Brynn H. Voy

The Collaborative Cross at Oak Ridge National Laboratory: developing a powerful resource for systems genetics.

Complex traits and disease comorbidity in humans and in model organisms are the result of naturally occurring polymorphisms that interact with each other and with the environment. To ensure the availability of resources needed to investigate biomolecular networks and systems-level phenotypes underlying complex traits, we have initiated breeding of a new genetic reference population of mice, the Collaborative Cross. This population has been designed to optimally support systems genetics analysis. Its novel and important features include a high level of genetic diversity, a large population size to ensure sufficient power in high-dimensional studies, and high mapping precision through accumulation of independent recombination events. Implementation of the Collaborative Cross has been ongoing at the Oak Ridge National Laboratory (ORNL) since May 2005. Production has been systematically managed using a software-assisted breeding program with fully traceable lineages, performed in a controlled environment. Currently, there are 650 lines in production, and close to 200 lines are now beyond their seventh generation of inbreeding. Retired breeders enter a high-throughput phenotyping protocol and DNA samples are banked for analyses of recombination history, allele drift and loss, and population structure. Herein we present a progress report of the Collaborative Cross breeding program at ORNL and a description of the kinds of investigations that this resource will support.

Imaging nanoparticles in cells by nanomechanical holography

Nanomaterials have potential medical applications, for example in the area of drug delivery, and their possible adverse effects and cytotoxicity are curently receiving attention. Inhalation of nanoparticles is of great concern, because nanoparticles can be easily aerosolized. Imaging techniques that can visualize local populations of nanoparticles at nanometre resolution within the structures of cells are therefore important. Here we show that cells obtained from mice exposed to single-walled carbon nanohorns can be probed using a scanning probe microscopy technique called scanning near field ultrasonic holography. The nanohorns were observed inside the cells, and this was further confirmed using micro Raman spectroscopy. Scanning near field ultrasonic holography is a useful technique for probing the interactions of engineered nanomaterials in biological systems, which will greatly benefit areas in drug delivery and nanotoxicology.

Genetic analysis in the Collaborative Cross breeding population

Genetic reference populations in model organisms are critical resources for systems genetic analysis of disease related phenotypes. The breeding history of these inbred panels may influence detectable allelic and phenotypic diversity. The existing panel of common inbred strains reflects historical selection biases, and existing recombinant inbred panels have low allelic diversity. All such populations may be subject to consequences of inbreeding depression. The Collaborative Cross (CC) is a mouse reference population with high allelic diversity that is being constructed using a randomized breeding design that systematically outcrosses eight founder strains, followed by inbreeding to obtain new recombinant inbred strains. Five of the eight founders are common laboratory strains, and three are wild-derived. Since its inception, the partially inbred CC has been characterized for physiological, morphological, and behavioral traits. The construction of this population provided a unique opportunity to observe phenotypic variation as new allelic combinations arose through intercrossing and inbreeding to create new stable genetic combinations. Processes including inbreeding depression and its impact on allelic and phenotypic diversity were assessed. Phenotypic variation in the CC breeding population exceeds that of existing mouse genetic reference populations due to both high founder genetic diversity and novel epistatic combinations. However, some focal evidence of allele purging was detected including a suggestive QTL for litter size in a location of changing allele frequency. Despite these inescapable pressures, high diversity and precision for genetic mapping remain. These results demonstrate the potential of the CC population once completed and highlight implications for development of related populations.

Extracting gene networks for low-dose radiation using graph theoretical algorithms.

Genes with common functions often exhibit correlated expression levels, which can be used to identify sets of interacting genes from microarray data. Microarrays typically measure expression across genomic space, creating a massive matrix of co-expression that must be mined to extract only the most relevant gene interactions. We describe a graph theoretical approach to extracting co-expressed sets of genes, based on the computation of cliques. Unlike the results of traditional clustering algorithms, cliques are not disjoint and allow genes to be assigned to multiple sets of interacting partners, consistent with biological reality. A graph is created by thresholding the correlation matrix to include only the correlations most likely to signify functional relationships. Cliques computed from the graph correspond to sets of genes for which significant edges are present between all members of the set, representing potential members of common or interacting pathways. Clique membership can be used to infer function about poorly annotated genes, based on the known functions of better-annotated genes with which they share clique membership (i.e., “guilt-by-association”). We illustrate our method by applying it to microarray data collected from the spleens of mice exposed to low-dose ionizing radiation. Differential analysis is used to identify sets of genes whose interactions are impacted by radiation exposure. The correlation graph is also queried independently of clique to extract edges that are impacted by radiation. We present several examples of multiple gene interactions that are altered by radiation exposure and thus represent potential molecular pathways that mediate the radiation response.

The Adipose Renin-Angiotensin System Modulates Systemic Markers of Insulin Sensitivity and Activates the Intrarenal Renin-Angiotensin System

Background. The adipose tissue renin-angiotensin system (RAS) contributes to regulation of fat mass and may also impact systemic functions such as blood pressure and metabolism. Methods and results. A panel of mouse models including mice lacking angiotensinogen, Agt (Agt-KO), mice expressing Agt solely in adipose tissue (aP2-Agt/Agt-KO), and mice overexpressing Agt in adipose tissue (aP2-Agt) was studied. Total body weight, epididymal fat pad weight, and circulating levels of leptin, insulin, and resistin were significantly decreased in Agt-KO mice, while plasma adiponectin levels were increased. aP2-Agt mice exhibited increased adiposity and plasma leptin and insulin levels compared to wild type (WT) controls. Angiotensinogen and type I Ang II receptor protein levels were also elevated in kidney of aP2-Agt mice. Conclusion. These findings demonstrate that alterations in adipose RAS activity significantly impact both local and systemic physiology in a way that may contribute to the detrimental health effects of obesity.

Overproduction of angiotensinogen from adipose Tissue Induces adipose Inflammation, Glucose Intolerance, and Insulin Resistance

Although obesity is associated with overactivation of the white adipose tissue (WAT) renin‐angiotensin system (RAS), a causal link between the latter and systemic insulin resistance is not established. We tested the hypothesis that overexpression of angiotensinogen (Agt) from WAT causes systemic insulin resistance via modulation of adipose inflammation. Glucose tolerance, systemic insulin sensitivity, and WAT inflammatory markers were analyzed in mice overexpressing Agt in the WAT (aP2‐Agt mice). Proteomic studies and in vitro studies using 3T3‐L1 adipocytes were performed to build a mechanistic framework. Male aP2‐Agt mice exhibited glucose intolerance, insulin resistance, and lower insulin‐stimulated glucose uptake by the skeletal muscle. The difference in glucose tolerance between genotypes was normalized by high‐fat (HF) feeding, and was significantly improved by treatment with angiotensin‐converting enzyme (ACE) inhibitor captopril. aP2‐Agt mice also had higher monocyte chemotactic protein‐1 (MCP‐1) and lower interleukin‐10 (IL‐10) in the WAT, indicating adipose inflammation. Proteomic studies in WAT showed that they also had higher monoglyceride lipase (MGL) and glycerol‐3‐phosphate dehydrogenase levels. Treatment with angiotensin II (Ang II) increased MCP‐1 and resistin secretion from adipocytes, which was prevented by cotreating with inhibitors of the nuclear factor‐κB (NF‐κB) pathway or nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In conclusion, we show for the first time that adipose RAS overactivation causes glucose intolerance and systemic insulin resistance. The mechanisms appear to be via reduced skeletal muscle glucose uptake, at least in part due to Ang II‐induced, NADPH oxidase and NFκB‐dependent increases in WAT inflammation.

Transcriptomic and metabolomic profiling of chicken adipose tissue in response to insulin neutralization and fasting

BackgroundDomestic broiler chickens rapidly accumulate adipose tissue due to intensive genetic selection for rapid growth and are naturally hyperglycemic and insulin resistant, making them an attractive addition to the suite of rodent models used for studies of obesity and type 2 diabetes in humans. Furthermore, chicken adipose tissue is considered as poorly sensitive to insulin and lipolysis is under glucagon control. Excessive fat accumulation is also an economic and environmental concern for the broiler industry due to the loss of feed efficiency and excessive nitrogen wasting, as well as a negative trait for consumers who are increasingly conscious of dietary fat intake. Understanding the control of avian adipose tissue metabolism would both enhance the utility of chicken as a model organism for human obesity and insulin resistance and highlight new approaches to reduce fat deposition in commercial chickens.ResultsWe combined transcriptomics and metabolomics to characterize the response of chicken adipose tissue to two energy manipulations, fasting and insulin deprivation in the fed state. Sixteen to 17 day-old commercial broiler chickens (ISA915) were fed ad libitum, fasted for five hours, or fed but deprived of insulin by injections of anti-insulin serum. Pair-wise contrasts of expression data identified a total of 2016 genes that were differentially expressed after correction for multiple testing, with the vast majority of differences due to fasting (1780 genes). Gene Ontology and KEGG pathway analyses indicated that a short term fast impacted expression of genes in a broad selection of pathways related to metabolism, signaling and adipogenesis. The effects of insulin neutralization largely overlapped with the response to fasting, but with more modest effects on adipose tissue metabolism. Tissue metabolomics indicated unique effects of insulin on amino acid metabolism.ConclusionsCollectively, these data provide a foundation for further study into the molecular basis for adipose expansion in commercial poultry and identify potential pathways through which fat accretion may be attenuated in the future through genetic selection or management practices. They also highlight chicken as a useful model organism in which to study the dynamic relationship between food intake, metabolism, and adipose tissue biology.

Elastic phase response of silica nanoparticles buried in soft matter

Tracking the uptake of nanomaterials by living cells is an important component in assessing both potential toxicity and in designing future materials for use in vivo. We show that the difference in the local elasticity at the site of silica (SiO2) nanoparticles confined within a macrophage enables functional ultrasonic interactions. By elastically exciting the cell, a phase perturbation caused by the buried SiO2 nanoparticles was detected and used to map the subsurface populations of nanoparticles. Localization and mapping of stiff chemically synthesized silica nanoparticles within the cellular structures of a macrophage are important in basic as well as applied studies.

n3 and n6 polyunsaturated fatty acids differentially modulate prostaglandin E secretion but not markers of lipogenesis in adipocytes

A dramatic rise in the incidence of obesity in the U.S. has accelerated the search for interventions that may impact this epidemic. One recently recognized target for such intervention is adipose tissue, which secretes a variety of bioactive substances including prostaglandins. Prostaglandin E2 (PGE2) has been shown to decrease lipolysis in adipocytes, but limited studies have explored alternative mechanisms by which PGE2 might impact obesity, such as adipogenesis or lipogenesis. Studies conducted on ApcMin/+ mice indicated that selective inhibition of the cyclooxygenase (COX)-2 enzyme led to significant reductions in fatty acid synthase (FAS) activity in adipose tissue suggesting lipogenic effects of PGE2. To further investigate whether these lipid mediators directly regulate lipogenesis, we used 3T3-L1 adipocytes to determine the impact of eicosapentaenoic acid (EPA) and celecoxib on PGE2 formation and FAS used as a lipogenic marker. Both arachidonic acid (AA) and EPA dose-dependently increased PGE secretion from adipocytes. AA was expectedly more potent and exhibiting at 150 uM dose a 5-fold increase in PGE2 secretion over EPA. Despite higher secretion of PGE by EPA and AA compared to control, neither PUFA significantly altered FAS activity. By contrast both AA and EPA significantly decreased FAS mRNA levels. Addition of celecoxib, a selective COX-2 inhibitor, significantly decreased PGE2 secretion (p < 0.05) versus control, and also significantly decreased FAS activity (p < 0.05). Unexpectedly, the combination of exogenous PGE2 and celecoxib further decreased the FAS activity compared to PGE2 alone or untreated controls. In conclusion, EPA-mediated inhibition of AA metabolism did not significantly alter FAS activity while both AA and EPA significantly decreased FAS mRNA expression. COX-2 inhibition significantly decreased PGE2 production resulting in a decrease in FAS activity and expression that was not reversed with the addition of exogenous PGE2, suggesting an additional mechanism that is independent of COX-2.

Assessing the pulmonary toxicity of single-walled carbon nanohorns

Previous studies have suggested that single-walled carbon nanotubes (SWCNTs) may pose a pulmonary hazard. We investigated the pulmonary toxicity of single-walled carbon nanohorns (SWCNHs), a relatively new carbon-based nanomaterial that is structurally similar to SWCNTs. Mice were exposed to 30 µg of surfactant-suspended SWCNHs or an equal volume of vehicle control by pharyngeal aspiration and sacrificed 24 hours or 7 days post-exposure. Total and differential cell counts and cytokine analysis of bronchoalveolar lavage fluid demonstrated a mild inflammatory response which was mitigated by day 7 post-exposure. Whole lung microarray analysis demonstrated that SWCNH-exposure did not lead to robust changes in gene expression. Finally, histological analysis showed no evidence of granuloma formation or fibrosis following SWCNH aspiration. These combined results suggest that SWCNH is a relatively innocuous nanomaterial when delivered to mice in vivo using aspiration as a delivery mechanism.