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.

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.

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.

When an essential amino acid is limited, a signaling cascade is triggered that leads to increased translation of the 'master regulator', activating transcription factor 4 (ATF4), and resulting in the induction of specific target genes. Binding of ATF4 to the amino acid response element (AARE) is an essential step in the transcriptional activation of CHOP (a CCAAT/enhancer-binding protein-related gene) by amino acid deprivation. We set out to identify proteins that interact with ATF4 and that play a role in the transcriptional activation of CHOP. Using a tandem affinity purification (TAP) tag approach, we identified p300/CBP-associated factor (PCAF) as a novel interaction partner of ATF4 in leucine-starved cells. We show that the N-terminal region of ATF4 is required for a direct interaction with PCAF and demonstrate that PCAF is involved in the full transcriptional response of CHOP by amino acid starvation. Chromatin immunoprecipitation analysis revealed that PCAF is engaged on the CHOP AARE in response to amino acid starvation and that ATF4 is essential for its recruitment. We also show that PCAF stimulates ATF4-driven transcription via its histone acetyltransferase domain. Thus PCAF acts as a coactivator of ATF4 and is involved in the enhancement of CHOP transcription following amino acid starvation.

The transcriptional activation of CHOP (a CCAAT/enhancer-binding protein-related gene) by amino acid deprivation involves the activating transcription factor 2 (ATF2) and the activating transcription factor 4 (ATF4) binding the amino acid response element (AARE) within the promoter. Using a chromatin immunoprecipitation approach, we report that in vivo binding of phospho-ATF2 and ATF4 to CHOP AARE are associated with acetylation of histones H4 and H2B in response to amino acid starvation. A time course analysis reveals that ATF2 phosphorylation precedes histone acetylation, ATF4 binding and the increase in CHOP mRNA. We also show that ATF4 binding and histone acetylation are two independent events that are required for the CHOP induction upon amino acid starvation. Using ATF2-deficient mouse embryonic fibroblasts, we demonstrate that ATF2 is essential in the acetylation of histone H4 and H2B in vivo. The role of ATF2 on histone H4 acetylation is dependent on its binding to the AARE and can be extended to other amino acid regulated genes. Thus, ATF2 is involved in promoting the modification of the chromatin structure to enhance the transcription of a number of amino acid-regulated genes.

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.

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.