Uranium (U) is a non-essential and toxic metal for plants. In Arabidopsis thaliana plants challenged with uranyl nitrate, we showed that U was mostly (64–71% of the total) associated with the root insoluble fraction containing membrane and cell wall proteins. Therefore, to uncover new molecular mechanisms related to U stress, we used label-free quantitative proteomics to analyze the responses of the root membrane- and cell wall-enriched proteome. Of the 2,802 proteins identified, 458 showed differential accumulation (≥1.5-fold change) in response to U. Biological processes affected by U include response to stress, amino acid metabolism, and previously unexplored functions associated with membranes and the cell wall. Indeed, our analysis supports a dynamic and complex reorganization of the cell wall under U stress, including lignin and suberin synthesis, pectin modification, polysaccharide hydrolysis, and Casparian strips formation. Also, the abundance of proteins involved in vesicular trafficking and water flux was significantly altered by U stress. Measurements of root hydraulic conductivity and leaf transpiration indicated that U significantly decreased the plant's water flux. This disruption in water balance is likely due to a decrease in PIP aquaporin levels, which may serve as a protective mechanism to reduce U toxicity. Finally, the abundance of transporters and metal-binding proteins was altered, suggesting that they may be involved in regulating the fate and toxicity of U in Arabidopsis. Overall, this study highlights how U stress impacts the insoluble root proteome, shedding light on the mechanisms used by plants to mitigate U toxicity.

Uranium (U) is a naturally occurring radioelement that is chemically toxic to all living organisms. We investigated the effect of U on Arabidopsis thaliana seed germination and the early stages of seedling development. We showed that U had very little effect on Arabidopsis seed germination, with germination rates above 90% at 500µM uranyl nitrate. However, U had a drastic effect on the Arabidopsis seedling development and the root was the most U-sensitive organ, with severe growth inhibition (>75%) starting at 20µM uranyl nitrate. We showed that the two soil bacterial strains Microbacterium sp. ViU2A and Stenotrophomonas bentonitica BII-R7, which are able to tolerate high concentrations of U, strongly reduced the toxic effects of U on seedling development. Thus, in the presence of both strains, root growth inhibition was prevented up to 100µM uranyl nitrate. This protection was specific to U-tolerant soil bacteria, as Escherichia coli was unable to protect seedlings. The analysis of U distribution between Arabidopsis seedlings and soil bacteria showed that the protective effect of bacteria was due to their ability to sequester U, either by biosorption at the cell surface and/or by intracellular or extracellular biomineralization. Also, the ability of both soil bacterial strains to prevent U toxicity was much less efficient when dead bacteria were used, suggesting the involvement of metabolism-dependent biological processes in seedling protection. Accordingly, we showed that the Uranium-induced protein A (UipA) contributes to the protection of Arabidopsis seedlings by Microbacterium sp. ViU2A. This study suggests that U-tolerant soil bacteria may be useful for phytostabilization strategies of U-contaminated soils and for limiting the entry of this toxic element into crops.

Uranium (U) contamination of terrestrial and aquatic ecosystems poses a significant threat to the environment and human health due to the chemotoxicity of this actinide. The characterization of organisms that tolerate and accumulate U is crucial to decipher the mechanisms evolved to cope with the radionuclide and to propose new effective strategies for the bioremediation of U-contaminated environments. Here, we isolated a unicellular green microalga of the genus Coelastrella from U-contaminated wastewater. We showed that Coelastrella sp. PCV is much more tolerant to U than Chlamydomonas reinhardtii and Chlorella vulgaris. Coelastrella sp. PCV is able to accumulate U very rapidly and then gradually release it into the medium, behaving as an excluder to limit the toxic effects of U. The ability of Coelastrella sp. PCV to accumulate U is remarkably high, with up to 240 mg of tightly bound U per g of dry biomass. Coelastrella sp. PCV is able to grow and maintain high photosynthesis in natural metal-contaminated waters from a wetland near a reclaimed U mine. In a single one-week growth cycle, Coelastrella sp. PCV is able to capture 25-55 % of the U from the contaminated waters and shows lipid droplet

Sanguina nivaloides is the main alga forming red snowfields in high mountains and Polar Regions. It is non-cultivable. Analysis of environmental samples by X-ray tomography, focused-ion-beam scanning-electron-microscopy, physicochemical and physiological characterization reveal adaptive traits accounting for algal capacity to reside in snow. Cysts populate liquid water at the periphery of ice, are photosynthetically active, can survive for months, and are sensitive to freezing. They harbor a wrinkled plasma membrane expanding the interface with environment. Ionomic analysis supports a cell efflux of K + , and assimilation of phosphorus. Glycerolipidomic analysis confirms a phosphate limitation. The chloroplast contains thylakoids oriented in all directions, fixes carbon in a central pyrenoid and produces starch in peripheral protuberances. Analysis of cells kept in the dark shows that starch is a short-term carbon storage. The biogenesis of cytosolic droplets shows that they are loaded with triacylglycerol and carotenoids for long-term carbon storage and protection against oxidative stress.

Uranium (U) is a naturally-occurring radionuclide that is toxic to living organisms. Given that proteins are primary targets of U(VI), their identification is an essential step towards understanding the mechanisms of radionuclide toxicity, and possibly detoxification. Here, we implemented a chromatographic strategy including immobilized metal affinity chromatography to trap protein targets of uranyl in Arabidopsis thaliana. This procedure allowed the identification of 38 uranyl-binding proteins (UraBPs) from root and shoot extracts. Among them, UraBP25, previously identified as plasma membrane-associated cation-binding protein 1 (PCaP1), was further characterized as a protein interacting in vitro with U(VI) and other metals using spectroscopic and structural approaches, and in planta through analyses of the fate of U(VI) in Arabidopsis lines with altered PCaP1 gene expression. Our results showed that recombinant PCaP1 binds U(VI) in vitro with affinity in the nM range, as well as Cu(II) and Fe(III) in high proportions, and that Ca(II) competes with U(VI) for binding. U(VI) induces PCaP1 oligomerization through binding at the monomer interface, at both the N-terminal structured domain and the C-terminal flexible region. Finally, U(VI) translocation in Arabidopsis shoots was affected in pcap1 null-mutant, suggesting a role for this protein in ion trafficking in planta.

Iron is an essential micronutrient for plant growth and development. Under low iron conditions, Arabidopsis plants take up soil iron using the root iron transporter IRT1. In addition to iron, IRT1 also transports others divalent metals, including cadmium, which consequently accumulates into plant tissues and enters the food chain. IRT1 expression was shown to be regulated at the transcriptional and post-translational levels by its essential metal substrates to maximize iron uptake while limiting the accumulation of zinc, manganese, or cobalt. Here, we characterized the regulation of IRT1 by cadmium. A short-term exposure to cadmium decreased the cell surface levels of IRT1 through endocytosis and degradation, but with a lower efficiency than observed for other IRT1 metal substrates. We demonstrated that IRT1 endocytosis in response to cadmium is mediated through the direct binding of cadmium to histidine residues within the regulatory loop of IRT1. However, we revealed that the affinity of the metal sensing motif is much lower for cadmium compared to other metal substrates of IRT1. Finally, we proved that cadmium-induced IRT1 degradation takes place through ubiquitin-mediated endocytosis driven by the UBC35/36 E2 ubiquitin-conjugating enzymes and the IDF1 E3 ubiquitin ligase. Altogether, this work sheds light on the mechanisms of cadmium-mediated downregulation of IRT1 and provides an additional molecular basis for cadmium accumulation and toxicity in plants.

Uranium (U) is a non-essential and toxic element that is taken up by plants from the environment. The assimilation pathway of U is still unknown in plants. In this study, we provide several evidences that U is taken up by the roots of Arabidopsis thaliana through Ca2+-permeable cation channels. First, we showed that deprivation of Arabidopsis plants with calcium induces a 1.5-fold increase in the capacity of roots to accumulate U, suggesting that calcium deficiency promotes the radionuclide import pathway. Second, we showed that external calcium inhibits U accumulation in roots, suggesting a common route for the uptake of both cations. Third, we found that gadolinium, nifedipine and verapamil inhibit the absorption of U, suggesting that different types of Ca2+-permeable channels serve as a route for U uptake. Last, we showed that U bioaccumulation in Arabidopsis mutants deficient for the Ca2+-permeable channels MCA1 and ANN1 is decreased by 40%. This suggests that MCA1 and ANN1 contribute to the absorption of U in different zones and cell layers of the root. Together, our results describe for the first time the involvement of Ca2+-permeable cation channels in the cellular uptake of U.

Background Early hyperphagia and hypothalamic inflammation encountered after Western diet (WD) are linked to rodent propensity to obesity. Inflammation in several brain structures has been associated with gut dysbiosis. Since gut microbiota is highly sensitive to dietary changes, we hypothesised that immediate gut microbiota adaptation to WD in rats is involved in inflammation-related hypothalamic modifications. Methods We evaluated short-term impact of WD consumption (2 h, 1, 2 and 4 days) on hypothalamic metabolome and caecal microbiota composition and metabolome. Data integration analyses were performed to uncover potential relationships among these three datasets. Finally, changes in hypothalamic gene expression in absence of gut microbiota were evaluated in germ-free rats fed WD for 2 days. Results WD quickly and profoundly affected the levels of several hypothalamic metabolites, especially oxidative stress markers. In parallel, WD consumption reduced caecal microbiota diversity, modified its composition towards pro-inflammatory profile and changed caecal metabolome. Data integration identified strong correlations between gut microbiota sub-networks, unidentified caecal metabolites and hypothalamic oxidative stress metabolites. Germ-free rats displayed reduced energy intake and no changes in redox homoeostasis machinery expression or pro-inflammatory cytokines after 2 days of WD, in contrast to conventional rats, which exhibited increased SOD2, GLRX and IL-6 mRNA levels. Conclusion A potentially pro-inflammatory gut microbiota and an early hypothalamic oxidative stress appear shortly after WD introduction. Tripartite data integration highlighted putative links between gut microbiota sub-networks and hypothalamic oxidative stress. Together with the absence of hypothalamic modifications in germ-free rats, this strongly suggests the involvement of the microbiota-hypothalamus axis in rat adaptation to WD introduction and in energy homoeostasis regulation.

Fructose is metabolized in the cytoplasm by the enzyme ketohexokinase (KHK), and excessive consumption may affect bone health. Previous work in calcium-restricted, growing mice demonstrated that fructose disrupted intestinal calcium transport. Thus, we hypothesized that the observed effects on bone were dependent on fructose metabolism and took advantage of a KHK knockout (KO) model to assess direct effects of high plasma fructose on the long bones of growing mice. Four groups (n = 12) of 4-week-old, male, C57Bl/6 background, congenic mice with intact KHK (wild-type, WT) or global knockout of both isoforms of KHK-A/C (KHK-KO), were fed 20% glucose (control diet) or fructose for 8 weeks. Dietary fructose increased by 40-fold plasma fructose in KHK-KO compared to the other three groups (p < 0.05). Obesity (no differences in epididymal fat or body weight) or altered insulin was not observed in either genotype. The femurs of KHK-KO mice with the highest levels of plasma fructose were shorter (2%). Surprisingly, despite the long-term blockade of KHK, fructose feeding resulted in greater bone mineral density, percent volume, and number of trabeculae as measured by µCT in the distal femur of KHK-KO. Moreover, higher plasma fructose concentrations correlated with greater trabecular bone volume, greater work-to-fracture in three-point bending of the femur mid-shaft, and greater plasma sclerostin. Since the metabolism of fructose is severely inhibited in the KHK-KO condition, our data suggest mechanism(s) that alter bone growth may be related to the plasma concentration of fructose.

Current fructose consumption levels often overwhelm the intestinal capacity to absorb fructose. We investigated the impact of fructose malabsorption on intestinal endocrine function and addressed the role of the microbiota in this process. To answer this question, a mouse model of moderate fructose malabsorption [ketohexokinase mutant (KHK)-/-] and wild-type (WT) littermate mice were used and received a 20%-fructose (KHK-F and WT-F) or 20%-glucose diet. Cholecystokinin (Cck) mRNA and protein expression in the ileum and cecum, as well as preproglucagon (Gcg) and neurotensin (Nts) mRNA expression in the cecum, increased in KHK-F mice. In KHK-F mice, triple-label immunohistochemistry showed major up-regulation of CCK in enteroendocrine cells (EECs) that were glucagon-like peptide-1 (GLP-1)+/Peptide YY (PYY-) in the ileum and colon and GLP-1-/PYY- in the cecum. The cecal microbiota composition was drastically modified in the KHK-F in association with an increase in glucose, propionate, succinate, and lactate concentrations. Antibiotic treatment abolished fructose malabsorption-dependent induction of cecal Cck mRNA expression and, in mouse GLUTag and human NCI-H716 cells, Cck mRNA expression levels increased in response to propionate, both suggesting a microbiota-dependent process. Fructose reaching the lower intestine can modify the composition and metabolism of the microbiota, thereby stimulating the production of CCK from the EECs possibly in response to propionate.-Zhang, X., Grosfeld, A., Williams, E., Vasiliauskas, D., Barretto, S., Smith, L., Mariadassou, M., Philippe, C., Devime, F., Melchior, C., Gourcerol, G., Dourmap, N., Lapaque, N., Larraufie, P., Blottière, H. M., Herberden, C., Gerard, P., Rehfeld, J. F., Ferraris, R. P., Fritton, J. C., Ellero-Simatos, S., Douard, V. Fructose malabsorption induces cholecystokinin expression in the ileum and cecum by changing microbiota composition and metabolism.

SCOPE:Food structure is a key factor controlling digestion and nutrient absorption. We test the hypothesis that protein emulsion structure in the diet may affect digestive and absorptive processes.METHODS & RESULTS:Rats (n = 40) are fed for 3 weeks with two diets chemically identical but based on lipid-protein liquid-fine (LFE) or gelled-coarse (GCE) emulsions that differ at the macro- and microstructure levels. After an overnight fasting, they ingest a 15 N-labeled LFE or GCE test meal and are euthanized 0, 15 min, 1 h, and 5 h later. 15 N enrichment in intestinal contents and blood are measured. Gastric emptying, protein digestion kinetics, 15 N absorption, and incorporation in blood protein and urea are faster with LFE than GCE. At 15 min time point, LFE group shows higher increase in GIP portal levels than GCE. Three weeks of dietary adaptation leads to higher expression of cationic amino acid transporters in ileum of LFE compared to GCE. LFE diet raises cecal butyrate and isovalerate proportion relative to GCE, suggesting increased protein fermentation. LFE diet increases fecal Parabacteroides relative abundance but decreases Bifidobacterium, Sutterella, Parasutterella genera, and Clostridium cluster XIV abundance.CONCLUSION:Protein emulsion structure regulates digestion kinetics and gastrointestinal physiology, and could be targeted to improve food health value.

Correction to: International Journal of Obesity advance online publication, 25 February 2014; doi:10.1038/ijo.2014.20 The original version of this article contained some errors in the figures. In Figure 1b, the x-axis labels ‘Visceral’ and ‘Epididymal’ were transposed. In Figure 2c, the second western blot image on the right was inverted and the images for Pax6, FOXA1 and CRIF1 were incorrectly represented.

BACKGROUND AND OBJECTIVES: Gut hormones secreted by enteroendocrine cells (EECs) play a major role in energy regulation. Differentiation of EEC is controlled by the expression of basic helix-loop-helix (bHLH) transcription factors. High-fat (HF) feeding alters gut hormone levels; however, the impact of HF feeding on bHLH transcription factors in mediating EEC differentiation and subsequent gut hormone secretion and expression is not known.METHODS: Outbred Sprague-Dawley rats were maintained on chow or HF diet for 12 weeks. Gene and protein expression of intestinal bHLH transcription factors, combined with immunofluorescence studies, were analyzed for both groups in the small intestine and colon. Gut permeability, intestinal lipid and carbohydrate transporters as well as circulating levels and intestinal protein expression of gut peptides were determined.RESULTS: We showed that HF feeding resulted in hyperphagia and increased adiposity. HF-fed animals exhibited decreased expression of bHLH transcription factors controlling EEC differentiation (MATH1, NGN3, NEUROD1) and increased expression of bHLH factors modulating enterocyte expression. Furthermore, HF-fed animals had decreased number of total EECs and L-cells. This was accompanied by increased gut permeability and expression of lipid and carbohydrate transporters, and a decrease in circulating and intestinal gut hormone levels.CONCLUSIONS: Taken together, our results demonstrate that HF feeding caused decreased secretory lineage (that is, EECs) differentiation through downregulation of bHLH transcription factors, resulting in reduced EEC number and gut hormone levels. Thus, impaired EEC differentiation pathways by HF feeding may promote hyperphagia and subsequent obesity.

Aberrations in gut microbiota are associated with metabolic disorders, including obesity. However, whether shifts in the microbiota profile during obesity are a characteristic of the phenotype or a consequence of obesogenic feeding remains elusive. Therefore, we aimed to determine differences in the gut microbiota of obese-prone (OP) and obese-resistant (OR) rats and examined the contribution of this microbiota to the behavioral and metabolic characteristics during obesity. We found that OP rats display a gut microbiota distinct from OR rats fed the same high-fat diet, with a higher Firmicutes-to-Bacteroidetes ratio and significant genera differences. Transfer of OP but not OR microbiota to germ-free (GF) mice replicated the characteristics of the OP phenotype, including reduced intestinal and hypothalamic satiation signaling, hyperphagia, increased weight gain and adiposity, and enhanced lipogenesis and adipogenesis. Furthermore, increased gut permeability through conventionalization resulted in inflammation by proinflammatory nuclear factor (NF)-κB/inhibitor of NF-κB kinase subunit signaling in adipose tissue, liver, and hypothalamus. OP donor and GF recipient animals harbored specific species from Oscillibacter and Clostridium clusters XIVa and IV that were completely absent from OR animals. In conclusion, susceptibility to obesity is characterized by an unfavorable microbiome predisposing the host to peripheral and central inflammation and promoting weight gain and adiposity during obesogenic feeding.

Recently, we showed that susceptibility to obesity is characterized by an unfavorable microbiome and that the gut chemosensory signature can be transferred via microbiota. To examine whether changes in the signaling pathways controlling differentiation of enteroendocrine cells (EEC) are transmitted through gut microbiota, we assessed gene and protein expression of intestinal bHLH transcription factors, in germ free mice conventionalized with microbiota from obese prone and resistant rats. Gut permeability, intestinal lipid and carbohydrates transporters as well as circulating levels and intestinal protein expression of gut peptides were determined. We showed that obese animals exhibited decreased expression of bHLH transcription factors controlling EECs differentiation (MATH1, NGN3, NEUROD1), increased expression of bHLH factors modulating absorptive enterocyte expression. and decreased number of total EECs L‐, I‐ and S‐cells. This was accompanied by increased gut permeability and expression of lipid and carbohydrates transporters. Transfer of obese gut microbiota replicated obesity‐associated decreased in bHLH transcription factors in germ free mice. These findings demonstrate that gut microbiota modulates complex molecular signaling machinery responsible for EECs differentiation pathways. It shows that susceptibility to obesity and associated microbiome impacts EEC differentiation through downregulation of bHLH transcription factors resulting in reduced EEC number and satiety gut hormone levels.

Obesity has been associated with activation of cellular stress signaling and inflammatory pathways. A key player in this cellular stress response is the endoplasmic reticulum (ER). Thus, this study examined the effect of high‐energy/high‐fat (HE/HF)‐induced obesity on ER stress activation. Using male Sprague Dawley rats, we showed that HF feeding activated ER stress response (UPR‐Unfolded Protein Response) in the small intestine, adipose tissue and liver, which was associated with altered gut peptides expression and release including glucagon‐like peptide (GLP‐1), polypeptide YY (PYY) and cholecystokinin (CCK). This effect paralleled increases in weight gain, increased gut permeability, elevated circulating inflammatory cytokines and adipokines levels and impaired calcium homeostasis through disruption of both Sarco(endo)plamsic Ca2+‐ATPase 2b (SERCA), Homocysteine‐inductible endoplasmic reticulum stress protein (HERP) and Der1‐Like Domain Family, Member 1 (DERL2). Further, HF‐diet‐induced ER stress increased systemic inflammation through proinflammatory IKKβ/NF‐κB signaling activation and diminishing insulin sensitivity in the liver tissue. Increased lipogenic and adipogenic enzyme expression were also observed in the liver and adipose tissue of HF‐fed animals. Our findings suggest that HF feeding results in profound dysfunctions of ER homeostasis promoting weight gain and increased adiposity, all of which may contribute to perpetuation of obesity.