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.

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.

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.

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.

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.

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.

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.

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.

Evidence for multiple signaling pathways in the regulation of gene expression by amino acids in human cell lines.

In mammals, plasma concentrations of amino acids (AA) are affected by nutritional or pathologic conditions. Alterations in AA profiles have been reported as a result of a deficiency of any one of the essential AA, a dietary imbalance of AA or an insufficient intake of protein. In recent years, evidence has accumulated that AA availability regulates the expression of several genes involved in the regulation of a number of cellular functions or AA metabolism. Nevertheless, the molecular mechanisms involved in the AA regulation of mammalian gene expression are limited, particularly the signaling pathways mediating the AA response. This work provides a better understanding of the signaling pathways involved in the AA control of gene expression. We studied the expression of C/EBP homologous protein (CHOP) and asparagine synthetase (AS) in response to deprivation of a single AA and investigated the possible link between protein synthesis inhibition due to amino acid limitation and gene expression. We have shown the following: 1) several mechanisms are involved in the AA control of gene expression. When omitted from the culture medium, each AA can activate one (or several) specific signaling pathways leading to the regulation of one specific pattern of genes. 2) AA limitation by itself can induce gene expression independently of a cellular stress due to protein synthesis inhibition. Together, these results suggest that AA control of gene expression involves several specific mechanisms by which one AA (or one group of AA) can activate one signaling pathway and thus alter one specific pattern of gene expression.

[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.

Amino acid limitation regulates gene expression.

In mammals, the plasma concentration of amino acids is affected by nutritional or pathological conditions. For example, an alteration in the amino acid profile has been reported when there is a deficiency of any one or more of the essential amino acids, a dietary imbalance of amino acids, or an insufficient intake of protein. We examined the role of amino acid limitation in regulating mammalian gene expression. Depletion of arginine, cystine and all essential amino acids leads to induction of insulin-like growth factor-binding protein-1 (IGFBP-1) mRNA and protein expression in a dose-dependent manner. Moreover, exposure of HepG2 cells to amino acids at a concentration reproducing the amino acid concentration found in portal blood of rats fed on a low-protein diet leads to a significantly higher (P < 0.0002) expression of IGFBP-1. Using CCAAT/enhancer-binding protein homologous protein (CHOP) induction by leucine deprivation as a model, we have characterized the molecular mechanisms involved in the regulation of gene expression by amino acids. We have shown that leucine limitation leads to induction of CHOP mRNA and protein. Elevated mRNA levels result from both an increase in the rate of CHOP transcription and an increase in mRNA stability. We have characterized two elements of the CHOP gene that are essential to the transcriptional activation produced by an amino acid limitation. These findings demonstrate that an amino acid limitation, as occurs during dietary protein deficiency, can induce gene expression. Thus, 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.

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 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.

[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.

Physiological concentration of amino acids regulates insulin-like-growth-factor-binding protein 1 expression.

Protein undernutrition is characterized by growth failure in young growing animals. Current evidence suggests that biosynthesis of insulin-like growth factor (IGF)-I and IGF-binding protein 1 (IGFBP-1) are key control points for nutritional regulation of growth. Here we examined the role of amino acid limitation in regulating the IGFBP-1 expression in the hepatic cell line. Our data show that leucine limitation strongly induces IGFBP-1 without affecting IGF-I and IGF-II expression in human HepG2 cells and in isolated rat hepatocytes. Depletion of arginine, cystine and all essential amino acids leads to induction of IGFBP-1 mRNA and protein expression in a dose-dependent manner. IGFBP-1 expression is significantly induced by leucine concentration in the range of that observed in the blood of rats fed a low-protein diet or in humans affected by kwashiorkor. Moreover, treatment of HepG2 cells with amino acids at a concentration reproducing the amino acid concentration found in portal blood of rats fed a low-protein diet leads to a significantly higher expression of IGFBP-1. These data represent the first demonstration that an amino acid limitation, as occurs during dietary protein deficiency, induces IGFBP-1 expression in hepatic cells. Therefore, amino acids by themselves can play, in concert with hormones, an important role in the control of gene expression.

Amino acid limitation induces expression of CHOP, a CCAAT/enhancer binding protein-related gene, at both transcriptional and post-transcriptional levels.

In mammals, plasma concentrations of amino acids are affected by nutritional or pathological conditions. Here we examined the role of amino acid limitation in regulating the expression of CHOP, a CCAAT/enhancer binding protein (C/EBP)-related gene. CHOP protein is capable of interacting with other C/EBPs to modify their DNA binding activities and may function as a negative regulator of these transcription factors. Our data show that leucine limitation in human cell lines leads to induction of CHOP mRNA and protein in a dose-dependent manner. CHOP mRNA induction is rapidly reversed by leucine replenishment. Elevated mRNA levels result from both an increase in the rate of CHOP transcription and an increase in the CHOP mRNA stability. Using a transient expression assay, we show that a promoter fragment, when linked to a reporter gene, is sufficient to mediate the regulation of CHOP expression by leucine starvation in HeLa cells. In addition, we found that decreasing amino acid concentration by itself can induce CHOP expression independently of a cellular stress due to protein synthesis inhibition. Moreover, CHOP expression is induced at leucine concentrations in the range of those observed in blood of protein-restricted animals suggesting that amino acids can participate, in concert with hormones, in the regulation of gene expression.

Multivariate analysis of pathophysiological factors in reflux oesophagitis.

BACKGROUND: Reflux oesophagitis is considered a multifactorial disease, but the respective roles of the main factors involved in its pathophysiology have not been clearly established. AIMS: To attempt to assign these roles by means of a multivariate logistic regression analysis of the main parameters associated with reflux oesophagitis. PATIENTS: Eighty seven patients with gastro-oesophageal reflux disease were studied: 41 without oesophagitis and 46 with reflux oesophagitis grade 1 to 3. METHODS: (1) Monovariate comparison of patients' characteristics and of parameters derived from in hospital 24 hour oesophageal pH monitoring, oesophageal manometry, double isotope gastric emptying studies, and basal and pentagastrin stimulated gastric acid and pepsin output determinations, between patients with and without oesophagitis. (2) Multivariate logistic regression analysis including the parameters significant in the monovariate analysis. RESULTS: Among the 16 significant parameters from monovariate analysis, three significant independent parameters were identified by multivariate logistic regression analysis: number of refluxes lasting more than five minutes, reflecting oesophageal acid clearance (p = 0.002); basal lower oesophageal sphincter pressure (p = 0.008); and peak acid output (p = 0.012). These three parameters were not correlated with each other. The multivariate model was highly discriminant (correct classification of 81.3% of the cases (95% confidence intervals 0.723, 0.903). Risk for oesophagitis increased as a function of the tercile threshold values of the three parameters. Odds ratios of the three parameters for oesophagitis risk were similar, regardless of whether they were calculated when the patients were compared as a function of oesophagitis grade or the presence or absence of oesophagitis. CONCLUSIONS: This multivariate approach adds evidence that impaired oesophageal acid clearance and hypotonic lower oesophageal sphincter are the two major independent pathophysiological factors of oesophagitis, but also showed that the acid secretion level is an independent pathophysiological factor.