Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens.

Inhibition of DYRK1A kinase, produced by chromosome 21 and consequently overproduced in trisomy 21 subjects, has been suggested as a therapeutic approach to treating the cognitive deficiencies observed in Down syndrome (DS). We now report the synthesis and potent DYRK1A inhibitory activities of fluoro derivatives of 3,5-di(polyhydroxyaryl)-7-azaindoles (F-DANDYs). One of these compounds (3-(4-fluorophenyl)-5-(3,4-dihydroxyphenyl)-1H-pyrrolo[2,3-b]pyridine, 5a) was selected for in vivo studies of cognitive rescuing effects in a standard mouse model of DS (Ts65Dn line). Using the Morris water maze task, Ts65Dn mice treated i.p. with 20 mg/kg of 5a performed significantly better than Ts65Dn mice treated with placebo, confirming the promnesiant effect of 5a in the trisomic mice. Overall, these results demonstrate for the first time that selective and competitive inhibition of DYRK1A kinase by the F-DANDY derivative 5a may provide a viable treatment strategy for combating the memory and learning deficiencies encountered in DS.

Abdominal aortic aneurysm (AAA) is an age-associated disease characterized by chronic inflammation, vascular cell apoptosis and metalloproteinase-mediated extracellular matrix degradation. Despite considerable progress in identifying targets involved in these processes, therapeutic approaches aiming to reduce aneurysm growth and rupture are still scarce. Indoleamine 2-3 dioxygenase 1 (IDO) is the first and rate-limiting enzyme involved in the conversion of tryptophan (Trp) into kynurenine (Kyn) pathway. In this study, we investigated the role of IDO in two different models of AAA in mice.

De novo pyrimidine biosynthesis is a key metabolic pathway involved in multiple biosynthetic processes. Here, we identified an original series of 3-(1H-indol-3-yl)-2,3-dihydro-4H-furo[3,2-c]chromen-4-one derivatives as a new class of pyrimidine biosynthesis inhibitors formed by two edge-fused polycyclic moieties. We show that identified compounds exhibit broad-spectrum antiviral activity and immunostimulatory properties, in line with recent reports linking de novo pyrimidine biosynthesis with innate defense mechanisms against viruses. Most importantly, we establish that pyrimidine deprivation can amplify the production of both type I and type III interferons by cells stimulated with retinoic acid-inducible gene 1 (RIG-I) ligands. Altogether, our results further expand the current panel of pyrimidine biosynthesis inhibitors and illustrate how the production of antiviral interferons is tightly coupled to this metabolic pathway. Functional and structural similarities between this new chemical series and dicoumarol, which was reported before to inhibit pyrimidine biosynthesis at the dihydroorotate dehydrogenase (DHODH) step, are discussed.

Hypothalamic lipotoxicity has been shown to induce central insulin resistance and dysregulation of glucose homeostasis; nevertheless, elucidation of the regulatory mechanisms remains incomplete. Here, we aimed to determine the role of de novo ceramide synthesis in hypothalamus on the onset of central insulin resistance and the dysregulation of glucose homeostasis induced by obesity.

Fatty acid biosynthesis (FASII) enzymes are considered valid targets for antimicrobial drug development against the human pathogen Staphylococcus aureus However, incorporation of host fatty acids confers FASII antibiotic adaptation that compromises prospective treatments. S. aureus adapts to FASII inhibitors by first entering a nonreplicative latency period, followed by outgrowth. Here, we used transcriptional fusions and direct metabolite measurements to investigate the factors that dictate the duration of latency prior to outgrowth. We show that stringent response induction leads to repression of FASII and phospholipid synthesis genes. (p)ppGpp induction inhibits synthesis of malonyl-CoA, a molecule that derepresses FapR, a key regulator of FASII and phospholipid synthesis. Anti-FASII treatment also triggers transient expression of (p)ppGpp-regulated genes during the anti-FASII latency phase, with concomitant repression of FapR regulon expression. These effects are reversed upon outgrowth. GTP depletion, a known consequence of the stringent response, also occurs during FASII latency, and is proposed as the common signal linking these responses. We next showed that anti-FASII treatment shifts malonyl-CoA distribution between its interactants FapR and FabD, toward FapR, increasing expression of the phospholipid synthesis genes plsX and plsC during outgrowth. We conclude that components of the stringent response dictate malonyl-CoA availability in S. aureus FASII regulation, and contribute to latency prior to anti-FASII-adapted outgrowth. A combinatory approach, coupling a (p)ppGpp inducer and an anti-FASII, blocks S. aureus outgrowth, opening perspectives for bi-therapy treatment.IMPORTANCEStaphylococcus aureus is a major human bacterial pathogen for which new inhibitors are urgently needed. Antibiotic development has centered on the fatty acid synthesis (FASII) pathway, which provides the building blocks for bacterial membrane phospholipids. However, S. aureus overcomes FASII inhibition and adapts to anti-FASII by using exogenous fatty acids that are abundant in host environments. This adaptation mechanism comprises a transient latency period followed by bacterial outgrowth. Here, we use metabolite sensors and promoter reporters to show that responses to stringent conditions and to FASII inhibition intersect, in that both involve GTP and malonyl-CoA. These two signaling molecules contribute to modulating the duration of latency prior to S. aureus adaptation outgrowth. We exploit these novel findings to propose a bi-therapy treatment against staphylococcal infections.

Variations in plasma fatty acid (FA) concentrations are detected by FA sensing neurons in specific brain areas such as the hypothalamus. These neurons play a physiological role in the control of food intake and the regulation of hepatic glucose production. Le Foll et al. previously showed in vitro that at least 50% of the FA sensing in ventromedial hypothalamic (VMH) neurons is attributable to the interaction of long chain FA with FA translocase/CD36 (CD36). The present work assessed whether in vivo effects of hypothalamic FA sensing might be partly mediated by CD36 or intracellular events such as acylCoA synthesis or β-oxidation. To that end, a catheter was implanted in the carotid artery toward the brain in male Wistar rats. After 1 wk recovery, animals were food-deprived for 5 h, then 10 min infusions of triglyceride emulsion, Intralipid +/- heparin (IL, IL(H), respectively) or saline/heparin (SH) were carried out and food intake was assessed over the next 5 h. Experimental groups included: 1) Rats previously injected in ventromedian nucleus (VMN) with shRNA against CD36 or scrambled RNA; 2) Etomoxir (CPT1 inhibitor) or saline co-infused with IL(H)/S(H); and 3) Triacsin C (acylCoA synthase inhibitor) or saline co-infused with IL(H)/S(H). IL(H) significantly lowered food intake during refeeding compared to S(H) (p<0.001). Five hours after refeeding, etomoxir did not affect this inhibitory effect of IL(H) on food intake while VMN CD36 depletion totally prevented it. Triacsin C also prevented IL(H) effects on food intake. In conclusion, the effect of FA to inhibit food intake is dependent on VMN CD36 and acylCoA synthesis but does not required FA oxidation.

Down syndrome is a common genetic disorder caused by trisomy of chromosome 21. Brain development in affected foetuses might be improved through prenatal treatment. One potential target is DYRK1A, a multifunctional kinase encoded by chromosome 21 that, when overexpressed, alters neuronal excitation-inhibition balance and increases GAD67 interneuron density. We used a green tea extract enriched in EGCG to inhibit DYRK1A function only during gestation of transgenic mice overexpressing Dyrk1a (mBACtgDyrk1a). Adult mice treated prenatally displayed reduced levels of inhibitory markers, restored VGAT1/VGLUT1 balance, and rescued density of GAD67 interneurons. Similar results for gabaergic and glutamatergic markers and interneuron density were obtained in Dp(16)1Yey mice, trisomic for 140 chromosome 21 orthologs; thus, prenatal EGCG exhibits efficacy in a more complex DS model. Finally, cognitive and behaviour testing showed that adult Dp(16)1Yey mice treated prenatally had improved novel object recognition memory but do not show improvement with Y maze paradigm. These findings provide empirical support for a prenatal intervention that targets specific neural circuitries.

Secretion of 17-β-estradiol (E2) by human granulosa cells can be disrupted by various environmental toxicants. In the current study, we investigated whether carbon black nanoparticles (CB NPs) affect the steroidogenic activity of cultured human granulosa cells. The human granulosa cell line KGN and granulosa cells from patients undergoing in vitro fertilization were treated with increasing concentrations of CB NPs (1 to 100 µg/mL) together or not with follicle-stimulating hormone (FSH). We observed that CB NPs are internalized in KGN cells without affecting cell viability. CB NPs could be localized in the cytoplasm, within mitochondria and in association with the outer face of the endoplasmic reticulum membrane. In both cell types, CB NPs reduced in a dose-dependent manner the activity of aromatase enzyme, as reflected by a decrease in E2 secretion. A significant decrease was observed in response to CB NPs concentrations from 25 and 50 µg/mL in KGN cell line and primary cultures, respectively. Furthermore, CB NPs decreased aromatase protein levels in both cells and reduced aromatase transcript levels in KGN cells. CB NPs rapidly activated extracellular signal-regulated kinase 1 and 2 in KGN cells and pharmacological inhibition of this signaling pathway using PD 98059 significantly attenuated the inhibitory effects of CB NPs on CYP19A1 gene expression and aromatase activity. CB NPs also inhibited the stimulatory effect of FSH on aromatase expression and activity. Altogether, our study on cultured ovarian granulosa cells reveals that CB NPs decrease estrogens production and highlights possible detrimental effect of these common NPs on female reproductive health.

Hyperhomocysteinemia, characterized by high plasma homocysteine levels, is recognized as an independent risk factor for cardiovascular diseases. The increased synthesis of homocysteine, a product of methionine metabolism involving B vitamins, and its slower intracellular utilization cause increased flux into the blood. Plasma homocysteine level is an important reflection of hepatic methionine metabolism and the rate of processes modified by B vitamins as well as different enzyme activity. Lowering homocysteine might offer therapeutic benefits. However, approximately 50% of hyperhomocysteinemic patients due to cystathionine-beta-synthase deficiency are biochemically responsive to pharmacological doses of B vitamins. Therefore, effective treatments to reduce homocysteine levels are needed, and gene therapy could provide a novel approach. We recently showed that hepatic expression of DYRK1A, a serine/threonine kinase, is negatively correlated with plasma homocysteine levels in cystathionine-beta-synthase deficient mice, a mouse model of hyperhomocysteinemia. Therefore, Dyrk1a is a good candidate for gene therapy to normalize homocysteine levels. We then used an adenoviral construct designed to restrict expression of DYRK1A to hepatocytes, and found decreased plasma homocysteine levels after hepatocyte-specific Dyrk1a gene transfer in hyperhomocysteinemic mice. The elevation of pyridoxal phosphate was consistent with the increase in cystathionine-beta-synthase activity. Commensurate with the decreased plasma homocysteine levels, targeted hepatic expression of DYRK1A resulted in elevated plasma paraoxonase-1 activity and apolipoprotein A-I levels, and rescued the Akt/GSK3 signaling pathways in aorta of mice, which can prevent homocysteine-induced endothelial dysfunction. These results demonstrate that hepatocyte-restricted Dyrk1a gene transfer can offer a useful therapeutic targets for the development of new selective homocysteine lowering therapy.

The aim of our study was to test the hypothesis that administration of Regenerating islet-derived protein 3α (Reg3α), a protein described as having protective effects against oxidative stress and anti-inflammatory activity, could participate in the control of glucose homeostasis and potentially be a new target of interest in the treatment of type 2 diabetes. To that end the recombinant human Reg3α protein was administered for one month in insulin-resistant mice fed high fat diet. We performed glucose and insulin tolerance tests, assayed circulating chemokines in plasma and measured glucose uptake in insulin sensitive tissues. We evidenced an increase in insulin sensitivity during an oral glucose tolerance test in ALF-5755 treated mice vs controls and decreased the pro-inflammatory cytokine C-X-C Motif Chemokine Ligand 5 (CXCL5). We also demonstrated an increase in glucose uptake in skeletal muscle. Finally, correlation studies using human and mouse muscle biopsies showed negative correlation between intramuscular Reg3α mRNA expression (or its murine isoform Reg3γ) and insulin resistance. Thus, we have established the proof of concept that Reg3α could be a novel molecule of interest in the treatment of T2D by increasing insulin sensitivity via a skeletal muscle effect.

Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein.

Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. It is considered as an "orphan" protein as few data are available on its physiological function(s) and spectral characteristics. Its only known substrates reported so far are unsaturated fatty acids such as arachidonic acid (AA), and, more recently, N-arachidonoylserotonin (AS) and some xenobiotics related to debrisoquine (Deb) and terfenadine.

Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer.

Protein tyrosine phosphatases (PTPs) are involved in numerous signaling pathways and dysfunctions of certain of these enzymes have been linked to several human diseases including cancer and autoimmune diseases. PTPN2 is a PTP mainly expressed in hematopoietic cells and involved in growth factor and JAK/STAT signaling pathways. Loss of function analyses in patients with mutation/deletion of the PTPN2 gene and knock-out mouse models indicate that PTPN2 acts as a tumor suppressor in T-cell malignancies and as a regulator of inflammation and immunity. The use of sensitive and quantitative assays is of prime importance to better characterize the biochemical properties of PTPN2 and its biological roles. We report a highly sensitive non-radioactive assay that allows the measurement of the activity of purified PTPN2 and of endogenous PTPN2 immunoprecipitated on agarose beads. The assay relies on separation and quantitation by reverse-phase ultra fast liquid chromatography (RP-UFLC) of a fluorescein-labeled phosphotyrosine peptide substrate derived from the sequence of STAT1. The applicability and reliability of this approach is supported by kinetic and mechanistic studies using PTP inhibitors. More broadly, our PTPN2 assay provides the basis for the design of flexible methods for the measurement of other PTPs.

SLC30A8 encodes a zinc transporter ZnT8 largely restricted to pancreatic islet β- and α-cells, and responsible for zinc accumulation into secretory granules. Although common SLC30A8 variants, believed to reduce ZnT8 activity, increase type 2 diabetes risk in humans, rare inactivating mutations are protective. To investigate the role of Slc30a8 in the control of glucagon secretion, Slc30a8 was inactivated selectively in α-cells by crossing mice with alleles floxed at exon 1 to animals expressing Cre recombinase under the pre-proglucagon promoter. Further crossing to Rosa26:tdRFP mice, and sorting of RFP(+): glucagon(+) cells from KO mice, revealed recombination in ∼ 30% of α-cells, of which ∼ 50% were ZnT8-negative (14 ± 1.8% of all α-cells). Although glucose and insulin tolerance were normal, female αZnT8KO mice required lower glucose infusion rates during hypoglycemic clamps and displayed enhanced glucagon release (p < 0.001) versus WT mice. Correspondingly, islets isolated from αZnT8KO mice secreted more glucagon at 1 mm glucose, but not 17 mm glucose, than WT controls (n = 5; p = 0.008). Although the expression of other ZnT family members was unchanged, cytoplasmic (n = 4 mice per genotype; p < 0.0001) and granular (n = 3, p < 0.01) free Zn(2+) levels were significantly lower in KO α-cells versus control cells. In response to low glucose, the amplitude and frequency of intracellular Ca(2+) increases were unchanged in α-cells of αZnT8KO KO mice. ZnT8 is thus important in a subset of α-cells for normal responses to hypoglycemia and acts via Ca(2+)-independent mechanisms.

Reproductive function is highly dependent on nutritional input. We recently provided evidence that the unsaturated ω6 fatty acid (FA), linoleic acid (linoleic), interferes with transcription and secretion of the gonadotropin LH, highlighting the existence of a lipid sensing in pituitary gonadotropes. Here, we show, using a combination of in vivo and in vitro models, that linoleic differentially regulates Lhb and Fshb expression. Central exposure of rats to linoleic over 7 days was associated with increase of Lhb but not Fshb transcript levels. Consistently, exposure of rat pituitary cells or LβT2 cells to linoleic increased Lhb, whereas it dramatically decreased Fshb transcript levels without affecting its stability. This effect was also induced by ω9 and ω3-polyunsaturated FA but not by saturated palmitic acid. Analysis of the underlying mechanisms in LβT2 cells using small interfering RNA revealed that early growth response protein 1 mediates linoleic stimulation of Lhb expression. Furthermore, we demonstrated that linoleic counteracts activin and bone morphogenetic protein-2 stimulation of Fshb expression. Using Western blotting and Smad-responsive reporter gene assays, linoleic was shown to decrease basal Smad2/3 phosphorylation levels as well as activin- and bone morphogenetic protein-2-dependent activation of Smad, uncovering a new FA-sensitive signaling cascade. Finally, the protein phosphatase magnesium-dependent 1A was shown to mediate linoleic inhibition of basal Smad phosphorylation and Fshb expression, identifying protein phosphatase magnesium-dependent 1A as a new target of FA in gonadotropes. Altogether, this study provides a novel mechanism by which FAs target gene expression and underlines the relevant role of pituitary gonadotropes in mediating the effects of nutritional FA on reproductive function.

Intracellular bacterial pathogens have developed a variety of strategies to avoid degradation by the host innate immune defense mechanisms triggered upon phagocytocis. Upon infection of mammalian host cells, the intracellular pathogen Francisella replicates exclusively in the cytosolic compartment. Hence, its ability to escape rapidly from the phagosomal compartment is critical for its pathogenicity. Here, we show for the first time that a glutamate transporter of Francisella (here designated GadC) is critical for oxidative stress defense in the phagosome, thus impairing intra-macrophage multiplication and virulence in the mouse model. The gadC mutant failed to efficiently neutralize the production of reactive oxygen species. Remarkably, virulence of the gadC mutant was partially restored in mice defective in NADPH oxidase activity. The data presented highlight links between glutamate uptake, oxidative stress defense, the tricarboxylic acid cycle and phagosomal escape. This is the first report establishing the role of an amino acid transporter in the early stage of the Francisella intracellular lifecycle.

The aim of the present study was to evaluate the potential antidiabetic effects of two-component drug Subetta and its components (release-active dilutions of antibodies to β -subunit insulin receptor (RAD of Abs to β -InsR) and to endothelial nitric oxide synthase (RAD of Abs to eNOS)) in Goto-Kakizaki (Paris colony) (GK/Par) diabetic rats. Subetta was administered orally for 28 days once daily (5 mL/kg) and compared to its two components (2.5 mL/kg), Rosiglitazone (5 mg/kg), and vehicle (5 mL water/kg). At day 28, fasting plasma glucose levels were significantly decreased only in Subetta and Rosiglitazone groups as compared to vehicle (P < 0.01): 147 ± 4 mg/dL and 145 ± 4 mg/dL and 165 ± 4 mg/dL, respectively. The data of glucose tolerance test showed that Subetta and RAD of Abs to β -InsR (similar to Rosiglitazone) prevented significantly (P < 0.01) the age-related spontaneous deterioration of glucose tolerance as seen in the control group. Subetta and RAD of Abs to β -InsR did not significantly modify the glucose-induced insulin secretion. Chronic administration of Subetta and RAD of Abs to β -InsR improves glucose control, to an extent similar to that of Rosiglitazone. We hypothesize that Subetta and RAD of Abs to β -InsR mostly act via an insulin-sensitizing effect upon target tissues.

Human arylamine N-acetyltransferase 1 (hNAT1) has become an attractive potential biomarker for estrogen-receptor-positive breast cancers. We describe here the mechanism of action of a selective non-covalent colorimetric biosensor for the recognition of hNAT1 and its murine homologue, mNat2, over their respective isoenzymes, leading to new opportunities in diagnosis. On interaction with the enzyme, the naphthoquinone probe undergoes an instantaneous and striking visible color change from red to blue. Spectroscopic, chemical, molecular modelling and biochemical studies reported here show that the color change is mediated by selective recognition between the conjugate base of the sulfonamide group within the probe and the conjugate acid of the arginine residue within the active site of both hNAT1 and mNat2. This represents a new mechanism for selective biomarker sensing and may be exploited as a general approach to the specific detection of biomarkers in disease.