method ([1-(13)C]phenylalanine) and muscle phosphorylation of protein S6 kinase, and protein S6 was measured in gastrocnemius muscle. Muscle proteolysis was assessed in vitro using the epitrochlearis muscle. Postabsorptive muscle protein synthesis and proteolysis were similar in NI and LGI. After food intake, muscle protein synthesis was significantly stimulated in NI but remained unresponsive in LGI. Muscle proteolysis was similar in both groups whatever the inflammation and/or the nutritional status. In conclusion, we showed that development of LGI during aging may be responsible, at least in part, for the defect in muscle protein synthesis stimulation induced by food intake in rats. Our results suggested that the control of LGI development in elderly improve meal effect on muscle protein synthesis and consequently slow down sarcopenia., (Copyright 2010 Elsevier Inc. All rights reserved.)

Ageing is characterized by a decline in muscle mass that could be explained by a defect in the regulation of postprandial muscle protein metabolism. Indeed, the stimulatory effect of food intake on protein synthesis and its inhibitory effect on proteolysis is blunted in old muscles from both animals and humans. Recently, low grade inflammation has been suspected to be one of the factors responsible for the decreased sensitivity of muscle protein metabolism to food intake. This study was undertaken to examine the effect of long-term prevention of low grade inflammation on muscle protein metabolism during ageing. Old rats (20 months of age) were separated into two groups: a control group and a group (IBU) in which low grade inflammation had been reduced with a non-steroidal anti inflammatory drug (ibuprofen). After 5 months of treatment, inflammatory markers and cytokine levels were significantly improved in treated old rats when compared with the controls: -22.3% fibrinogen, -54.2% alpha2-macroglobulin, +12.6% albumin, -59.6% IL(6) and -45.9% IL(1beta) levels. As expected, food intake had no effect on muscle protein synthesis or muscle proteolysis in controls whereas it significantly increased muscle protein synthesis by 24.8% and significantly decreased proteolysis in IBU rats. The restoration of muscle protein anabolism at the postprandial state by controlling the development of low grade inflammation in old rats significantly decreased muscle mass loss between 20 and 25 months of age. In conclusion, the observations made in this study have identified low grade inflammation as an important target for pharmacological, nutritional and lifestyle interventions that aim to limit sarcopenia and muscle weakness in the rapidly growing elderly population in Europe and North America.

Immobilization produces morphological, physiological, and biochemical alterations in skeletal muscle leading to muscle atrophy and long periods of recovery. Muscle atrophy during disuse results from an imbalance between protein synthesis and proteolysis but also between apoptosis and regeneration processes. This work aimed to characterize the mechanisms underlying muscle atrophy and recovery following immobilization by studying the regulation of the mitochondria-associated apoptotic and the ubiquitin-proteasome-dependent proteolytic pathways. Animals were subjected to hindlimb immobilization for 4-8 days (I4 to I8) and allowed to recover after cast removal for 10-40 days (R10 to R40). Soleus and gastrocnemius muscles atrophied from I4 to I8 to a greater extent than extensor digitorum longus and tibialis anterior muscles. Gastrocnemius muscle atrophy was first stabilized at R10 before being progressively reduced until R40. Polyubiquitinated proteins accumulated from I4, whereas the increased ubiquitination rates and chymotrypsin-like activity of the proteasome were detectable from I6 to I8. Apoptosome and caspase-3 or -9 activities increased at I6 and I8, respectively. The ubiquitin-proteasome-dependent pathway was normalized early when muscle stops to atrophy (R10). By contrast, the mitochondria-associated apoptotic pathway was first downregulated below basal levels when muscle started to recover at R15 and completely normalized at R20. Myf 5 protein levels decreased from I4 to I8 and were normalized at R10. Altogether, our results suggest a two-stage process in which the ubiquitin-proteasome pathway is rapidly up- and downregulated when muscle atrophies and recovers, respectively, whereas apoptotic processes may be involved in the late stages of atrophy and recovery.

A dramatic overexpression of IGFBP-1 is responsible for growth inhibition, in response to a low-protein diet feeding. It has been demonstrated that a fall in the amino acid concentration was directly responsible for IGFBP-1 induction. In this report, we sought to determine the mechanism by which amino acid limitation upregulates IGFBP-1 expression. Our results show that both transcriptional activation and mRNA stabilization are involved. We also demonstrate that (i) the mGCN2/ATF4 pathway is not involved in this regulation and (ii) the 3'UTR of IGFBP-1 mRNA is responsible for its destabilization and regulates its stability in response to amino acid starvation.

To insure an adequate supply of nutrients, omnivores choose among available food sources. This process is exemplified by the well-characterized innate aversion of omnivores to otherwise nutritious foods of imbalanced amino acid content. We report that brain-specific inactivation of GCN2, a ubiquitously expressed protein kinase that phosphorylates translation initiation factor 2 alpha (eIF2alpha) in response to intracellular amino acid deficiency, impairs this aversive response. GCN2 inactivation also diminishes phosphorylated eIF2alpha levels in the mouse anterior piriform cortex following consumption of an imbalanced meal. An ancient intracellular signal transduction pathway responsive to amino acid deficiency thus affects feeding behavior by activating a neuronal circuit that biases consumption against imbalanced food sources.

The CHOP gene is transcriptionally induced by amino acid starvation. We have previously identified a genomic cis-acting element (amino acid response element (AARE)) involved in the transcriptional activation of the human CHOP gene by leucine starvation and shown that it binds the activating transcription factor 2 (ATF2). The present study was designed to identify other transcription factors capable of binding to the CHOP AARE and to establish their role with regard to induction of the gene by amino acid deprivation. Electrophoretic mobility shift assay and transient transfection experiments show that several transcription factors that belong to the C/EBP or ATF families bind the AARE sequence and activate transcription. Among all these transcription factors, only ATF4 and ATF2 are involved in the amino acid control of CHOP expression. We show that inhibition of ATF2 or ATF4 expression impairs the transcriptional activation of CHOP by amino acid starvation. The transacting capacity of ATF4 depends on its expression level and that of ATF2 on its phosphorylation state. In response to leucine starvation, ATF4 expression and ATF2 phosphorylation are increased. However, induction of ATF4 expression by the endoplasmic reticulum stress pathway does not fully activate the AARE-dependent transcription. Taken together our results demonstrate that at least two pathways, one leading to ATF4 induction and one leading to ATF2 phosphorylation, are necessary to induce CHOP expression by amino acid starvation. This work was extended to the regulation of other amino acid regulated genes and suggests that ATF4 and ATF2 are key components of the amino acid control of gene expression.