Loss of insulin synthesis and beta cell survival induced by oxidized LDL require activation of reticulum endoplasmic stress: a mechanism countered by HDL

Elevated circulating concentrations in modified LDL-cholesterol particles (e.g. oxidised LDL) and low levels in HDL increase not only the risk for diabetic patients to develop cardiovascular diseases but also may contribute to development and progression of diabetes by directly having adverse effects on β-cells. Chronic exposure of β-cells to 2 mM human oxidised LDL-cholesterol (oxLDL) increases the rate of apoptosis, reduce insulin biosynthesis and the secretory capacity of the cells in response to nutrients. In line with the protective role, HDL efficiently antagonised the harmful effects of ox- LDL, suggesting that low levels of HDL would be inefficient to protect β-cells against oxLDL attack in patients. Activation of endoplasmic reticulum (ER) stress is pointed out to contribute to β-cell dysfunction elicited by environmental stressors. In this study we investigated whether activation of ER stress is required for oxLDL to mediate detrimental effects on β-cells and we tested the potential antagonist properties of HDL: The mouse MIN6 insulin-secreting cells were cultured with 2 mM of LDL-cholesterol preparation (native or in vitro oxidized) in the presence or absence of 1 mM of HDL-cholesterol or the ER stress inhibitor 4-phenylbutyrate (4-PBA): Prolonged exposure of MIN6 cells to 2 mM oxLDL-cholesterol for 48 hours led to an increase in expression of ER stress markers such as ATF4, CHOP and p58 and stimulated the splicing of XBP-1 whereas, induction of these markers was not observable in the cells cultured with native LDL. Treatment of the cells with the 4-PBA chemical chaperone molecule efficiently blocked activation of the ER stress markers induced by oxLDL. The latter mediates β-cell dysfunction and apoptosis by diminishing the expression of islet brain 1 (IB1) and Bcl2. The levels of these two proteins were preserved in the cells that were co-treated with oxLDL and the 4-PBA. Consistent with this result we found that blockade of ER stress activation alleviated the loss of insulin synthesis and abolished apoptosis evoked by oxLDL. However incubation of the cells with 4-PBA did not prevent impairment of insulin secretion elicited by oxLDL, indicating that ER stress is not responsible for the oxLDL-mediated defect of insulin secretion. Co-incubation of the cells with HDL mimicked the effects of 4-PBA on the expression of IB1 and Blc2 and thereby counteracted oxLDL attacks on insulin synthesis and cell survivals. We found that HDL efficiently inhibited activation of the ER stress mediated by oxLDL: These data highlight the contribution of the ER stress in the defects of insulin synthesis and cell survivals induced by oxLDL and emphasize the potent role of HDL to counter activation of the oxLDL-mediated ER-stress activation:

Função ovariana de novilhas Nelore alimentadas com dieta suplementada com gordura protegida ruminal

O objetivo deste trabalho foi avaliar o efeito da suplementação alimentar com gordura protegida ruminal sobre as estruturas ovarianas e sobre a concentração sérica de progesterona, em novilhas Nelore mantidas em pasto. Quarenta novilhas foram divididas em dois grupos: um suplementado com gordura protegida Megalac-E (G); e outro sem suplementação de gordura (C). O grupos foram avaliados em delineamento "crossover". Utilizaram-se dietas isoenergéticas e isoproteicas. Após 15 dias de suplementação, os animais foram submetidos a um protocolo hormonal, para avaliação da influência da suplementação com gordura no metabolismo da progesterona. Para isto, em um dia aleatório do ciclo (D0), inseriu-se um implante intravaginal de liberação de progesterona (CIDR), e aplicou-se prostaglandina F2α (PGF2α, i.m.). No D7, o implante foi retirado, e outra aplicaηão de PGF2α foi realizada. No D18, foi feita uma nova aplicaηão de PGF2α e, então, foram observados diariamente os exames ultrassonográficos ovarianos e a ocorrência de estro. Para o ensaio com progesterona, colheu-se sangue 4 dias após a inserção do implante e, novamente, 7 e 14 dias após a ovulação. A concentração de progesterona sérica no D4 foi maior no grupo G. Não houve diferença nas concentrações séricas de progesterona 7 e 14 dias após a ovulação, nem no diâmetro do folículo ovulatório, nem no volume luteal. A suplementação com Megalac-E altera o metabolismo de progesterona, mas não altera a função ovariana em novilhas zebuínas em pasto.

Cocoa flavonoid-enriched diet modulates systemic and intestinal immunoglobulin synthesis in adult Lewis rats

Previous studies have reported that a diet containing 10% cocoa, a rich source of flavonoids, has immunomodulatory effects on rats and, among others effects, is able to attenuate the immunoglobulin (Ig) synthesis in both systemic and intestinal compartments. The purpose of the present study was focused on investigating whether these effects were attributed exclusively to the flavonoid content or to other compounds present in cocoa. To this end, eight-week-old Lewis rats were fed, for two weeks, either a standard diet or three isoenergetic diets containing increasing proportions of cocoa flavonoids from different sources: one with 0.2% polyphenols from conventional defatted cocoa, and two others with 0.4% and 0.8% polyphenols, respectively, from non-fermented cocoa. Diet intake and body weight were monitored and fecal samples were obtained throughout the study to determine fecal pH, IgA, bacteria proportions, and IgA-coated bacteria. Moreover, IgG and IgM concentrations in serum samples collected during the study were quantified. At the end of the dietary intervention no clear changes of serum IgG or IgM concentrations were quantified, showing few effects of cocoa polyphenol diets at the systemic level. However, in the intestine, all cocoa polyphenol-enriched diets attenuated the age-related increase of both fecal IgA and IgA-coated bacteria, as well as the proportion of bacteria in feces. As these effects were not dependent on the dose of polyphenol present in the diets, other compounds and/or the precise polyphenol composition present in cocoa raw material used for the diets could be key factors in this effect.

Fructose induces synthesis and reduces oxidation of liver fatty acids through ChREBP activation

Introduction and aims. During last few decades, the prevalence of obesity, metabolic syndrome and insulin resistance, among other metabolic disturbances, has raised considerably in many countries worldwide. Environmental factors (diet, physical activity), in tandem with predisposing genetic factors, may be responsible for this trend. Along with an increase in total energy consumption during recent decades, there has also been a shift in the type of nutrients, with an increased consumption of fructose, largely attributable to a greater intake of beverages containing high levels of fructose...

Fructose induces synthesis and reduces oxidation of liver fatty acids through ChREBP activation

Introduction and aims. During last few decades, the prevalence of obesity, metabolic syndrome and insulin resistance, among other metabolic disturbances, has raised considerably in many countries worldwide. Environmental factors (diet, physical activity), in tandem with predisposing genetic factors, may be responsible for this trend. Along with an increase in total energy consumption during recent decades, there has also been a shift in the type of nutrients, with an increased consumption of fructose, largely attributable to a greater intake of beverages containing high levels of fructose...

Minimal requirements for parameters to be used for the development of predictive models for microbial source tracking. The example of the pair somatic coliphages and phages infecting Bacteroides

Several methods and approaches for measuring parameters to determine fecal sources of pollution in water have been developed in recent years. No single microbial or chemical parameter has proved sufficient to determine the source of fecal pollution. Combinations of parameters involving at least one discriminating indicator and one universal fecal indicator offer the most promising solutions for qualitative and quantitative analyses. The universal (nondiscriminating) fecal indicator provides quantitative information regarding the fecal load. The discriminating indicator contributes to the identification of a specific source. The relative values of the parameters derived from both kinds of indicators could provide information regarding the contribution to the total fecal load from each origin. It is also essential that both parameters characteristically persist in the environment for similar periods. Numerical analysis, such as inductive learning methods, could be used to select the most suitable and the lowest number of parameters to develop predictive models. These combinations of parameters provide information on factors affecting the models, such as dilution, specific types of animal source, persistence of microbial tracers, and complex mixtures from different sources. The combined use of the enumeration of somatic coliphages and the enumeration of Bacteroides-phages using different host specific strains (one from humans and another from pigs), both selected using the suggested approach, provides a feasible model for quantitative and qualitative analyses of fecal source identification.

IL-10 controls cystatin C synthesis and blood concentration in response to inflammation through regulation of IFN regulatory factor 8 expression.

Cystatin C (CstC) is a cysteine protease inhibitor of major clinical importance. Low concentration of serum CstC is linked to atherosclerosis. CstC can prevent formation of amyloid β associated with Alzheimer's disease and can itself form toxic aggregates. CstC regulates NO secretion by macrophages and is a TGF-β antagonist. Finally, the serum concentration of CstC is an indicator of kidney function. Yet, little is known about the regulation of CstC expression in vivo. In this study, we demonstrate that the transcription factor IFN regulatory factor 8 (IRF-8) is critical for CstC expression in primary dendritic cells. Only those cells with IRF-8 bound to the CstC gene promoter expressed high levels of the inhibitor. Secretion of IL-10 in response to inflammatory stimuli downregulated IRF-8 expression and consequently CstC synthesis in vivo. Furthermore, the serum concentration of CstC decreased in an IL-10-dependent manner in mice treated with the TLR9 agonist CpG. CstC synthesis is therefore more tightly regulated than hitherto recognized. The mechanisms involved in this regulation might be targeted to alter CstC production, with potential therapeutic value. Our results also indicate that caution should be exerted when using the concentration of serum CstC as an indicator of kidney function in conditions in which inflammation may alter CstC production.

Design, synthesis, and biological and crystallographic evaluation of novel inhibitors of Plasmodium falciparum enoyl-ACP-reductase (PfFabI).

Malaria, a disease of worldwide significance, is responsible for over one million deaths annually. The liver-stage of Plasmodium's life cycle is the first, obligatory, but clinically silent step in malaria infection. The P. falciparum type II fatty acid biosynthesis pathway (PfFAS-II) has been found to be essential for complete liver-stage development and has been regarded as a potential antimalarial target for the development of drugs for malaria prophylaxis and liver-stage eradication. In this paper, new coumarin-based triclosan analogues are reported and their biological profile is explored in terms of inhibitory potency against enzymes of the PfFAS-II pathway. Among the tested compounds, 7 and 8 showed the highest inhibitory potency against Pf enoyl-ACP-reductase (PfFabI), followed by 15 and 3. Finally, we determined the crystal structures of compounds 7 and 11 in complex with PfFabI to identify their mode of binding and to confirm outcomes of docking simulations.

Convenient synthesis of C75, an inhibitor of FAS and CPT1

C75 is a synthetic racemic α-methylene-γ-butyrolactone exhibiting anti-tumoral properties in vitro and in vivo as well as inducing hypophagia and weight loss in rodents. These interesting properties are thought to be a consequence of the inhibition of the key enzymes FAS and CPT1 involved in lipid metabolism. The need for larger amounts of this compound for biological evaluation prompted us to develop a convenient and reliable route to multigram quantities of C75 from easily available ethyl penta-3,4-dienoate 6. A recently described protocol for the addition of 6 to a mixture of dicyclohexylborane and nonanal followed by acidic treatment of the crude afforded lactone 8, as a mixture of cis and trans isomers, in good yield. The DBU-catalyzed isomerization of the methyl esters 9 arising from 8 gave a 10:1 trans/cis mixture from which the trans isomer was isolated and easily transformed into C75. The temporary transformation of C75 into a phenylseleno ether derivative makes its purification, manipulation and storage easier.

Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants.

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.

Microbial communities in Cerrado soils under native vegetation subjected to prescribed fire and under pasture

The objective of this work was to evaluate the effects of fire regimes and vegetation cover on the structure and dynamics of soil microbial communities, through phospholipid fatty acid (PLFA) analysis. Comparisons were made between native areas with different woody covers ("cerrado stricto sensu" and "campo sujo"), under different fire regimes, and a 20-year-old active palisadegrass pasture in the Central Plateau of Brazil. Microbial biomass was higher in the native plots than in the pasture, and the highest monthly values were observed during the rainy season in the native plots. No significant differences were observed between fire regimes or between communities from the two native vegetation types. However, the principal component (PC) analysis separated the microbial communities by vegetation cover (native x pasture) and season (wet x dry), accounting for 45.8% (PC1 and PC3) and 25.6% (PC2 and PC3), respectively, of the total PLFA variability. Changes in land cover and seasonal rainfall in Cerrado ecosystems have significant effects on the total density of soil microorganisms and on the abundance of microbial groups, especially Gram-negative and Gram-positive bacteria.

Foraging behavior and ruminal fermentation of dairy cows grazing ryegrass pasture alone or with white clover

The objective of this work was to evaluate the influence of pasture composition and regrowth age on the relationship between feeding behavior and ruminal fermentation in dairy cows grazing perennial ryegrass with or without white clover. The experiment was carried out in a 2x2 factorial arrangement, with two sward types and two ages of regrowth. Swards of perennial ryegrass sown alone (PRG) and of perennial ryegrass mixed with white clover (GC) were evaluated. Twelve late-lactation Holstein cows, fistulated at the rumen, were distributed in a 4x4 latin square experimental design with four 12-day periods. Daily distribution of grazing was similar in the PRG and the GC swards, but the concentration of rumen volatile fatty acids (VFA) was higher and the proportion of propionate was lower on mixed swards during the day. Daily distribution of grazing was similar in pastures of different ages. However, in the oldest swards, rumen fluid pH increased and VFA concentration decreased after evening milking. Time spent grazing does not influence ruminal fermentation, which depends on the changes that occur as different sward layers are grazed.

Synthesis of polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae by using intermediates of fatty acid beta-oxidation.

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the beta-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.

Context-dependent dual role of SKI8 homologs in mRNA synthesis and turnover.

Eukaryotic mRNA transcription and turnover is controlled by an enzymatic machinery that includes RNA polymerase II and the 3' to 5' exosome. The activity of these protein complexes is modulated by additional factors, such as the nuclear RNA polymerase II-associated factor 1 (Paf1c) and the cytoplasmic Superkiller (SKI) complex, respectively. Their components are conserved across uni- as well as multi-cellular organisms, including yeast, Arabidopsis, and humans. Among them, SKI8 displays multiple facets on top of its cytoplasmic role in the SKI complex. For instance, nuclear yeast ScSKI8 has an additional function in meiotic recombination, whereas nuclear human hSKI8 (unlike ScSKI8) associates with Paf1c. The Arabidopsis SKI8 homolog VERNALIZATION INDEPENDENT 3 (VIP3) has been found in Paf1c as well; however, whether it also has a role in the SKI complex remains obscure so far. We found that transgenic VIP3-GFP, which complements a novel vip3 mutant allele, localizes to both nucleus and cytoplasm. Consistently, biochemical analyses suggest that VIP3-GFP associates with the SKI complex. A role of VIP3 in the turnover of nuclear encoded mRNAs is supported by random-primed RNA sequencing of wild-type and vip3 seedlings, which indicates mRNA stabilization in vip3. Another SKI subunit homolog mutant, ski2, displays a dwarf phenotype similar to vip3. However, unlike vip3, it displays neither early flowering nor flower development phenotypes, suggesting that the latter reflect VIP3's role in Paf1c. Surprisingly then, transgenic ScSKI8 rescued all aspects of the vip3 phenotype, suggesting that the dual role of SKI8 depends on species-specific cellular context.

Toxicity of triclosan, penconazole and metalaxyl on Caulobacter crescentus and a freshwater microbial community as assessed by flow cytometry.

Biocides are widely used for domestic hygiene, agricultural and industrial applications. Their widespread use has resulted in their introduction into the environment and raised concerns about potential deleterious effects on aquatic ecosystems. In this study, the toxicity of the biocides triclosan, penconazole and metalaxyl were evaluated with the freshwater bacterium Caulobacter crescentus and with a freshwater microbial community using a combination of single- and double-stain flow cytometric assays. Growth of C.  crescentus and the freshwater community were repressed by triclosan but not by penconazole or metalaxyl at concentrations up to 250 μM. The repressive effect of triclosan was dependent on culture conditions. Caulobacter crescentus was more sensitive to triclosan when grown with high glucose at high cell density than when grown directly in sterilized lake water at low cell density. This suggests that the use of conventional growth conditions may overestimate biocide toxicity. Additional experiments showed that the freshwater community was more sensitive to triclosan than C.  crescentus, with 10 nM of triclosan being sufficient to repress growth and change the phylogenetic composition of the community. These results demonstrate that isolate-based assays may underestimate biocide toxicity and highlight the importance of assessing toxicity directly on natural microbial communities. Because 10 nM of triclosan is within the range of concentrations observed in freshwater systems, these results also raise concerns about the risk of introducing triclosan into the environment.