Trans-10, cis-12-conjugated linoleic acid alters hepatic gene expression in a polygenic obese line of mice displaying hepatic lipidosis.

The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) causes a rapid reduction of body and adipose mass in mice. In addition to changes in adipose tissue, numerous studies have reported alterations in hepatic lipid metabolism. Livers of CLA-fed mice gain mass, partly due to lipid accumulation; however, the precise molecular mechanisms are unknown. To elucidate these mechanisms, we examined fatty acid composition and gene expression profiles of livers from a polygenic obese line of mice fed 1% trans-10, cis-12-CLA for 14 days. Analysis of gene expression data led to the identification of 1393 genes differentially expressed in the liver of CLA-fed male mice at a nominal P value of .01, and 775 were considered significant using a false discovery rate (FDR) threshold of .05. While surprisingly few genes in lipid metabolism were impacted, pathway analysis found that protein kinase A (PKA) and cyclic adenosine monophosphate (cAMP) pathways signaling pathways were affected by CLA treatment and 98 of the 775 genes were found to be regulated by hepatocyte nuclear factor 4alpha, a transcription factor important in controlling liver metabolic status.

The contribution of epistatic pleiotropy to the genetic architecture of covariation among polygenic traits in mice.

The contribution that pleiotropic effects of individual loci make to covariation among traits is well understood theoretically and is becoming well documented empirically. However, little is known about the role of epistasis in determining patterns of covariation among traits. To address this problem we combine a quantitative trait locus (QTL) analysis with a two-locus model to assess the contribution of epistasis to the genetic architecture of variation and covariation of organ weights and limb bone lengths in a backcross population of mice created from the M16i and CAST/Ei strains. Significant epistasis was exhibited by 14 pairwise combinations of QTL for organ weights and 10 combinations of QTL for limb bone lengths, which contributed, on average, about 5% of the variation in organ weights and 8% in limb bone lengths beyond that of single-locus QTL effects. Epistatic pleiotropy was much more common in the limb bones (seven of 10 epistatic combinations affecting limb bone lengths were pleiotropic) than the organs (three of the 14 epistatic combinations affecting organ weights were pleiotropic). In both cases, epistatic pleiotropy was less common than single-locus pleiotropy. Epistatic pleiotropy accounted for an average of 6% of covariation among organ weights and 21% of covariation among limb bone lengths, which represented an average of one-fifth (for organ weights) and one-third (for limb bone lengths) of the total genetic covariance between traits. Thus, although epistatic pleiotropy made a smaller contribution than single-locus pleiotropy, it clearly made a significant contribution to the genetic architecture of variation/covariation.

Bayesian analyses of multiple epistatic QTL models for body weight and body composition in mice.

To comprehensively investigate the genetic architecture of growth and obesity, we performed Bayesian analyses of multiple epistatic quantitative trait locus (QTL) models for body weights at five ages (12 days, 3, 6, 9 and 12 weeks) and body composition traits (weights of two fat pads and five organs) in mice produced from a cross of the F1 between M16i (selected for rapid growth rate) and CAST/Ei (wild-derived strain of small and lean mice) back to M16i. Bayesian model selection revealed a temporally regulated network of multiple QTL for body weight, involving both strong main effects and epistatic effects. No QTL had strong support for both early and late growth, although overlapping combinations of main and epistatic effects were observed at adjacent ages. Most main effects and epistatic interactions had an opposite effect on early and late growth. The contribution of epistasis was more pronounced for body weights at older ages. Body composition traits were also influenced by an interacting network of multiple QTLs. Several main and epistatic effects were shared by the body composition and body weight traits, suggesting that pleiotropy plays an important role in growth and obesity.

Genomic mapping of direct and correlated responses to long-term selection for rapid growth rate in mice.

Understanding the genetic architecture of traits such as growth, body composition, and energy balance has become a primary focus for biomedical and agricultural research. The objective of this study was to map QTL in a large F(2) (n = 1181) population resulting from an intercross between the M16 and ICR lines of mice. The M16 line, developed by long-term selection for 3- to 6-week weight gain, is larger, heavier, fatter, hyperphagic, and diabetic relative to its randomly selected control line of ICR origin. The F(2) population was phenotyped for growth and energy intake at weekly intervals from 4 to 8 weeks of age and for body composition and plasma levels of insulin, leptin, TNFalpha, IL6, and glucose at 8 weeks and was genotyped for 80 microsatellite markers. Since the F(2) was a cross between a selection line and its unselected control, the QTL identified likely represent genes that contributed to direct and correlated responses to long-term selection for rapid growth rate. Across all traits measured, 95 QTL were identified, likely representing 19 unique regions on 13 chromosomes. Four chromosomes (2, 6, 11, and 17) harbored loci contributing disproportionately to selection response. Several QTL demonstrating differential regulation of regional adipose deposition and age-dependent regulation of growth and energy consumption were identified.

Functional genomic characterization of delipidation elicited by trans-10, cis-12-conjugated linoleic acid (t10c12-CLA) in a polygenic obese line of mice.

Gene expression was measured during t10c12-CLA-induced body fat reduction in a polygenic obese line of mice. Adult mice (n = 185) were allotted to a 2 x 2 factorial experiment consisting of either nonobese (ICR-control) or obese (M16-selected) mice fed a 7% fat, purified diet containing either 1% linoleic acid (LA) or 1% t10c12-CLA. Body weight (BW) by day 14 was 12% lower in CLA- compared with LA-fed mice (P < 0.0001). By day 14, t10c12-CLA reduced weights of epididymal, mesenteric, and brown adipose tissues, as a percentage of BW, in both lines by 30, 27, and 58%, respectively, and increased liver weight/BW by 34% (P < 0.0001). Total RNA was isolated and pooled (4 pools per tissue per day) from epididymal adipose (days 5 and 14) of the obese mice to analyze gene expression profiles using Agilent mouse oligo microarray slides representing > 20,000 genes. Numbers of genes differentially expressed by greater than or equal to twofold in epididymal adipose (days 5 and 14) were 29 and 125, respectively. It was concluded that, in adipose tissue, CLA increased expression of uncoupling proteins (1 and 2), carnitine palmitoyltransferase system, tumor necrosis factor-alpha (P < 0.05), and caspase-3 but decreased expression of peroxisome proliferator-activated receptor-gamma, glucose transporter-4, perilipin, caveolin-1, adiponectin, resistin, and Bcl-2 (P < 0.01). In conclusion, this experiment has revealed candidate genes that will be useful in elucidating mechanisms of adipose delipidation.

Bayesian model selection for genome-wide epistatic quantitative trait loci analysis.

The problem of identifying complex epistatic quantitative trait loci (QTL) across the entire genome continues to be a formidable challenge for geneticists. The complexity of genome-wide epistatic analysis results mainly from the number of QTL being unknown and the number of possible epistatic effects being huge. In this article, we use a composite model space approach to develop a Bayesian model selection framework for identifying epistatic QTL for complex traits in experimental crosses from two inbred lines. By placing a liberal constraint on the upper bound of the number of detectable QTL we restrict attention to models of fixed dimension, greatly simplifying calculations. Indicators specify which main and epistatic effects of putative QTL are included. We detail how to use prior knowledge to bound the number of detectable QTL and to specify prior distributions for indicators of genetic effects. We develop a computationally efficient Markov chain Monte Carlo (MCMC) algorithm using the Gibbs sampler and Metropolis-Hastings algorithm to explore the posterior distribution. We illustrate the proposed method by detecting new epistatic QTL for obesity in a backcross of CAST/Ei mice onto M16i.

Fine mapping of a QTL region with large effects on growth and fatness on mouse chromosome 2.

We combined the use of a congenic line and recombinant progeny testing (RPT) to characterize and fine map a previously identified region of distal mouse chromosome 2 (MMU2) harboring quantitative trait loci (QTL) with large effects on growth and fatness. The congenic line [M16i.B6-(D2Mit306-D2Mit52); MB2] was created using an inbred line (M16i) derived from a line that had undergone long-term selection for rapid weight gain (M16) as the recipient for an approximately 38-cM region on MMU2 from the inbred line C57BL/6J. A large F2 cohort (1,200 mice) originating from a cross between MB2 and M16i was created, and 40 F2 males with defined recombinations within the QTL region were used to produce 665 segregating progeny. Linkage analysis of the F2 population detected QTL with very large effects on body weight, body fat, lean tissue mass, bone mineral density, and liver weight. Confidence intervals of the QTL were narrowed to regions of 1.5-4.5 cM. Analysis of progeny of the recombinant F2 males confirmed the existence of the QTL and further contributed to localization of their map positions. These efforts confirmed the presence of QTL with major effect on MMU2, narrowed the estimated region harboring the QTL from 38 to 12 cM, and further characterized phenotypic effects of the QTL, effectively culminating in a significantly decreased pool of positional candidate genes potentially representing these genes controlling predisposition to growth and fatness.

The M16 mouse: an outbred animal model of early onset polygenic obesity and diabesity.

OBJECTIVE: To characterize the phenotypic consequences of long-term selective breeding for rapid weight gain, with an emphasis on obesity and obesity-induced diabetes (diabesity). RESEARCH METHODS AND PROCEDURES: M16 is the result of long-term selection for 3- to 6-week weight gain from an ICR base population. Experiment 1 characterized males from both lines for body weights (3, 6, and 8 weeks), feed (4 to 8 weeks) and H(2)O (6 to 8 weeks) consumption, and heat loss, body composition, and levels of several plasma proteins at 8 weeks of age. Experiment 2 characterized differences between lines for both sexes at three ages (6, 8, and 16 weeks) and fed two diets (high and normal fat). Body weight, composition, blood glucose, and plasma insulin and leptin levels were evaluated after an 8-hour fast. RESULTS: At all ages measured, M16 mice were heavier, fatter, hyperphagic, hyperinsulinemic, and hyperleptinemic relative to ICR. M16 males and females were hyperglycemic relative to ICR, with 56% and 22% higher fasted blood glucose levels at 8 weeks of age. DISCUSSION: M16 mice represent an outbred animal model to facilitate gene discovery and pathway regulation controlling early onset polygenic obesity and type 2 diabetic phenotypes. Phenotypes prevalent in the M16 model, with obesity and diabesity exhibited at a young age, closely mirror current trends in human populations.

Characterization of QTL with major effects on fatness and growth on mouse chromosome 2.

OBJECTIVE: To isolate and characterize a region on mouse chromosome 2 harboring quantitative trait loci with large influences on growth and fatness. RESEARCH METHODS AND PROCEDURES: A congenic line [M16i.B6-(D2Mit306-D2Mit52); MB2] was created using the polygenic obese M16i selection line as the recipient for an approximately 38-centimorgan region from C57BL/6J. Males and females from M16i and MB2 were compared for body weight, body composition, feed consumption, and additional traits at 6, 15, and 24 weeks. Interactions of genotype and environment (low and high dietary fat) were investigated. Males (8 weeks) were evaluated for fatty acid profiles in liver and for transcriptional profiles in liver and adipose. RESULTS: Consequences of replacing M16i alleles with C57BL/6J alleles in MB2 were maximized at 15 weeks. MB2 mice were up to 15% lighter than M16i at this age, with no differences in feed consumption. As a percentage of body weight, MB2 had dramatically less epididymal (males) or perimetrial (females) fat (1.17% vs. 2.79% pooled across sex) and lower total lipids (16.1% vs. 23.3%) than M16i. Decreased adiposity in MB2 was not dependent on gender or diet. MB2 mice also had significant decreases in levels of leptin, insulin, and glucose, decreased de novo synthesis of hepatic fatty acid, and transcriptional changes for many genes both within, and external to, the congenic region. DISCUSSION: Results confirm the presence and large effects of mouse chromosome 2 quantitative trait loci and further define their phenotypic consequences related to energy balance. The MB2 congenic line is a powerful resource for eventual identification of pathways and mutations within genes regulating predisposition to growth and obesity.

A large-sample QTL study in mice: I. Growth.

By use of long-term selection lines for high and low growth, a large-sample (n = approximately 1,000 F2) experiment was conducted in mice to further understand the genetic architecture of complex polygenic traits. In combination with previous work, we conclude that QTL analysis has reinforced classic polygenic paradigms put in place prior to molecular analysis. Composite interval mapping revealed large numbers of QTL for growth traits with an exponential distribution of magnitudes of effects and validated theoretical expectations regarding gene action. Of particular significance, large effects were detected on Chromosome (Chr) 2. Regions on Chrs 1, 3, 6, 10, 11, and 17 also harbor loci with significant contributions to phenotypic variation for growth. Despite the large sample size, average confidence intervals of approximately 20 cM exhibit the poor resolution for initial estimates of QTL location. Analysis with genome-wide and chromosomal polygenic models revealed that, under certain assumptions, large fractions of the genome may contribute little to phenotypic variation for growth. Only a few epistatic interactions among detected QTL, little statistical support for gender-specific QTL, and significant age effects on genetic architecture were other primary observations from this study.

A large-sample QTL study in mice: II. Body composition.

Using lines of mice having undergone long-term selection for high and low growth, a large-sample (n = approximately 1,000 F2) experiment was conducted to gain further understanding of the genetic architecture of complex polygenic traits. Composite interval mapping on data from male F2 mice (n = 552) detected 50 QTL on 15 chromosomes impacting weights of various organ and adipose subcomponents of growth, including heart, liver, kidney, spleen, testis, and subcutaneous and epididymal fat depots. Nearly all aggregate growth QTL could be interpreted in terms of the organ and fat subcomponents measured. More than 25% of QTL detected map to MMU2, accentuating the relevance of this chromosome to growth and fatness in the context of this cross. Regions of MMU7, 15, and 17 also emerged as important obesity "hot-spots." Average degrees of directional dominance are close to additivity, matching expectations for body composition traits. A strong QTL congruency is evident among heart, liver, kidney, and spleen weights. Liver and testis are organs whose genetic architectures are, respectively, most and least aligned with that for aggregate body weight. In this study, growth and body weight are interpreted in terms of organ subcomponents underlying the macro aggregate traits, and anchored on the corresponding genomic locations.

A large-sample QTL study in mice: III. Reproduction.

Using lines of mice having undergone long-term selection for high and low growth, a large-sample (n approximately to 1000 F2) experiment was conducted to gain further understanding of the genetic architecture of complex polygenic traits. Composite interval mapping on data from 10-week-old F2 females (n = 439) detected 15 quantitative trait loci (QTLs) on 5 chromosomes that influence reproduction traits characterized at day 16 of gestation. These QTL are broadly categorized into two groups: those where effects on the number of live fetuses (LF) were accompanied by parallel effects on the number of dead fetuses (DF), and those free of such undesirable effects. QTL for ovulation rate (OR) did not overlap with QTL for litter size, potentially indicating the importance of uterine capacity. Large dominance effects were identified for most QTL detected, and overdominance was also present. The QTL of largest effects were detected in regions of Chromosome 2, where large QTL effects for growth and fatness have also been found and where corroborating evidence from other studies exists. Considerable overlap between locations of QTL for reproductive traits and for growth traits corresponds well with the positive correlations usually observed among these sets of phenotypes. Some support for the relevance of QTL x genetic background interactions in reproduction was detected. Traits with low heritability demand considerably larger sample sizes to achieve effective power of QTL detection. This is unfortunate as traits with low heritability are among those that could most benefit from QTL-complemented breeding and selection strategies in food animal production.