The effect of high-fat diet and inhibition of ceramide production on insulin action in liver

Liver, as one of the most important organs involved in lipids and glucose metabolism, is perceived as a key tissue for pharmacotherapy of insulin resistance (IRes) and type 2 diabetes. Ceramides (Cer) are biologically active lipids, which accumulation is associated with the induction of muscle IRes. We sought to determine the role of intrahepatic bioactive lipids production on insulin action in liver of insulin-resistant rats and after myriocin administration. The experiments were conducted on male Wistar rats divided into three groups: Control, fed high-fat diet (HFD), and fed HFD and treated with myriocin (HFD/Myr). Before sacrifice, the animals were infused with a [U-¹³C]palmitate to calculate lipid synthesis rate by means of tracer incorporation technique in particular lipid groups. Liver Cer, diacylglycerols (DAG), acyl-carnitine concentration, and isotopic enrichment were analyzed by LC/MS/MS. Proteins involved in lipid metabolism and insulin pathway were analyzed by western blot analysis. An OGTT and ITT was also performed. HFD-induced IRes and increased both the synthesis rate and the content of DAG and Cer, which was accompanied by inhibition of an insulin pathway. Interestingly, myriocin treatment reduced synthesis rate not only of Cer but also DAG and improved insulin sensitivity. We conclude that the insulin-sensitizing action of myriocin in the liver is a result of the lack of inhibitory effect of lipids on the insulin pathway, due to the reduction of their synthesis rate. This is the first study showing how the synthesis rate of individual lipid groups in liver changes after myriocin administration.     

NMR and interval PLS as reliable methods for determination of cholesterol in rodent lipoprotein fractions

Risk of cardiovascular disease is related to cholesterol distribution in different lipoprotein fractions. Lipoproteins in rodent model studies can only reliably be measured by time- and plasma-consuming fractionation. An alternative method to measure cholesterol distribution in the lipoprotein fractions in rat plasma is presented in this paper. Plasma from two rat studies (n = 68) was used in determining the lipoprotein profile by an established ultracentrifugation method and proton nuclear magnetic resonance (NMR) spectra of replicate samples was obtained. From the ultracentrifugation reference data and the NMR spectra, an interval partial least-square (iPLS) regression model to predict the amount of cholesterol in the different lipoprotein fractions was developed. The relative errors of the prediction models were between 12 and 33% and had correlation coefficients (r) between 0.96 and 0.84. The models were tested with an independent test set giving prediction errors between 19 and 46% and r between 0.96 and 0.76. Prediction of High, Low and Very Low Density Lipoprotein (HDL, LDL and VLDL) and total cholesterol was conducted in a study where rats had been supplemented with two doses of air-dried apple-powder. No significant difference in LDL, VLDL and total cholesterol was observed between the groups. The high apple-powder (20%) group had significantly lower HDL cholesterol (11%, P = 0.0452) than the control group. It is concluded that the iPLS approach yielded excellent regression models and thus univocal established chemometric analysis of NMR spectra of rat plasma as a strong and efficient way to quantify lipoprotein fractions in rat studies.     

In vivo assessment and potential diagnosis of xenobiotics that perturb the thyroid pathway: Proteomic analysis of Xenopus laevis brain tissue following exposure to model T4 inhibitors

As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression patterns in the brain of the amphibian model (Xenopus laevis) following in vivo exposure to a suite of T4 synthesis inhibitors. We specifically address the application of Two Dimensional Polyacrylamide Gel Electrophoresis (2D PAGE), Isobaric Tags for Relative and Absolute Quantitation (iTRAQ®) and LC–MS/MS to assess changes in relative protein expression levels. 2D PAGE and iTRAQ proved to be effective complementary techniques for distinguishing protein changes in the developing amphibian brain in response to T4 synthesis inhibition. This information served to evaluate the use of distinctive protein profiles as a potential mechanism to screen chemicals for endocrine activity in anurans. Regulatory pathways associated with proteins expressed as a result of chemical effect are reported. To our knowledge, this is also the first account of the anuran larvae brain proteome characterization using proteomic technologies. Correlation of protein changes to other cellular and organism-level responses will aid in the development of a more rapid and cost-effective, non-mammalian screening assay for thyroid axis-disrupting chemicals.     

Controlling mosquitoes through innovative and collaborative wetland management practices in the Pacific Northwest

Wetlands Ecology and Management - When developing a plan to restore or modify a wetland within the Pacific Northwest of the United States (PNW), land managers must consider all of the potential...     

Patterns of DNA Barcode Variation in Canadian Marine Molluscs

Background

Molluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area.

Methodology/Principal Findings

This study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0–26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%–46.5%), and showed a significant positive correlation with nearest neighbour distances.

Conclusions/Significance

DNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.     

Expression of miRNAs in ovine fetal gonads: potential role in gonadal differentiation

Background  

Gonadal differentiation in the mammalian fetus involves a complex dose-dependent genetic network. Initiation and progression of fetal ovarian and testicular pathways are accompanied by dynamic expression patterns of thousands of genes. We postulate these expression patterns are regulated by small non-coding RNAs called microRNAs (miRNAs). The aim of this study was to identify the expression of miRNAs in mammalian fetal gonads using sheep as a model.     

Synthetic CpG oligonucleotides induce a genetic profile ameliorating murine myocardial I/R injury

We previously demonstrated that pre‐conditioning with CpG oligonucleotide (ODN) 1668 induces quick up‐regulation of gene expression 3 hours post‐murine myocardial ischaemia/reperfusion (I/R) injury, terminating inflammatory processes that sustain I/R injury. Now, performing comprehensive microarray and biocomputational analyses, we sought to further enlighten the “black box” beyond these first 3 hours. C57BL/6 mice were pretreated with either CpG 1668 or with control ODN 1612, respectively. Sixteen hours later, myocardial ischaemia was induced for 1 hour in a closed‐chest model, followed by reperfusion for 24 hours. RNA was extracted from hearts, and labelled cDNA was hybridized to gene microarrays. Data analysis was performed with BRB ArrayTools and Ingenuity Pathway Analysis. Functional groups mediating restoration of cellular integrity were among the top up‐regulated categories. Genes known to influence cardiomyocyte survival were strongly induced 24 hours post‐I/R. In contrast, proinflammatory pathways were down‐regulated. Interleukin‐10, an upstream regulator, suppressed specifically selected proinflammatory target genes at 24 hours compared to 3 hours post‐I/R. The IL1 complex is supposed to be one regulator of a network increasing cardiovascular angiogenesis. The up‐regulation of numerous protective pathways and the suppression of proinflammatory activity are supposed to be the genetic correlate of the cardioprotective effects of CpG 1668 pre‐conditioning.     

Source of nitrogen as a factor limiting saponin production by hairy root and suspension cultures of <Emphasis Type="Italic">Calendula officinalis</Emphasis> L.

Triterpenic saponins represented in Calendula officinalis L. by oleanolic acid (OA) glycosides are pentacyclic triterpene compounds with a wide range of biological and medicinal properties. This report demonstrates nitrogen source impact on growth, saponin accumulation, and secretion in hairy root and suspension cultures of marigold. Hairy roots preferred nitrate as a mineral source of nitrogen, but its impact on growth, OA glycosides accumulation, and secretion were line-dependent. The best productivity of OA glycosides was found in CC16 line (74.86 mg flask^?1) in ½ MS medium modified by 2.5× KNO₃ and ammonium elimination with 2.5 g l^?1 peptone. Organic nitrogen source at 27.5-g l^?1 impairs the growth rate of hairy roots. Its effect on saponin accumulation and secretion to the surrounding medium depended on line and media composition. Nitrate:ammonium ratio of 4:2 for CC16 resulted in 5.7-fold increment of saponin secretion comparing to the standard medium. Embryo roots, apical bud, and hypocotyls explants were crucial for induction of suspension culture synthesizing saponins; however, effect of mineral form of nitrogen in cultivating medium had to be considered. The highest OA glycosides level (171.97 μg g^?1 of dry weight) was recorded in the root derived culture with nitrate as a sole mineral form of nitrogen. Peptone from lactalbumin decidedly inhibited the saponin formation; however, it was essential for culture initiation, proliferation, and organ differentiation.