Calvin Pan

Genetic Control of Obesity and Gut Microbiota Composition in Response to High-Fat, High-Sucrose Diet in Mice

Obesity is a highly heritable disease driven by complex interactions between genetic and environmental factors. Human genome-wide association studies (GWAS) have identified a number of loci contributing to obesity; however, a major limitation of these studies is the inability to assess environmental interactions common to obesity. Using a systems genetics approach, we measured obesity traits, global gene expression, and gut microbiota composition in response to a high-fat/high-sucrose (HF/HS) diet of more than 100 inbred strains of mice. Here we show that HF/HS feeding promotes robust, strain-specific changes in obesity that are not accounted for by food intake and provide evidence for a genetically determined set point for obesity. GWAS analysis identified 11 genome-wide significant loci associated with obesity traits, several of which overlap with loci identified in human studies. We also show strong relationships between genotype and gut microbiota plasticity during HF/HS feeding and identify gut microbial phylotypes associated with obesity.

A high-resolution association mapping panel for the dissection of complex traits in mice.

Systems genetics relies on common genetic variants to elucidate biologic networks contributing to complex disease-related phenotypes. Mice are ideal model organisms for such approaches, but linkage analysis has been only modestly successful due to low mapping resolution. Association analysis in mice has the potential of much better resolution, but it is confounded by population structure and inadequate power to map traits that explain less than 10% of the variance, typical of mouse quantitative trait loci (QTL). We report a novel strategy for association mapping that combines classic inbred strains for mapping resolution and recombinant inbred strains for mapping power. Using a mixed model algorithm to correct for population structure, we validate the approach by mapping over 2500 cis-expression QTL with a resolution an order of magnitude narrower than traditional QTL analysis. We also report the fine mapping of metabolic traits such as plasma lipids. This resource, termed the Hybrid Mouse Diversity Panel, makes possible the integration of multiple data sets and should prove useful for systems-based approaches to complex traits and studies of gene-by-environment interactions.

Genetic and environmental control of host-gut microbiota interactions

Genetics provides a potentially powerful approach to dissect host-gut microbiota interactions. Toward this end, we profiled gut microbiota using 16s rRNA gene sequencing in a panel of 110 diverse inbred strains of mice. This panel has previously been studied for a wide range of metabolic traits and can be used for high-resolution association mapping. Using a SNP-based approach with a linear mixed model, we estimated the heritability of microbiota composition. We conclude that, in a controlled environment, the genetic background accounts for a substantial fraction of abundance of most common microbiota. The mice were previously studied for response to a high-fat, high-sucrose diet, and we hypothesized that the dietary response was determined in part by gut microbiota composition. We tested this using a cross-fostering strategy in which a strain showing a modest response, SWR, was seeded with microbiota from a strain showing a strong response, A×B19. Consistent with a role of microbiota in dietary response, the cross-fostered SWR pups exhibited a significantly increased response in weight gain. To examine specific microbiota contributing to the response, we identified various genera whose abundance correlated with dietary response. Among these, we chose Akkermansia muciniphila, a common anaerobe previously associated with metabolic effects. When administered to strain A×B19 by gavage, the dietary response was significantly blunted for obesity, plasma lipids, and insulin resistance. In an effort to further understand host-microbiota interactions, we mapped loci controlling microbiota composition and prioritized candidate genes. Our publicly available data provide a resource for future studies.

Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis

We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions.

Network for Activation of Human Endothelial Cells by Oxidized Phospholipids: A Critical Role of Heme Oxygenase 1

Rationale: Oxidized palmitoyl arachidonyl phosphatidylcholine (Ox-PAPC) accumulates in atherosclerotic lesions, is proatherogenic, and influences the expression of more than 1000 genes in endothelial cells. Objective: To elucidate the major pathways involved in Ox-PAPC action, we conducted a systems analysis of endothelial cell gene expression after exposure to Ox-PAPC. Methods and Results: We used the variable responses of primary endothelial cells from 149 individuals exposed to Ox-PAPC to construct a network that consisted of 11 groups of genes, or modules. Modules were enriched for a broad range of Gene Ontology pathways, some of which have not been identified previously as major Ox-PAPC targets. Further validating our method of network construction, modules were consistent with relationships established by cell biology studies of Ox-PAPC effects on endothelial cells. This network provides novel hypotheses about molecular interactions, as well as candidate molecular regulators of inflammation and atherosclerosis. We validated several hypotheses based on network connections and genomic association. Our network analysis predicted that the hub gene CHAC1 (cation transport regulator homolog 1) was regulated by the ATF4 (activating transcription factor 4) arm of the unfolded protein response pathway, and here we showed that ATF4 directly activates an element in the CHAC1 promoter. We showed that variation in basal levels of heme oxygenase 1 (HMOX1) contribute to the response to Ox-PAPC, consistent with its position as a hub in our network. We also identified G-protein–coupled receptor 39 (GPR39) as a regulator of HMOX1 levels and showed that it modulates the promoter activity of HMOX1. We further showed that OKL38/OSGN1 (oxidative stress–induced growth inhibitor), the hub gene in the blue module, is a key regulator of both inflammatory and antiinflammatory molecules. Conclusions: Our systems genetics approach has provided a broad view of the pathways involved in the response of endothelial cells to Ox-PAPC and also identified novel regulatory mechanisms.

Identification of CAD candidate genes in GWAS loci and their expression in vascular cells

Recent genome-wide association studies (GWAS) have identified 35 loci that significantly associate with coronary artery disease (CAD) susceptibility. The majority of the genes represented in these loci have not previously been studied in the context of atherosclerosis. To characterize the roles of these candidate genes in the vessel wall, we determined their expression levels in endothelial, smooth muscle, and macrophage cells isolated from healthy, prelesioned, and lesioned mouse aortas. We also performed expression quantitative locus (eQTL) mapping of these genes in human endothelial cells under control and proatherogenic conditions. Of the 57 genes studied, 31 were differentially expressed in one or more cell types in disease state in mice, and the expression levels of 8 were significantly associated with the CAD SNPs in human cells, 7 of which were also differentially expressed in mice. By integrating human and mouse results, we predict that PPAP2B, GALNT4, MAPKAPK5, TCTN1, SRR, SNF8, and ICAM1 play a causal role in the susceptibility to atherosclerosis through a role in the vasculature. Additionally, we highlight the genetic complexity of a subset of CAD loci through the differential expression of multiple candidate genes per locus and the involvement of genes that lie outside linkage disequilibrium blocks.

Transgenic 6F tomatoes act on the small intestine to prevent systemic inflammation and dyslipidemia caused by Western diet and intestinally derived lysophosphatidic acid

We recently reported that levels of unsaturated lysophosphatidic acid (LPA) in the small intestine significantly correlated with the extent of aortic atherosclerosis in LDL receptor-null (LDLR−/−) mice fed a Western diet (WD). Here we demonstrate that WD increases unsaturated (but not saturated) LPA levels in the small intestine of LDLR−/− mice and causes changes in small intestine gene expression. Confirmation of microarray analysis by quantitative RT-PCR showed that adding transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to WD prevented many WD-mediated small intestine changes in gene expression. If instead of feeding WD, unsaturated LPA was added to chow and fed to the mice: i) levels of LPA in the small intestine were similar to those induced by feeding WD; ii) gene expression changes in the small intestine mimicked WD-mediated changes; and iii) changes in plasma serum amyloid A, total cholesterol, triglycerides, HDL-cholesterol levels, and the fast-performance liquid chromatography lipoprotein profile mimicked WD-mediated changes. Adding Tg6F (but not control tomatoes) to LPA-supplemented chow prevented the LPA-induced changes. We conclude that: i) WD-mediated systemic inflammation and dyslipidemia may be in part due to WD-induced increases in small intestine LPA levels; and ii) Tg6F reduces WD-mediated systemic inflammation and dyslipidemia by preventing WD-induced increases in LPA levels in the small intestine.

Genetic regulation of human adipose microRNA expression and its consequences for metabolic traits

The genetics of messenger RNA (mRNA) expression has been extensively studied in humans and other organisms, but little is known about genetic factors contributing to microRNA (miRNA) expression. We examined natural variation of miRNA expression in adipose tissue in a population of 200 men who have been carefully characterized for metabolic syndrome (MetSyn) phenotypes as part of the Metabolic Syndrome in Men (METSIM) study. We genotyped the subjects using high-density single-nucleotide polymorphism microarrays and quantified the mRNA abundance using genome-wide expression arrays and miRNA abundance using next-generation sequencing. We reliably quantified 356 miRNA species that were expressed in human adipose tissue, a limited number of which made up most of the expressed miRNAs. We mapped the miRNA abundance as an expression quantitative trait and determined cis regulation of expression for nine of the miRNAs and of the processing of one miRNA (miR-28). The degree of genetic variation of miRNA expression was substantially less than that of mRNAs. For the majority of the miRNAs, genetic regulation of expression was independent of the expression of mRNA from which the miRNA is transcribed. We also showed that for 108 miRNAs, mapped reads displayed widespread variation from the canonical sequence. We found a total of 24 miRNAs to be significantly associated with MetSyn traits. We suggest a regulatory role for miR-204-5p which was predicted to inhibit acetyl coenzyme A carboxylase β, a key fatty acid oxidation enzyme that has been shown to play a role in regulating body fat and insulin resistance in adipose tissue.

Genetic Architecture of Insulin Resistance in the Mouse

Insulin resistance (IR) is a complex trait with multiple genetic and environmental components. Confounded by large differences between the sexes, environment, and disease pathology, the genetic basis of IR has been difficult to dissect. Here we examine IR and related traits in a diverse population of more than 100 unique male and female inbred mouse strains after feeding a diet rich in fat and refined carbohydrates. Our results show dramatic variation in IR among strains of mice and widespread differences between sexes that are dependent on genotype. We uncover more than 15 genome-wide significant loci and validate a gene, Agpat5, associated with IR. We also integrate plasma metabolite levels and global gene expression from liver and adipose tissue to identify metabolite quantitative trait loci (mQTL) and expression QTL (eQTL), respectively. Our results provide a resource for analysis of interactions between diet, sex, and genetic background in IR.

Influenza Virus Affects Intestinal Microbiota and Secondary Salmonella Infection in the Gut through Type I Interferons.

Human influenza viruses replicate almost exclusively in the respiratory tract, yet infected individuals may also develop gastrointestinal symptoms, such as vomiting and diarrhea. However, the molecular mechanisms remain incompletely defined. Using an influenza mouse model, we found that influenza pulmonary infection can significantly alter the intestinal microbiota profile through a mechanism dependent on type I interferons (IFN-Is). Notably, influenza-induced IFN-Is produced in the lungs promote the depletion of obligate anaerobic bacteria and the enrichment of Proteobacteria in the gut, leading to a “dysbiotic” microenvironment. Additionally, we provide evidence that IFN-Is induced in the lungs during influenza pulmonary infection inhibit the antimicrobial and inflammatory responses in the gut during Salmonella-induced colitis, further enhancing Salmonella intestinal colonization and systemic dissemination. Thus, our studies demonstrate a systemic role for IFN-Is in regulating the host immune response in the gut during Salmonella-induced colitis and in altering the intestinal microbial balance after influenza infection.