The peroxisome proliferator-activated receptor alpha regulates amino acid metabolism.

The peroxisome proliferator-activated receptor alpha is a ligand-activated transcription factor that plays an important role in the regulation of lipid homeostasis. PPARalpha mediates the effects of fibrates, which are potent hypolipidemic drugs, on gene expression. To better understand the biological effects of fibrates and PPARalpha, we searched for genes regulated by PPARalpha using oligonucleotide microarray and subtractive hybridization. By comparing liver RNA from wild-type and PPARalpha null mice, it was found that PPARalpha decreases the mRNA expression of enzymes involved in the metabolism of amino acids. Further analysis by Northern blot revealed that PPARalpha influences the expression of several genes involved in trans- and deamination of amino acids, and urea synthesis. Direct activation of PPARalpha using the synthetic PPARalpha ligand WY14643 decreased mRNA levels of these genes, suggesting that PPARalpha is directly implicated in the regulation of their expression. Consistent with these data, plasma urea concentrations are modulated by PPARalpha in vivo. It is concluded that in addition to oxidation of fatty acids, PPARalpha also regulates metabolism of amino acids in liver, indicating that PPARalpha is a key controller of intermediary metabolism during fasting.

Positive regulation of the peroxisomal beta-oxidation pathway by fatty acids through activation of peroxisome proliferator-activated receptors (PPAR).

Peroxisome proliferators regulate the transcription of genes by activating ligand-dependent transcription factors, which, due to their structure and function, can be assigned to the superfamily of nuclear hormone receptors. Three such peroxisome proliferator-activated receptors (PPAR alpha, beta, and gamma) have been cloned in Xenopus laevis. Their mRNAs are expressed differentially; xPPAR alpha and beta but not xPPAR gamma are expressed in oocytes and embryos. In the adult, expression of xPPAR alpha and beta appears to be ubiquitous, and xPPAR gamma is mainly observed in adipose tissue and kidney. Immunocytochemical analysis revealed that PPARs are nuclear proteins, and that their cytoplasmic-nuclear translocation is independent of exogenous activators. A target gene of PPARs is the gene encoding acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in the peroxisomal beta-oxidation of fatty acids. A peroxisome proliferator response element (PPRE), to which PPARs bind, has been identified within the promoter of the ACO gene. Besides the known xenobiotic activators of PPARs, such as hypolipidemic drugs, natural activators have been identified. Polyunsaturated fatty acids at physiological concentrations are efficient activators of PPARs, and 5,8,11,14-eicosatetraynoic acid (ETYA), which is the alkyne homolog of arachidonic acid, is the most potent activator of xPPAR alpha described to date. Taken together, our data suggest that PPARs have an important role in lipid metabolism.

Regulation of phosphate starvation responses in plants: signaling players and cross-talks.

Phosphate (Pi) availability is a major factor limiting growth, development, and productivity of plants. In both ecological and agricultural contexts, plants often grow in soils with low soluble phosphate content. Plants respond to this situation by a series of developmental and metabolic adaptations that are aimed at increasing the acquisition of this vital nutrient from the soil, as well as to sustain plant growth and survival. The development of a comprehensive understanding of how plants sense phosphate deficiency and coordinate the responses via signaling pathways has become of major interest, and a number of signaling players and networks have begun to surface for the regulation of the phosphate-deficiency response. In practice, application of such knowledge to improve plant Pi nutrition is hindered by complex cross-talks, which are emerging in the face of new data, such as the coordination of the phosphate-deficiency signaling networks with those involved with hormones, photo-assimilates (sugar), as well as with the homeostasis of other ions, such as iron. In this review, we focus on these cross-talks and on recent progress in discovering new signaling players involved in the Pi-starvation responses, such as proteins having SPX domains.

Up-regulation of the levels of expression and function of a constitutively active mutant of the hamster alpha1B-adrenoceptor by ligands that act as inverse agonists.

The alpha1-adrenergic agonist phenylephrine stimulated phospholipase D (PLD) activity in Rat 1 fibroblasts transfected to express either the wild-type hamster alpha1B-adrenoceptor or a constitutively active mutant (CAM) form of this receptor. The EC50 for agonist stimulation of PLD activity was substantially lower at the CAM receptor than at the wild-type receptor as previously noted for phenylephrine stimulation of phosphoinositidase C activity. Sustained treatment of cells expressing the CAM alpha1B-adrenoceptor with phentolamine resulted in a marked up-regulation in levels of this receptor with half-maximal effects produced within 24 h and with an EC50 of approx. 40 nM. Such an up-regulation could be produced with a range of other ligands generally viewed as alpha1-adrenoceptor antagonists but equivalent treatment of cells expressing the wild-type alpha1B-adrenoceptor was unable to mimic these effects. After sustained treatment of the CAM alpha1B-adrenoceptor expressing cells with phentolamine, basal PLD activity was increased and phenylephrine was now able to stimulate PLD activity to greater levels than in vehicle-treated CAM alpha1B-adrenoceptor-expressing cells. The EC50 for phenylephrine stimulation of PLD activity was not altered, however, by phentolamine pretreatment and the associated up-regulation of the receptor. After phentolamine-induced up-regulation of basal PLD activity, a range of alpha1-antagonists were shown to possess the characteristics of inverse agonists of the CAM alpha1B-adrenoceptor as they were able to substantially decrease the elevated basal PLD activity.

Role and regulation of polyphosphate in leishmania parasites

Le protozoaire unicellulaire Leishmania est l'agent responsable de la leishmaniose, une maladie parasitaire humaine qui se manifeste par des lésions de la peau, se résolvant le plus souvent spontanément, jusqu'à des lésions viscérales fatales. Le parasite est transmis de l'insecte à l'hôte mammifère lors d'un repas sanguin de la mouche des sables et y réside respectivement sous formes extra- et intracellulaires. On estime que cette maladie touche environ 12 millions de personnes dans 98 pays. Etant donné que les médicaments disponibles à ce jour sont faiblement efficaces et/ou hautement toxiques, il est indispensable de consolider les connaissances sur le fonctionnement et la survie du parasite pour pouvoir développer de nouvelles stratégies de traitements et de préventions. Tous les organismes vivants, dont Leishmania, contiennent du polyphopshate (polyP). Cette molécule chargée négativement est constituée de trois jusqu'à plusieurs centaines de résidus de phosphates reliées par des liaisons à haute énergie. Le polyP sert donc de source d'énergie et de réservoir de phosphate; dans certaines espèces, il joue aussi un rôle dans l'adaptation au stress et la virulence de pathogènes. Ceci nous a amené à étudier le rôle du polyP dans le parasite Leishmania. L'enzyme responsable de la synthèse de polyP a été identifié récemment dans la levure : il s'agit de la chaperone de transport vacuolaire 4 (Vtc4). Nous avons identifié un homologue de Vtc4 chez les Trypanosomatidae, et avons donc décidé d'examiner sa fonction dans le métabolisme du polyP chez Leishmania. En éliminant l'expression de Vtc4 chez L. major et L. guyanensis, nous avons pu démontrer qu'il est indispensable pour la production de polyP chez Leishmania. De plus, nous avons constaté que ces parasites possèdent des chaînes de polyP allant de trois jusqu'à environ 300 résidus de phosphate. Le taux de polyP dans la cellule est précisément régulé et varie entre un très haut niveau durant la phase proliférative des promastigotes à un niveau bas en phase stationnaire tardive, alors que l'expression de Vtc4p reste stable. Dans les amastigotes intracellulaires, seulement des petites quantités de polyP et de Vtc4p sont détectées. En outre, l'absence de Vc4p et de polyP n'a pas d'effet significatif sur les infections in vivo de souris, ce qui indique que le polyP n'est pas nécessaire au développement de la leishmaniose. Ceci suggère que Vtc4p n 'est pas une bonne cible pour le développement de nouveaux traitements contre Leishmania. "Néanmoins, la présence du polyP favorise fortement la survie du parasite suite à un choc de température (37°C) et aide ainsi à sa persistance intracellulaire pendant les premiers jours d'infection de macrophages. En résumé, nos résultats indiquent que si le polyP a peu d'importance pendant l'infection et le développement de la leishmaniose chez la souris, il est par contre crucial pour l'adaptation à des situations de stress comme l'augmentation de la température. Le fait que le polyP a été conservé dans tous les organismes durant l'évolution suggère toutefois que cette molécule joue un rôle fondamental. Etant donné que l'absence de polyP n'a pas d'effet sur la survie des amastigotes, il pourrait être plus important dans la forme promastigote infectant la mouche des sables. - The unicellular protozoan parasite Leishmania is the causative agent of the human disease leishmaniasis, which can range from self-healing skin lesions to fatal visceral lesions. The parasite is transmitted from the insect vector to the mammalian host when the sand fly takes its blood meal and exists in an extra- and an intracellular form, respectively. The disease is estimated to affect 12 million people in 98 countries and currently available drug treatments are of relatively low potency and/or high toxicity. Thus, investigating parasite survival mechanisms and parasite adaptation to the two host environments contributes to the general understanding of Leishmania propagation and might therefore help to develop future treatments or preventions. All living cells, including Leishmania, contain a negatively charged polymer of a few up to several hundred phosphate residues. These so-called polyphosphates (polyPs) serve as an energy source and phosphate reservoir. In some organisms, polyP is also involved in adaptation to stresses and virulence of pathogens. Therefore we were interested in investigating the importance of polyP in Leishmania parasites. Recently, an eukaryotic enzyme responsible for polyP synthesis has been identified as the vacuolar transporter chaperone 4 (Vtc4) in yeast. We, and others, found a Vtc4 homologue in trypanosomatids and decided to examine its potential function in polyP metabolism. By generating VTC4 knock-out cell lines in L. major and Vtc4 knock-down cell lines in L. guyanensis, we were able to demonstrate that Vtc4p is responsible for the total amount of cellular polyP. We also observed that Leishmania polyP chain length ranges from a few up to around 300 residues and that its level is tightly regulated. PolyP abundance is highest during the logarithmic proliferating phase of promastigotes and decreases in the stationary phase, while Vtc4 protein expression remains stable during both phases. In the intracellular amastigote form, only low amounts of polyP and Vtc4p were detectable. Furthermore, absence of Vtc4p and polyP did not have a significant effect on in vivo mouse infections, indicating that polyP is not necessary for Leishmania disease progression. This suggests that Vtc4p would be a poor drug target against Leishmania infection. However, presence of the polymer strongly supported parasite survival during heat shock (37°C) and thereby promoted intracellular persistence during the first days of macrophage infections. Taken together, we found that polyP has little importance in Leishmania {in vivo) infection but that it plays a crucial role during adaptation to stress, such as heat shock. Given that polyP has been preciously conserved in all organisms during evolution it seems to play a fundamental role. Since absence of polyP does not affect amastigote survival, it might be significant for promastigote existence in the sand fly vector.

Peroxisome proliferator activated receptors: transcriptional regulators of adipogenesis, lipid metabolism and more....

The recent discovery of lipid-activatable transcription factors that regulate the genes controlling lipid metabolism and adipogenesis has provided insight into the way that organisms sense and respond to lipid levels. Identification of the signaling pathways in which these receptors are involved will help us to understand the control of energy balance and the molecular defects underlying its disorders.

Peroxisome proliferator-activated receptors and lipid metabolism.

PPARs are nuclear hormone receptors which, like the retinoid, thyroid hormone, vitamin D, and steroid hormone receptors, are ligand-activated transcription factors mediating the hormonal control of gene expression. Two lines of evidence indicate that PPARs have an important function in fatty acid metabolism. First, PPARs are activated by hypolipidemic drugs and physiological concentrations of fatty acids, and second, PPARs control the peroxisomal beta-oxidation pathway of fatty acids through transcriptional induction of the gene encoding the acyl-CoA oxidase (ACO), which is the rate-limiting enzyme of the pathway. Furthermore, the PPAR signaling pathway appears to converge with the 9-cis retinoic acid receptor (RXR) signaling pathway in the regulation of the ACO gene because heterodimerization between PPAR and RXR is essential for in vitro binding to the PPRE and because the strongest stimulation of this gene is observed when both receptors are exposed simultaneously to their activators. Thus, it appears that PPARs are involved in the 9-cis retinoic acid signaling pathway and that they play a pivotal role in the hormonal control of lipid metabolism.

Three small RNAs jointly ensure secondary metabolism and biocontrol in Pseudomonas fluorescens CHA0.

In many Gram-negative bacteria, the GacS/GacA two-component system positively controls the expression of extracellular products or storage compounds. In the plant-beneficial rhizosphere bacterium Pseudomonas fluorescens CHA0, the GacS/GacA system is essential for the production of antibiotic compounds and hence for biological control of root-pathogenic fungi. The small (119-nt) RNA RsmX discovered in this study, together with RsmY and RsmZ, forms a triad of GacA-dependent small RNAs, which sequester the RNA-binding proteins RsmA and RsmE and thereby antagonize translational repression exerted by these proteins in strain CHA0. This small RNA triad was found to be both necessary and sufficient for posttranscriptional derepression of biocontrol factors and for protection of cucumber from Pythium ultimum. The same three small RNAs also positively regulated swarming motility and the synthesis of a quorum-sensing signal, which is unrelated to N-acyl-homoserine lactones, and which autoinduces the Gac/Rsm cascade. Expression of RsmX and RsmY increased in parallel throughout cell growth, whereas RsmZ was produced during the late growth phase. This differential expression is assumed to facilitate fine tuning of GacS/A-controlled cell population density-dependent regulation in P. fluorescens.

Genetic and hormonal regulation of cambial development.

The stems and roots of most dicot plants increase in diameter by radial growth, due to the activity of secondary meristems. Two types of meristems function in secondary plant body formation: the vascular cambium, which gives rise to secondary xylem and phloem, and the cork cambium, which produces a bark layer that replaces the epidermis and protects the plant stem from mechanical damage and pathogens. Cambial development, the initiation and activity of the vascular cambium, leads to an accumulation of wood, the secondary xylem tissue. The thick, cellulose-rich cell walls of wood provide a source of cellulose and have the potential to be used as a raw material for sustainable and renewable energy production. In this review, we will discuss what is known about the mechanisms regulating the cambium and secondary tissue development.

A link between FXYD3 (Mat-8)-mediated Na,K-ATPase regulation and differentiation of Caco-2 intestinal epithelial cells.

FXYD3 (Mat-8) proteins are regulators of Na,K-ATPase. In normal tissue, FXYD3 is mainly expressed in stomach and colon, but it is also overexpressed in cancer cells, suggesting a role in tumorogenesis. We show that FXYD3 silencing has no effect on cell proliferation but promotes cell apoptosis and prevents cell differentiation of human colon adenocarcinoma cells (Caco-2), which is reflected by a reduction in alkaline phosphatase and villin expression, a change in several other differentiation markers, and a decrease in transepithelial resistance. Inhibition of cell differentiation in FXYD3-deficient cells is accompanied by an increase in the apparent Na+ and K+ affinities of Na,K-ATPase, reflecting the absence of Na,K-pump regulation by FXYD3. In addition, we observe a decrease in the maximal Na,K-ATPase activity due to a decrease in its turnover number, which correlates with a change in Na,K-ATPase isozyme expression that is characteristic of cancer cells. Overall, our results suggest an important role of FXYD3 in cell differentiation of Caco-2 cells. One possibility is that FXYD3 silencing prevents proper regulation of Na,K-ATPase, which leads to perturbation of cellular Na+ and K+ homeostasis and changes in the expression of Na,K-ATPase isozymes, whose functional properties are incompatible with Caco-2 cell differentiation.

Glucose transporters in the regulation of intestinal, renal, and liver glucose fluxes.

Five functional mammalian facilitated hexose carriers (GLUTs) have been characterized by molecular cloning. By functional expression in heterologous systems, their specificity and affinity for different hexoses have been defined. There are three high-affinity transporters (GLUT-1, GLUT-3 and GLUT-4) and one low-affinity transporter (GLUT-2), and GLUT-5 is primarily a fructose carrier. Because their Michaelis constants (Km) are below the normal blood glucose concentration, the high-affinity transporters function at rates close to maximal velocity. Thus their level of cell surface expression greatly influences the rate of glucose uptake into the cells. In contrast, the rate of glucose uptake by GLUT-2 (Km = 17 mM) increases in parallel with the rise in blood glucose over the physiological concentration range. High-affinity transporters are found in almost every tissue, but their expression is higher in cells with high glycolytic activity. Glut-2, however, is found in tissues carrying large glucose fluxes, such as intestine, kidney, and liver. As an adaptive response to variations in metabolic conditions, the expression of these transporters is regulated by glucose and different hormones. Thus, because of their specific characteristics and regulated expression, the facilitated glucose transporters control fundamental aspects of glucose homeostasis. I review data pertaining to the structure and regulated expression of the glucose carriers present in intestine, kidney, and liver and discuss their role in the control of glucose flux into or out of these different tissues.

Differential regulation of the expression of two high-affinity sulfate transporters, SULTR1.1 and SULTR1.2, in Arabidopsis

The molecular mechanisms regulating the initial uptake of inorganic sulfate in plants are still largely unknown. The current model for the regulation of sulfate uptake and assimilation attributes positive and negative regulatory roles to O-acetyl-serine (O-acetyl-Ser) and glutathione, respectively. This model seems to suffer from exceptions and it has not yet been clearly validated whether intracellular O-acetyl-Ser and glutathione levels have impacts on regulation. The transcript level of the two high-affinity sulfate transporters SULTR1.1 and SULTR1.2 responsible for sulfate uptake from the soil solution was compared to the intracellular contents of O-acetyl-Ser, glutathione, and sulfate in roots of plants submitted to a wide diversity of experimental conditions. SULTR1.1 and SULTR1.2 were differentially expressed and neither of the genes was regulated in accordance with the current model. The SULTR1.1 transcript level was mainly altered in response to the sulfur-related treatments. Split-root experiments show that the expression of SULTR1.1 is locally regulated in response to sulfate starvation. In contrast, accumulation of SULTR1.2 transcripts appeared to be mainly related to metabolic demand and is controlled by photoperiod. On the basis of the new molecular insights provided in this study, we suggest that the expression of the two transporters depends on different regulatory networks. We hypothesize that interplay between SULTR1.1 and SULTR1.2 transporters could be an important mechanism to regulate sulfate content in the roots

Brain lactate metabolism in humans with subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Lactate is central for the regulation of brain metabolism and is an alternative substrate to glucose after injury. Brain lactate metabolism in patients with subarachnoid hemorrhage has not been fully elucidated. METHODS: Thirty-one subarachnoid hemorrhage patients monitored with cerebral microdialysis (CMD) and brain oxygen (PbtO(2)) were studied. Samples with elevated CMD lactate (>4 mmol/L) were matched to PbtO(2) and CMD pyruvate and categorized as hypoxic (PbtO(2) <20 mm Hg) versus nonhypoxic and hyperglycolytic (CMD pyruvate >119 μmol/L) versus nonhyperglycolytic. RESULTS: Median per patient samples with elevated CMD lactate was 54% (interquartile range, 11%-80%). Lactate elevations were more often attributable to cerebral hyperglycolysis (78%; interquartile range, 5%-98%) than brain hypoxia (11%; interquartile range, 4%-75%). Mortality was associated with increased percentage of samples with elevated lactate and brain hypoxia (28% [interquartile range 9%-95%] in nonsurvivors versus 9% [interquartile range 3%-17%] in survivors; P=0.02) and lower percentage of elevated lactate and cerebral hyperglycolysis (13% [interquartile range, 1%-87%] versus 88% [interquartile range, 27%-99%]; P=0.07). Cerebral hyperglycolytic lactate production predicted good 6-month outcome (odds ratio for modified Rankin Scale score, 0-3 1.49; CI, 1.08-2.05; P=0.016), whereas increased lactate with brain hypoxia was associated with a reduced likelihood of good outcome (OR, 0.78; CI, 0.59-1.03; P=0.08). CONCLUSIONS: Brain lactate is frequently elevated in subarachnoid hemorrhage patients, predominantly because of hyperglycolysis rather than hypoxia. A pattern of increased cerebral hyperglycolytic lactate was associated with good long-term recovery. Our data suggest that lactate may be used as an aerobic substrate by the injured human brain.