BackgroundThe choline‐derived metabolite trimethylamine N‐oxide (TMAO) has been demonstrated to contribute to atherosclerosis and is associated with coronary artery disease risk.Methods and ResultsWe explored the impact of TMAO on endothelial and smooth muscle cell function in vivo, focusing on disease‐relevant outcomes for atherogenesis. Initially, we observed that aortas of LDLR −/− mice fed a choline diet showed elevated inflammatory gene expression compared with controls. Acute TMAO injection at physiological levels was sufficient to induce the same inflammatory markers and activate the well‐known mitogen‐activated protein kinase, extracellular signal–related kinase, and nuclear factor‐κB signaling cascade. These observations were recapitulated in primary human aortic endothelial cells and vascular smooth muscle cells. We also found that TMAO promotes recruitment of activated leukocytes to endothelial cells. Through pharmacological inhibition, we further showed that activation of nuclear factor‐κB signaling was necessary for TMAO to induce inflammatory gene expression in both of these relevant cell types as well as endothelial cell adhesion of leukocytes.ConclusionsOur results suggest a likely contributory mechanism for TMAO‐dependent enhancement in atherosclerosis and cardiovascular risks.

A major task in dissecting the genetics of complex traits is to identify causal genes for disease phenotypes. We previously developed a method to infer causal relationships among genes through the integration of DNA variation, gene transcription, and phenotypic information. Here we validated our method through the characterization of transgenic and knockout mouse models of candidate genes that were predicted to be causal for abdominal obesity. Perturbation of eight out of the nine genes, with Gas7, Me1 and Gpx3 being novel, resulted in significant changes in obesity related traits. Liver expression signatures revealed alterations in common metabolic pathways and networks contributing to abdominal obesity and overlapped with a macrophage-enriched metabolic network module that is highly associated with metabolic traits in mice and humans. Integration of gene expression in the design and analysis of traditional F2 intercross studies allows high confidence prediction of causal genes and identification of involved pathways and networks.

Inbred strains of mice are strikingly different in susceptibility to obesity-driven diabetes. For instance, deficiency in leptin receptor (db/db) leads to hyperphagia and obesity in both C57BL/6 and DBA/2 mice, but only on the DBA/2 background do the mice develop beta-cell loss leading to severe diabetes, while C57BL/6 mice are relatively resistant. To further investigate the genetic factors predisposing to diabetes, we have studied leptin receptor-deficient offspring of an F2 cross between C57BL/6J (db/ϩ) males and DBA/2J females. The results show that the genetics of diabetes susceptibility are enormously complex and a number of quantitative trait loci (QTL) contributing to diabetesrelated traits were identified, notably on chromosomes 4, 6, 7, 9, 10, 11, 12, and 19. The Chr. 4 locus is likely due to a disruption of the Zfp69 gene in C57BL/6J mice. To identify candidate genes and to model coexpression networks, we performed global expression array analysis in livers of the F2 mice. Expression QTL (eQTL) were identified and used to prioritize candidate genes at clinical trait QTL. In several cases, clusters of eQTLs colocalized with clinical trait QTLs, suggesting a common genetic basis. We constructed coexpression networks for both 5 and 12 wk old mice and identified several modules significantly associated with clinical traits. One module in 12 wk old mice was associated with several measures of hepatic fat content as well as with other lipid-and diabetes-related traits. These results add to the understanding of the complex genetic interactions contributing to obesity-induced diabetes. leptin receptor; leptin signaling; C57BLKS; Zfp69; coexpression networks INBRED STRAINS OF MICE EXHIBIT variable susceptibility to Type 2 diabetes. A classic approach demonstrating the role of genetic background on the development of obesity-associated diabetes has been the introgression of diabetes (db) mutation (9) into various inbred strains (4). The db mutation disrupts leptin signaling by abrogating the function of leptin receptor and results in hyperphagia and obesity regardless of strain background. In contrast, inbred mouse strains exhibit profound metabolic differences in response to obesity induced by db. Some strains, such as DBA/2 (DBA)-db/db and C57BLKS (BKS)-db/db, develop severe diabetes, whereas others, including C57BL6 (B6)-db/db and 129-db/db, are resistant to the disease (15). Although the role of genetic factors in the development of obesity-associated diabetes has long been appreciated, the genes responsible for differences in diabetes susceptibility remain largely uncharacterized.In the present study, we examined obese (db/db) F2 mice from a cross between B6 and DBA to identify loci that contribute to diabetic progression, insulin resistance, and lipid accumulation in the liver. We characterized two cohorts of mice: one at 5 wk of age prior to anticipated islet depletion and another cohort at 12 wk when BKS db/db begin to show decreased insulin production but prior to the onset of significant diabetic com...

Objective-We previously reported the mapping of a quantitative trait locus (QTL) on chromosome 15 contributing to hyperlipidemia in a cross between inbred strains MRL/MpJ (MRL) and BALB/cJ (BALB). Using marker-assisted breeding, we constructed a congenic strain in which chromosome 15 interval from MRL is placed on the genetic background of BALB. The congenic allowed us to confirm the QTL result and to further characterize the properties and location of the underlying gene. Methods and Results-On chow and high-fat (atherogenic) diets, the congenic mice exhibited higher levels of plasma triglycerides and cholesterol than BALB mice. In response to the atherogenic diet, the congenic mice but not BALB mice exhibited a dramatic Ϸ30-fold increase in atherogenic lesions accompanied by Ϸ2-fold decrease in high-density lipoprotein cholesterol levels. With respect to atherosclerotic lesions and some lipid parameters, this chromosome 15 gene, designated Hyplip2, exhibited dominant inheritance. Expression array analyses suggested that Hyplip2 may influence inflammatory and bile acid synthesis pathways. Finally, we demonstrated the usefulness of subcongenic strains to narrow the locus (50 Mbp) with the goal of positionally cloning Hyplip2. Conclusions-Our data demonstrate that the Hyplip2 gene significantly contributes to combined hyperlipidemia and increased atherosclerosis in mice.