Amino acid deprivation regulates the stress-inducible gene p8 via the GCN2/ATF4 pathway.

In mammals, the GCN2/ATF4 pathway has been described as the main pathway involved in the regulation of gene expression upon amino acid limitation. This regulation is notably conferred by the presence of a cis-element called Amino Acid Response Element (AARE) in the promoter of specific genes. In vivo, the notion of amino acid limitation is not limited to nutritional context, indeed several pathological situations are associated with alteration of endogenous amino acid availability. This is notably true in the context of tumour in which the alteration of the microenvironment can lead to a perturbation in nutrient availability. P8 is a small weakly folded multifunctional protein that is overexpressed in several kinds of cancers and whose expression is induced by different stresses. In this study we have demonstrated that amino acid starvation was also able to induce p8 expression. Moreover, we brought the evidence, in vitro and in vivo, that the GCN2/ATF4 pathway is involved in this regulation through the presence of an AARE in p8 promoter.

Specificity of amino acid regulated gene expression: analysis of genes subjected to either complete or single amino acid deprivation.

Amino acid deprivation activates the amino acid response (AAR) pathway that enhances transcription of genes containing an amino acid response element (AARE). The present data reveal a quantitative difference in the response to deprivation of individual amino acids. The AAR leads to increased eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and ATF4 translation. When HepG2 cells were deprived of an individual essential amino acid, p-eIF2alpha and activating transcription factor 4 were increased, but the correlation was relatively weak. Complete amino acid starvation in either Earle's balanced salt solution or Krebs-Ringer bicarbonate buffer (KRB) resulted in activation of transcription driven by a SNAT2 genomic fragment that contained an AARE. However, for the KRB, a proportion of the transcription was AARE-independent suggesting that amino acid-independent mechanisms were responsible. Therefore, activation of AARE-driven transcription is triggered by a deficiency in any one of the essential amino acids, but the response is not uniform. Furthermore, caution must be exercised when using a medium completely devoid of amino acids.

Amino acids control mammalian gene transcription: activating transcription factor 2 is essential for the amino acid responsiveness of the CHOP promoter.

In mammals, plasma concentration of amino acids is affected by nutritional or pathological conditions. It has been well established that nutrients, and particularly amino acids, are involved in the control of gene expression. Here we examined the molecular mechanisms involved in the regulation of CHOP (a CCAAT/enhancer-binding protein [C/EBP]-related gene) expression upon amino acid limitation. We have previously shown that regulation of CHOP mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. We report the analysis of cis- and trans-acting elements involved in the transcriptional activation of the human CHOP gene by leucine starvation. Using a transient expression assay, we show that a cis-positive element is essential for amino acid regulation of the CHOP promoter. This sequence is the first described that can regulate a basal promoter in response to starvation for several individual amino acids and therefore can be called an amino acid response element (AARE). In addition, we show that the CHOP AARE is related to C/EBP and ATF/CRE binding sites and binds in vitro the activating transcription factor 2 (ATF-2) in starved and unstarved conditions. Using ATF-2-deficient mouse embryonic fibroblasts and an ATF-2-dominant negative mutant, we demonstrate that expression of this transcription factor is essential for the transcriptional activation of CHOP by leucine starvation. Altogether, these results suggest that ATF-2 may be a member of a cascade of molecular events by which the cellular concentration of amino acids can regulate mammalian gene expression.

Inhibition of CHOP translation by a peptide encoded by an open reading frame localized in the chop 5'UTR.

Chop is a ubiquitously expressed mammalian gene encoding a small nuclear protein related to the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. CHOP protein plays an important role in various cellular processes such as growth, differentiation and programmed cell death. CHOP expression is strongly increased in response to a large variety of stresses including perturbation of the endoplasmic reticulum function, DNA damage and nutrient deprivation. Multiple mechanisms including transcriptional and post-transcriptional controls are involved in the regulation of CHOP expression. We show here that the 5'UTR of the Chop transcript plays an important role in controlling the synthesis of CHOP protein. In particular, the 5'UTR contains a conserved uORF which encodes a 31 amino acid peptide that inhibits the expression of the downstream ORF. Mutational analysis of the 5' leader region and peptide coding sequences suggests that the peptide itself inhibits expression of the downstream ORF. Such results suggest a role for uORF in limiting ribosomal access to downstream initiation sites. With respect to the importance of CHOP protein in the regulation of cellular functions, the mechanisms that regulate its basal level are of considerable interest.

Amino acid limitation regulates CHOP expression through a specific pathway independent of the unfolded protein response.

The gene encoding CHOP (C/EBP-homologous protein) is transcriptionally activated by many stimuli and by amino acid deprivation. CHOP induction was considered to be due to an accumulation of unfolded protein into the ER (unfolded protein response (UPR)). We investigate the role of the UPR in the induction of CHOP by amino acid deprivation and show that this induction is not correlated with BiP expression (an UPR marker). Moreover, amino acid deprivation and UPR inducers regulate the CHOP promoter activity using distinct cis elements. We conclude that amino acid deprivation does not activate the UPR and regulates CHOP expression through a pathway that is independent of the UPR.

Short-term protein and energy supplementation activates nitrogen kinetics and accretion in poorly nourished elderly subjects.

BACKGROUND: An increase in protein intake exerts a stimulating effect on protein kinetics in children, young adults, and healthy elderly persons. However, there are few data on the response to such dietary changes in malnourished elderly subjects, despite important medical implications in this population. OBJECTIVE: The objective of this study was to determine the metabolic response to short-term nutritional supplementation in moderately malnourished elderly subjects. DESIGN: The influence of 10 d of supplementation (1.67 MJ/d and 30 g protein/d) on body composition, resting energy expenditure, and whole-body protein kinetics was studied in 17 malnourished elderly patients and 12 healthy young adults. A control group of 6 malnourished elderly patients received no supplementation. RESULTS: Supplemented elderly subjects had a significantly greater fat-free mass gain than did unsupplemented elderly subjects (1.3 and 0.1 kg, respectively; age effect, P < 0.05; diet effect, P < 0.02) and a significantly greater increase in fasting rate of protein synthesis than did young supplemented subjects (0.6 and 0.2 g*kg FFM(-1)*11 h(-1); age effect, P < 0.05). The net protein balance in the supplemented elderly subjects in the fed state was positively correlated with protein intake (r(2) = 0.46) and in the fasted state was negatively correlated with protein intake (r(2) = 0.27). The sum of these regressions is a line with increasingly positive net diurnal protein balance produced by increasing protein intake. CONCLUSION: These data provide evidence of a short-term anabolic response of protein metabolism to dietary supplementation in malnourished elderly patients that is likely to improve muscle strength and functional status.

Intronic and 5' flanking sequences of the Drosophila beta 3 tubulin gene are essential to confer ecdysone responsiveness.

The expression of the beta 3 tubulin gene is regulated, at the transcriptional level, by the steroid hormone ecdysone, in Drosophila Kc cells. Using a transient expression assay, we show that 360 bp from the first intron of the beta 3 tubulin gene, associated with the 5' flanking sequences, are essential to confer ecdysone inducibility on a minimum promoter driving the chloramphenicol acetyl transferase (CAT) gene. The 5' flanking region contains ecdysone-independent cis-positive elements located in proximity to the promoter. Deletion analysis of the 360 bp intronic region reveals that a fragment of 57 bp is crucial for the ecdysone response of the beta 3 tubulin gene. This fragment contains 5'-TGA(A/C)C-3' motifs homologous to ecdysone responsive elements (EcRE) half sites. Band shift assays show that this 57-bp fragment is bound by three specific complexes. One of them appears to be involved in the level of the ecdysone response.

Enhancer and silencer elements within the first intron mediate the transcriptional regulation of the beta 3 tubulin gene by 20-hydroxyecdysone in Drosophila Kc cells.

We have studied the transcriptional regulation of the beta 3 tubulin gene by the steroid hormone 20-hydroxyecdysone (20-E) in Drosophila Kc cells. A series of hybrid genes, with different fragments of the beta 3 tubulin gene driving the bacterial chloramphenicol acetyl transferase (CAT) gene were constructed. The promoter activity was assayed after transient expression in Kc cells, in the presence and the absence of 20-E. Constructs with 0.91 kb upstream from the transcription start site and 360 bp from the first large intron allowed the hormonal regulation, i.e. a repression in the absence of 20-E and a derepression-activation in the presence of the hormone. This 360 bp fragment contains several enhancers and silencer(s) sequences. The regulation of the expression of the beta 3 tubulin gene results from the combined activity of all the positive and negative regulatory sequences of the first intron, and a dialogue with the promoter sequences. The nucleotide sequence of this intronic regulatory-fragment has been established and we have identified several EcRE (ecdysone responsive element) consensus sequences.

Variability of blood pressure response to orthostatism and reproducibility of the diagnosis of orthostatic hypotension in elderly subjects.

BACKGROUND: Orthostatic hypotension (OH) is a major problem in the elderly population. Its diagnosis is based on measurement of the blood pressure (BP) response to orthostatism (BPRO). This study investigates the within-day and day-to-day variability of the BPRO and the reproducibility of the diagnosis of OH in this population. METHODS: BP was measured in the supine position and after 1 and 2 minutes of orthostatism in 53 consecutive elderly patients (43 women and 10 men aged 83.7 +/- 9.5 years) of an intermediate care geriatric ward. BPRO was assessed 4 times on the same day (8-9 AM, 10-11 AM, 1-2 PM, and 5-6 PM) and twice more on another day of the same week (8-9 AM and 1-2 PM). RESULTS: There were significant within-day differences between the four orthostatic changes in systolic BP (OCs, supine minus standing systolic BP) after 1 minute or 2 minutes (p < .05). Day-to-day differences between the OCs measured at the same times were not significant. OH defined as an OCs of 20 mm Hg or more at 1 or 2 minutes of orthostatism, was found in ten cases (19%) in the initial set of measurements on the first day. A cumulative diagnosis of OH after the six BPRO tests was found in 23 cases (43%). The reproducibility of the diagnosis of OH was mild or poor (all kappa values were below 0.47). CONCLUSIONS: BPRO exhibits significant within-day variability in elderly patients. Within-day and day-to-day reproducibility of the diagnosis of OH, based on conventional criteria, were found to be poor.

Nutritional status after short-term dietary supplementation in hospitalized malnourished geriatric patients.

AIM: To examine the evolution of different parameters of the nutritional status after short-term oral protein-energy supplementation in moderately malnourished geriatric patients. METHODS: Seventeen hospitalized malnourished elderly patients and 12 healthy adults received dietary supplements for 10 days. A group of six malnourished elderly subjects served as controls. Spontaneous oral intakes, biological and biophysical markers of the nutritional status were measured. Fat-free mass (FFM) was assessed using Dual energy X-ray absorptiometry (DXA), bio-impedance analysis (BIA) and anthropometry. RESULTS: In elderly subjects, the supplementation significantly increased both dietary intake (energy +32%, protein +65%) and FFM (+1.3 kg, P<0.001) as assessed using DXA. BIA and anthropometric data correlated with DXA measurements in the elderly (BIA: r=0.68--0.80, anthropometry: r=0.80--0.89), but failed to reflect accurately the changes measured in FFM. Supplementation had no notable effect on biological markers in any of the groups. IGF-I and hand-grip strength were not significantly influenced by the supplementation despite trends towards an improvement. CONCLUSIONS: Monitoring short-term changes in nutritional status in malnourished elderly individuals is a problem in routine clinical management. Our data put in the limelight the changes in IGF-I values related to dietary supplementation, and, chiefly, suggest a prime role for the assessment of dietary intake and FFM, as assessed by DXA, as indicators of short-term efficacy of refeeding. Nevertheless larger studies are necessary to confirm the clinical and prognostic significance of the changes.

[Nutritional support in elderly malnourished patients]. / L'assistance nutritionnelle chez les malades âgés dénutris.

ASSESSMENT OF NUTRITIONAL STATUS: Protein-calorie malnutrition is frequent and often severe in fragile hospitalized or institutionalized elderly subjects. Underdiagnosis and undertreatment is commonplace. Since under- or malnutrition is more difficult to correct in the elderly than in the young subject, a careful assessment of the nutritional status must be made in order to recognize any nutritional disorder early and initiate proper treatment early. NUTRITIONAL ASSISTANCE: High-protein oral supplementation and complementary enteral nutrition can improve the clinical status of certain under- or malnourished elderly subjects. It is particularly important to initiate nutritional assistance early and provide effective treatment for any recognized surgical or medical cause of malnutrition. Patient cooperation is crucial. EXPECTED RESULTS: Associated early with rehabilitation therapy, nutritional assistance can be expected to improve the functional independence of the elderly patient. Treatment efficacy and patient tolerance must be evaluated regularly to adapt nutritional assistance to the patient's particular clinical situation. Intensive enteral nutrition is not appropriate for elderly patients with severe malnutrition whose quality of life is compromised by motor, psycho-cognitive, or sensorial handicaps.

[Quality of the information collected during admission to a hospital geriatric service: importance of a structured medical record]. / Qualité du recueil d'information à l'admission en service hospitalier de gériatrie: intérêt d'un dossier médical structuré.

OBJECTIVES: The medical record (MR) is a key document for hospitalized patients. Several audits have however demonstrated that much important information is often missing in hospital MR. We conducted this survey with the aim of improving the quality of MR in a geriatric unit. PATIENTS AND METHODS: A structured MR was elaborated and implemented in order to guide and record the assessment of patients admitted in our geriatric ward. MR of 54 consecutive patients admitted after implementation of the structured MR were studied and compared to those of 108 consecutive patients admitted on the preceeding year (classical MR). Quality of data collected at admission was assessed using a 33-item guide, proposed by 3 experts in geriatric medicine unaware of the structured MR studies. For each item, a binary score (present/absent) and a precision score were used. A validation study was conducted using the same methods in another geriatric ward which has not participated to development of the structured charts MR studied. RESULTS: For most items studied, information was present in a significantly higher proportion in structured MR than in classical MR. Likewise, the precision score was significantly higher in structured MR. The validation survey found analogous results. CONCLUSION: Use of a structured MR significantly improves the quality of data collection at admission in geriatric units. This improvement appears to be related more to the use of the structured MR than the effect of developing a new tool.

Recent advances on molecular mechanisms involved in amino acid control of gene expression.

In mammals, the impact of nutrients on gene expression has become an important area of research. Because amino acids have multiple and important functions, their homeostasis has to be finely maintained. However, amino acidaemia can be affected by certain nutritional conditions or various forms of aggression. It follows that mammals have to adjust several of their physiological functions involved in the adaptation to amino acid availability by regulating the expression of numerous genes. It has been shown that amino acids by themselves can modify the expression of target genes. However, the current understanding of amino acid-dependent control of gene expression has just started to emerge. This review focuses on the recent advances on mechanisms involved in the amino acids control of gene expression. Several examples discussed in this paper demonstrate that amino acids regulate gene expression at the level of transcription, messenger RNA stability and translation.

In vivo estradiol-dependent dephosphorylation of the repressor MDBP-2-H1 correlates with the loss of in vitro preferential binding to methylated DNA.

We have previously shown that estradiol treatment of roosters resulted in a rapid loss of binding activity of the repressor MDBP-2-H1 (a member of the histone H1 family) to methylated DNA that was not due to a decrease in MDBP-2-H1 concentration. Here we demonstrate that MDBP-2-H1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamino acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phosphopeptide analysis show that MDBP-2-H1 is phosphorylated at several sites. Treatment of roosters with estradiol triggers a dephosphorylation of at least two sites in the protein. Phosphatase treatment of purified rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to methylated DNA and that the dephosphorylation can occur on the protein bound to methylated DNA causing its release from DNA. Thus, these results suggest that in vivo modification of the phosphorylation status of MDBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA.

Phosphorylation/dephosphorylation of the repressor MDBP-2-H1 selectively affects the level of transcription from a methylated promoter in vitro.

We have previously shown that in vivo estradiol-dependent dephosphorylation of MDBP-2-H1 (a member of the histone H1 family) correlates with the loss of in vitro preferential binding to methylated DNA. To study the effects of the phosphorylation/dephosphorylation of MDBP-2-H1 on the expression of the avian vitellogenin II gene, we optimised an in vitro transcription system using HeLa nuclear extracts. We show that in the absence of the phosphorylated form of MDBP-2-H1 from rooster, methylation of the vitellogenin II promoter does not affect the transcription. Addition of purified MDBP-2-H1 from rooster to the in vitro transcription system inhibits transcription more efficiently from a methylated than an unmethylated DNA template. Dephosphorylation of rooster MDBP-2-H1 by phosphatase treatment or estradiol treatment of rooster lead to the loss of inhibitory activity of the protein when added to the in vitro transcription assays. These findings indicate that the phosphorylation of MDBP-2-H1 is essential for the repression of the transcription. Taken together these results establish the relationship between the dephosphorylation of MDBP-2-H1 caused by estradiol, the down regulation of its binding activity to methylated DNA and the derepression of vitellogenin II transcription.

Amino acid regulation of gene expression.

In mammals, the plasma concentration of amino acids is affected by nutritional or pathological conditions. For example, an amino acid profile alteration has been reported as a result of a deficiency of any one of the essential amino acids, a dietary imbalance of amino acids or an insufficient intake of protein. Amino acid availability regulates the expression of several genes involved in the regulation of growth, cellular function or amino acid metabolism. A limitation of several amino acids strongly increases the expression of insulin-like growth factor binding protein CHOP and asparagine synthetase genes. Elevated messenger RNA levels result from both an increase in the rate of transcription and an increase in messenger RNA stability. DNA amino acid response elements have been characterized in the promoter of CHOP and asparagine synthetase genes. The underlying mechanisms of gene regulation by amino acid limitation are not yet completely understood. The results discussed in this review demonstrate that amino acids by themselves can play, in concert with hormones, an important role in the control of gene expression.

Amino acid regulation of gene expression.

The impact of nutrients on gene expression in mammals has become an important area of research. Nevertheless, the current understanding of the amino acid-dependent control of gene expression is limited. Because amino acids have multiple and important functions, their homoeostasis has to be finely maintained. However, amino-acidaemia can be affected by certain nutritional conditions or various forms of stress. It follows that mammals have to adjust several of their physiological functions involved in the adaptation to amino acid availability by regulating the expression of numerous genes. The aim of the present review is to examine the role of amino acids in regulating mammalian gene expression and protein turnover. It has been reported that some genes involved in the control of growth or amino acid metabolism are regulated by amino acid availability. For instance, limitation of several amino acids greatly increases the expression of the genes encoding insulin-like growth factor binding protein-1, CHOP (C/EBP homologous protein, where C/EBP is CCAAT/enhancer binding protein) and asparagine synthetase. Elevated mRNA levels result from both an increase in the rate of transcription and an increase in mRNA stability. Several observations suggest that the amino acid regulation of gene expression observed in mammalian cells and the general control process described in yeast share common features. Moreover, amino acid response elements have been characterized in the promoters of the CHOP and asparagine synthetase genes. Taken together, the results discussed in the present review demonstrate that amino acids, by themselves, can, in concert with hormones, play an important role in the control of gene expression.