Intestinal microbiota contributes to individual susceptibility to alcoholic liver disease.

OBJECTIVE: There is substantial inter-individual diversity in the susceptibility of alcoholics to liver injury. Alterations of intestinal microbiota (IM) have been reported in alcoholic liver disease (ALD), but the extent to which they are merely a consequence or a cause is unknown. We aimed to demonstrate that a specific dysbiosis contributes to the development of alcoholic hepatitis (AH). DESIGN: We humanised germ-free and conventional mice using human IM transplant from alcoholic patients with or without AH. The consequences on alcohol-fed recipient mice were studied. RESULTS: A specific dysbiosis was associated with ALD severity in patients. Mice harbouring the IM from a patient with severe AH (sAH) developed more severe liver inflammation with an increased number of liver T lymphocyte subsets and Natural Killer T (NKT) lymphocytes, higher liver necrosis, greater intestinal permeability and higher translocation of bacteria than mice harbouring the IM from an alcoholic patient without AH (noAH). Similarly, CD45+ lymphocyte subsets were increased in visceral adipose tissue, and CD4(+)T and NKT lymphocytes in mesenteric lymph nodes. The IM associated with sAH and noAH could be distinguished by differences in bacterial abundance and composition. Key deleterious species were associated with sAH while the Faecalibacterium genus was associated with noAH. Ursodeoxycholic acid was more abundant in faeces from noAH mice. Additionally, in conventional mice humanised with the IM from an sAH patient, a second subsequent transfer of IM from an noAH patient improved alcohol-induced liver lesions. CONCLUSIONS: Individual susceptibility to ALD is substantially driven by IM. It may, therefore, be possible to prevent and manage ALD by IM manipulation.

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.

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 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.

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.

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.

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.