Mining cholesterol genes from thousands of mouse livers identifies aldolase C as a regulator of cholesterol biosynthesis.

The availability of genome-wide transcriptomic and proteomic datasets is ever-increasing and often not used beyond initial publication. Here, we applied module-based coexpression network analysis to a comprehensive catalog of 35 mouse genome-wide liver expression datasets (encompassing more than 3800 mice) with the goal of identifying and validating unknown genes involved in cholesterol metabolism. From these 35 datasets, we identified a conserved module of genes enriched with cholesterol biosynthetic genes. Using a systematic approach across the 35 datasets, we identified three genes (Rdh11, Echdc1, and Aldoc) with no known role in cholesterol metabolism. We then performed functional validation studies and show that each gene is capable of regulating cholesterol metabolism. For the glycolytic gene, Aldoc, we demonstrate that it contributes to de novo cholesterol biosynthesis and regulates cholesterol and triglyceride levels in mice. As Aldoc is located within a genome-wide significant genome-wide association studies locus for human plasma cholesterol levels, our studies establish Aldoc as a causal gene within this locus. Through our work, we develop a framework for leveraging mouse genome-wide liver datasets for identifying and validating genes involved in cholesterol metabolism.

Lipidomic QTL in Diversity Outbred mice identifies a novel function for α/ß hydrolase domain 2 (Abhd2) as an enzyme that metabolizes phosphatidylcholine and cardiolipin.

We and others have previously shown that genetic association can be used to make causal connections between gene loci and small molecules measured by mass spectrometry in the bloodstream and in tissues. We identified a locus on mouse chromosome 7 where several phospholipids in liver showed strong genetic association to distinct gene loci. In this study, we integrated gene expression data with genetic association data to identify a single gene at the chromosome 7 locus as the driver of the phospholipid phenotypes. The gene encodes α/ß-hydrolase domain 2 (Abhd2), one of 23 members of the ABHD gene family. We validated this observation by measuring lipids in a mouse with a whole-body deletion of Abhd2. The Abhd2KO mice had a significant increase in liver levels of phosphatidylcholine and phosphatidylethanolamine. Unexpectedly, we also found a decrease in two key mitochondrial lipids, cardiolipin and phosphatidylglycerol, in male Abhd2KO mice. These data suggest that Abhd2 plays a role in the synthesis, turnover, or remodeling of liver phospholipids.

Lipidomic QTL in Diversity Outbred mice identifies a novel function for α/ß hydrolase domain 2 ( Abhd2 ) as an enzyme that metabolizes phosphatidylcholine and cardiolipin.

We and others have previously shown that genetic association can be used to make causal connections between gene loci and small molecules measured by mass spectrometry in the bloodstream and in tissues. We identified a locus on mouse chromosome 7 where several phospholipids in liver showed strong genetic association to distinct gene loci. In this study, we integrated gene expression data with genetic association data to identify a single gene at the chromosome 7 locus as the driver of the phospholipid phenotypes. The gene encodes α/ß-hydrolase domain 2 ( Abhd2 ), one of 23 members of the ABHD gene family. We validated this observation by measuring lipids in a mouse with a whole-body deletion of Abhd2 . The Abhd2 KO mice had a significant increase in liver levels of phosphatidylcholine and phosphatidylethanolamine. Unexpectedly, we also found a decrease in two key mitochondrial lipids, cardiolipin and phosphatidylglycerol, in male Abhd2 KO mice. These data suggest that Abhd2 plays a role in the synthesis, turnover, or remodeling of liver phospholipids.

"Humanizing" mouse environments: Humidity, diurnal cycles and thermoneutrality.

Thermoneutral housing has been shown to promote more accurate and robust development of several pathologies in mice. Raising animal housing temperatures a few degrees may create a relatively straightforward opportunity to improve translatability of mouse models. In this commentary, we discuss the changes of physiology induced in mice housed at thermoneutrality, and review techniques for measuring systemic thermogenesis, specifically those affecting storage and mobilization of lipids in adipose depots. Environmental cues are a component of the information integrated by the brain to calculate food consumption and calorie deposition. We show that relative humidity is one of those cues, inducing a rapid sensory response that is converted to a more chronic susceptibility to obesity. Given high inter-institutional variability in the regulation of relative humidity, study reproducibility may be improved by consideration of this factor. We evaluate a "humanized" environmental cycling protocol, where mice sleep in warm temperature housing, and are cool during the wake cycle. We show that this protocol suppresses adaptation to cool exposure, with consequence for adipose-associated lipid storage. To evaluate systemic cues in mice housed at thermoneutral temperatures, we characterized the circulating lipidome, and show that sera are highly depleted in some HDL-associated phospholipids, specifically phospholipids containing the essential fatty acid, 18:2 linoleic acid, and its derivative, arachidonic acid (20:4) and related ether-phospholipids. Given the role of these fatty acids in inflammatory responses, we propose they may underlie the differences in disease progression observed at thermoneutrality.

Pcpe2, a Novel Extracellular Matrix Protein, Regulates Adipocyte SR-BI-Mediated High-Density Lipoprotein Uptake.

Objective: To investigate the role of adipocyte Pcpe2 (procollagen C-endopeptidase enhancer 2) in SR-BI (scavenger receptor class BI)-mediated HDL-C (high-density lipoprotein cholesterol) uptake and contributions to adipose lipid storage. Approach and Results: Pcpe2, a glycoprotein devoid of intrinsic proteolytic activity, is believed to participate in extracellular protein-protein interactions, supporting SR-BI- mediated HDL-C uptake. In published studies, Pcpe2 deficiency increased the development of atherosclerosis by reducing SR-BI-mediated HDL-C catabolism, but the biological impact of this deficiency on adipocyte SR-BI-mediated HDL-C uptake is unknown. Differentiated cells from Ldlr-/-/Pcpe2-/- (Pcpe2-/-) mouse adipose tissue showed elevated SR-BI protein levels, but significantly reduced HDL-C uptake compared to Ldlr-/- (control) adipose tissue. SR-BI-mediated HDL-C uptake was restored by preincubation of cells with exogenous Pcpe2. In diet-fed mice lacking Pcpe2, significant reductions in visceral, subcutaneous, and brown adipose tissue mass were observed, despite elevations in plasma triglyceride and cholesterol concentrations. Significant positive correlations exist between adipose mass and Pcpe2 expression in both mice and humans. Conclusions: Overall, these findings reveal a novel and unexpected function for Pcpe2 in modulating SR-BI expression and function as it relates to adipose tissue expansion and cholesterol balance in both mice and humans.

Cross-species data integration to prioritize causal genes in lipid metabolism.

PURPOSE OF REVIEW: More than one hundred loci have been identified from human genome-wide association studies (GWAS) for blood lipids. Despite the success of GWAS in identifying loci, subsequent prioritization of causal genes related to these loci remains a challenge. To address this challenge, recent work suggests that candidate causal genes within loci can be prioritized through cross-species integration using genome-wide data from the mouse. RECENT FINDINGS: Mouse model systems provide unparalleled access to primary tissues, like the liver, that are not readily available for human studies. Given the key role the liver plays in controlling blood lipid levels and the wealth of liver genome-wide transcript and protein data available in the mouse, these data can be leveraged. Using coexpression network analysis approaches with mouse genome-wide data, coupled with cross-species analysis of human lipid GWAS, causal genes within lipid loci can be prioritized. Prioritization through both mouse and human along with biochemical validation provide a systematic and valuable method to discover lipid metabolism genes. SUMMARY: The prioritization of causal lipid genes within GWAS loci is a challenging process requiring a multidisciplinary approach. Integration of data types across species, such as the mouse, can aid in causal gene prioritization.

Genetic regulation of liver lipids in a mouse model of insulin resistance and hepatic steatosis.

To elucidate the contributions of specific lipid species to metabolic traits, we integrated global hepatic lipid data with other omics measures and genetic data from a cohort of about 100 diverse inbred strains of mice fed a high-fat/high-sucrose diet for 8 weeks. Association mapping, correlation, structure analyses, and network modeling revealed pathways and genes underlying these interactions. In particular, our studies lead to the identification of Ifi203 and Map2k6 as regulators of hepatic phosphatidylcholine homeostasis and triacylglycerol accumulation, respectively. Our analyses highlight mechanisms for how genetic variation in hepatic lipidome can be linked to physiological and molecular phenotypes, such as microbiota composition.

The Genetic Architecture of Carbon Tetrachloride-Induced Liver Fibrosis in Mice.

BACKGROUND & AIMS: Liver fibrosis is a multifactorial trait that develops in response to chronic liver injury. Our aim was to characterize the genetic architecture of carbon tetrachloride (CCl4)-induced liver fibrosis using the Hybrid Mouse Diversity Panel, a panel of more than 100 genetically distinct mouse strains optimized for genome-wide association studies and systems genetics. METHODS: Chronic liver injury was induced by CCl4 injections twice weekly for 6 weeks. Four hundred thirty-seven mice received CCl4 and 256 received vehicle, after which animals were euthanized for liver histology and gene expression. Using automated digital image analysis, we quantified fibrosis as the collagen proportionate area of the whole section, excluding normal collagen. RESULTS: We discovered broad variation in fibrosis among the Hybrid Mouse Diversity Panel strains, demonstrating a significant genetic influence. Genome-wide association analyses revealed significant and suggestive loci underlying susceptibility to fibrosis, some of which overlapped with loci identified in mouse crosses and human population studies. Liver global gene expression was assessed by RNA sequencing across the strains, and candidate genes were identified using differential expression and expression quantitative trait locus analyses. Gene set enrichment analyses identified the underlying pathways, of which stellate cell involvement was prominent, and coexpression network modeling identified modules associated with fibrosis. CONCLUSIONS: Our results provide a rich resource for the design of experiments to understand mechanisms underlying fibrosis and for rational strain selection when testing antifibrotic drugs.

Integrating Mouse and Human Genetic Data to Move beyond GWAS and Identify Causal Genes in Cholesterol Metabolism.

Identifying the causal gene(s) that connects genetic variation to a phenotype is a challenging problem in genome-wide association studies (GWASs). Here, we develop a systematic approach that integrates mouse liver co-expression networks with human lipid GWAS data to identify regulators of cholesterol and lipid metabolism. Through our approach, we identified 48 genes showing replication in mice and associated with plasma lipid traits in humans and six genes on the X chromosome. Among these 54 genes, 25 have no previously identified role in lipid metabolism. Based on functional studies and integration with additional human lipid GWAS datasets, we pinpoint Sestrin1 as a causal gene associated with plasma cholesterol levels in humans. Our validation studies demonstrate that Sestrin1 influences plasma cholesterol in multiple mouse models and regulates cholesterol biosynthesis. Our results highlight the power of combining mouse and human datasets for prioritization of human lipid GWAS loci and discovery of lipid genes.

PGC-1a integrates a metabolism and growth network linked to caloric restriction.

Deleterious changes in energy metabolism have been linked to aging and disease vulnerability, while activation of mitochondrial pathways has been linked to delayed aging by caloric restriction (CR). The basis for these associations is poorly understood, and the scope of impact of mitochondrial activation on cellular function has yet to be defined. Here, we show that mitochondrial regulator PGC-1a is induced by CR in multiple tissues, and at the cellular level, CR-like activation of PGC-1a impacts a network that integrates mitochondrial status with metabolism and growth parameters. Transcriptional profiling reveals that diverse functions, including immune pathways, growth, structure, and macromolecule homeostasis, are responsive to PGC-1a. Mechanistically, these changes in gene expression were linked to chromatin remodeling and RNA processing. Metabolic changes implicated in the transcriptional data were confirmed functionally including shifts in NAD metabolism, lipid metabolism, and membrane lipid composition. Delayed cellular proliferation, altered cytoskeleton, and attenuated growth signaling through post-transcriptional and post-translational mechanisms were also identified as outcomes of PGC-1a-directed mitochondrial activation. Furthermore, in vivo in tissues from a genetically heterogeneous mouse population, endogenous PGC-1a expression was correlated with this same metabolism and growth network. These data show that small changes in metabolism have broad consequences that arguably would profoundly alter cell function. We suggest that this PGC-1a sensitive network may be the basis for the association between mitochondrial function and aging where small deficiencies precipitate loss of function across a spectrum of cellular activities.

Gene-by-Sex Interactions in Mitochondrial Functions and Cardio-Metabolic Traits.

We studied sex differences in over 50 cardio-metabolic traits in a panel of 100 diverse inbred strains of mice. The results clearly showed that the effects of sex on both clinical phenotypes and gene expression depend on the genetic background. In support of this, genetic loci associated with the traits frequently showed sex specificity. For example, Lyplal1, a gene implicated in human obesity, was shown to underlie a sex-specific locus for diet-induced obesity. Global gene expression analyses of tissues across the panel implicated adipose tissue "beiging" and mitochondrial functions in the sex differences. Isolated mitochondria showed gene-by-sex interactions in oxidative functions, such that some strains (C57BL/6J) showed similar function between sexes, whereas others (DBA/2J and A/J) showed increased function in females. Reduced adipose mitochondrial function in males as compared to females was associated with increased susceptibility to obesity and insulin resistance. Gonadectomy studies indicated that gonadal hormones acting in a tissue-specific manner were responsible in part for the sex differences.

DNA Methylation Changes More Slowly Than Physiological States in Response to Weight Loss in Genetically Diverse Mouse Strains.

Responses to a high fat, high sucrose (HFHS) diet vary greatly among inbred strains of mice. We sought to examine the epigenetic (DNA methylation) changes underlying these differences as well as variation in weight loss when switched to a low-fat chow diet. We surveyed DNA methylation from livers of 45 inbred mouse strains fed a HFHS diet for 8 weeks using reduced-representation bisulfite sequencing (RRBS). We observed a total of 1,045,665 CpGs of which 83 candidate sites were significantly associated with HFHS diet. Many of these CpGs correlated strongly with gene expression or clinical traits such as body fat percentage and plasma glucose. Five inbred strains were then studied in the context of weight loss to test for evidence of epigenetic "memory." The mice were first fed a HFHS diet for 6 weeks followed by a low-fat chow diet for 4 weeks. Four of the five strains returned to initial levels of body fat while one strain, A/J, retained almost 50% of the fat gained. A total of 36 of the HFHS diet responsive CpGs exhibited evidence of persistent epigenetic modifications following weight normalization, including CpGs near the genes Scd1 and Cdk1. Our study identifies DNA methylation changes in response to a HFHS diet challenge that revert more slowly than overall body fat percentage in weight loss and provides evidence for epigenetic mediated "memory."

Regulator of Calcineurin 1 helps coordinate whole-body metabolism and thermogenesis.

Increasing non-shivering thermogenesis (NST), which expends calories as heat rather than storing them as fat, is championed as an effective way to combat obesity and metabolic disease. Innate mechanisms constraining the capacity for NST present a fundamental limitation to this approach, yet are not well understood. Here, we provide evidence that Regulator of Calcineurin 1 (RCAN1), a feedback inhibitor of the calcium-activated protein phosphatase calcineurin (CN), acts to suppress two distinctly different mechanisms of non-shivering thermogenesis (NST): one involving the activation of UCP1 expression in white adipose tissue, the other mediated by sarcolipin (SLN) in skeletal muscle. UCP1 generates heat at the expense of reducing ATP production, whereas SLN increases ATP consumption to generate heat. Gene expression profiles demonstrate a high correlation between Rcan1 expression and metabolic syndrome. On an evolutionary timescale, in the context of limited food resources, systemic suppression of prolonged NST by RCAN1 might have been beneficial; however, in the face of caloric abundance, RCAN1-mediated suppression of these adaptive avenues of energy expenditure may now contribute to the growing epidemic of obesity.

A Strategy for Discovery of Endocrine Interactions with Application to Whole-Body Metabolism.

Inter-tissue communication via secreted proteins has been established as a vital mechanism for proper physiologic homeostasis. Here, we report a bioinformatics framework using a mouse reference population, the Hybrid Mouse Diversity Panel (HMDP), which integrates global multi-tissue expression data and publicly available resources to identify and functionally annotate novel circuits of tissue-tissue communication. We validate this method by showing that we can identify known as well as novel endocrine factors responsible for communication between tissues. We further show the utility of this approach by identification and mechanistic characterization of two new endocrine factors. Adipose-derived Lipocalin-5 is shown to enhance skeletal muscle mitochondrial function, and liver-secreted Notum promotes browning of white adipose tissue, also known as "beiging." We demonstrate the general applicability of the method by providing in vivo evidence for three additional novel molecules mediating tissue-tissue interactions.

Genetic, dietary, and sex-specific regulation of hepatic ceramides and the relationship between hepatic ceramides and IR.

Elevated hepatic ceramide levels have been implicated in both insulin resistance (IR) and hepatic steatosis. To understand the factors contributing to hepatic ceramide levels in mice of both sexes, we have quantitated ceramides in a reference population of mice, the Hybrid Mouse Diversity Panel that has been previously characterized for a variety of metabolic syndrome traits. We observed significant positive correlations between Cer(d18:1/16:0) and IR/hepatic steatosis, consistent with previous findings, although the relationship broke down between sexes, as females were less insulin resistant, but had higher Cer(d18:1/16:0) levels than males. The sex difference was due in part to testosterone-mediated repression of ceramide synthase 6. One ceramide species, Cer(d18:1/20:0), was present at higher levels in males and was associated with IR only in males. Clear evidence of gene-by-sex and gene-by-diet interactions was observed, including sex-specific genome-wide association study results. Thus, our studies show clear differences in how hepatic ceramides are regulated between the sexes, which again suggests that the physiological roles of certain hepatic ceramides differ between the sexes.

Genetic Regulation of Adipose Gene Expression and Cardio-Metabolic Traits.

Subcutaneous adipose tissue stores excess lipids and maintains energy balance. We performed expression quantitative trait locus (eQTL) analyses by using abdominal subcutaneous adipose tissue of 770 extensively phenotyped participants of the METSIM study. We identified cis-eQTLs for 12,400 genes at a 1% false-discovery rate. Among an approximately 680 known genome-wide association study (GWAS) loci for cardio-metabolic traits, we identified 140 coincident cis-eQTLs at 109 GWAS loci, including 93 eQTLs not previously described. At 49 of these 140 eQTLs, gene expression was nominally associated (p < 0.05) with levels of the GWAS trait. The size of our dataset enabled identification of five loci associated (p < 5 × 10-8) with at least five genes located >5 Mb away. These trans-eQTL signals confirmed and extended the previously reported KLF14-mediated network to 55 target genes, validated the CIITA regulation of class II MHC genes, and identified ZNF800 as a candidate master regulator. Finally, we observed similar expression-clinical trait correlations of genes associated with GWAS loci in both humans and a panel of genetically diverse mice. These results provide candidate genes for further investigation of their potential roles in adipose biology and in regulating cardio-metabolic traits.

Genetic and hormonal control of hepatic steatosis in female and male mice.

The etiology of nonalcoholic fatty liver disease is complex and influenced by factors such as obesity, insulin resistance, hyperlipidemia, and sex. We now report a study on sex difference in hepatic steatosis in the context of genetic variation using a population of inbred strains of mice. While male mice generally exhibited higher concentration of hepatic TG levels on a high-fat high-sucrose diet, sex differences showed extensive interaction with genetic variation. Differences in percentage body fat were the best predictor of hepatic steatosis among the strains and explained about 30% of the variation in both sexes. The difference in percent gonadal fat and HDL explained 9.6% and 6.7% of the difference in hepatic TGs between the sexes, respectively. Genome-wide association mapping of hepatic TG revealed some striking differences in genetic control of hepatic steatosis between females and males. Gonadectomy increased the hepatic TG to body fat percentage ratio among male, but not female, mice. Our data suggest that the difference between the sexes in hepatic TG can be partly explained by differences in body fat distribution, plasma HDL, and genetic regulation. Future studies are required to understand the molecular interactions between sex, genetics, and the environment.

Hypothalamic transcriptomes of 99 mouse strains reveal trans eQTL hotspots, splicing QTLs and novel non-coding genes.

Previous studies had shown that the integration of genome wide expression profiles, in metabolic tissues, with genetic and phenotypic variance, provided valuable insight into the underlying molecular mechanisms. We used RNA-Seq to characterize hypothalamic transcriptome in 99 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP), a reference resource population for cardiovascular and metabolic traits. We report numerous novel transcripts supported by proteomic analyses, as well as novel non coding RNAs. High resolution genetic mapping of transcript levels in HMDP, reveals both local and trans expression Quantitative Trait Loci (eQTLs) demonstrating 2 trans eQTL 'hotspots' associated with expression of hundreds of genes. We also report thousands of alternative splicing events regulated by genetic variants. Finally, comparison with about 150 metabolic and cardiovascular traits revealed many highly significant associations. Our data provide a rich resource for understanding the many physiologic functions mediated by the hypothalamus and their genetic regulation.

Chromatin variation associated with liver metabolism is mediated by transposable elements.

BACKGROUND: Functional regulatory regions in eukaryotic genomes are characterized by the disruption of nucleosomes leading to accessible chromatin. The modulation of chromatin accessibility is one of the key mediators of transcriptional regulation, and variation in chromatin accessibility across individuals has been linked to complex traits and disease susceptibility. While mechanisms responsible for chromatin variation across individuals have been investigated, the overwhelming majority of chromatin variation remains unexplained. Furthermore, the processes through which the variation of chromatin accessibility contributes to phenotypic diversity remain poorly understood. RESULTS: We profiled chromatin accessibility in liver from seven strains of mice with phenotypic diversity in response to a high-fat/high-sucrose (HF/HS) diet and identified reproducible chromatin variation across the individuals. We found that sites of variable chromatin accessibility were more likely to coincide with particular classes of transposable elements (TEs) than sites with common chromatin signatures. Evolutionarily younger long interspersed nuclear elements (LINEs) are particularly likely to harbor variable chromatin sites. These younger LINEs are enriched for binding sites of immune-associated transcription factors, whereas older LINEs are enriched for liver-specific transcription factors. Genomic region enrichment analysis indicates that variable chromatin sites at TEs may function to regulate liver metabolic pathways. CRISPR-Cas9 deletion of a number of variable chromatin sites at TEs altered expression of nearby metabolic genes. Finally, we show that polymorphism of TEs and differential DNA methylation at TEs can both influence chromatin variation. CONCLUSIONS: Our results demonstrate that specific classes of TEs show variable chromatin accessibility across strains of mice that display phenotypic diversity in response to a HF/HS diet. These results indicate that chromatin variation at TEs is an important contributor to phenotypic variation among populations.

Sex differences and hormonal effects on gut microbiota composition in mice.

We previously reported quantitation of gut microbiota in a panel of 89 different inbred strains of mice, and we now examine the question of sex differences in microbiota composition. When the total population of 689 mice was examined together, several taxa exhibited significant differences in abundance between sexes but a larger number of differences were observed at the single strain level, suggesting that sex differences can be obscured by host genetics and environmental factors. We also examined a subset of mice on chow and high fat diets and observed sex-by-diet interactions. We further investigated the sex differences using gonadectomized and hormone treated mice from 3 different inbred strains. Principal coordinate analysis with unweighted UniFrac distances revealed very clear effects of gonadectomy and hormone replacement on microbiota composition in all 3 strains. Moreover, bile acid analyses showed gender-specific differences as well as effects of gonodectomy, providing one possible mechanism mediating sex differences in microbiota composition.