Imbalances in lipid storage and secretion lead to hepatic steatosis, the accumulation of lipid droplets in hepatocytes 1,2 . Our understanding of the mechanisms that govern the channelling of neutral lipids in hepatocytes towards cytosolic lipid droplets or secreted lipoproteins remains incomplete 3,4 . Here we performed a series of CRISPR–Cas9 screens under different metabolic states that led to the identification of CLCC1 as a critical regulator of neutral lipid storage and secretion in hepatocytes. Loss of CLCC1 resulted in the buildup of large lipid droplets in hepatoma cells and Clcc1 knockout in mice caused liver steatosis. Lipid droplets were present in the lumen of the endoplasmic reticulum of the Clcc1 -knockout hepatocytes and exhibited properties of lipoproteins, indicating a profound shift in neutral lipid flux. The loss of CLCC1 also led to the accumulation of nuclear membrane herniations accompanied by a reduction in nuclear pores. Remote homology searches identified a domain in CLCC1 that is homologous to yeast Brl1 and Brr6, factors that promote nuclear envelope fusion during nuclear pore complex assembly. Molecular dynamics simulations and mutagenesis studies support a model in which CLCC1 mediates membrane bending and fusion. We propose that CLCC1 mediates membrane fusion to promote hepatic neutral lipid flux and nuclear pore complex assembly.

Hypercholesterolemia contributes to the development of atherosclerosis and is a major risk factor for cardiovascular diseases (CVD). Dietary fiber can attenuate CVD, at least in part, by serving as a fermentable substrate for gut bacteria, leading to the production of short-chain fatty acids (SCFAs), such as butyrate and propionate, which have been linked to atheroprotective effects. SCFAs are sensed by G-protein coupled receptors including GPR41, GPR43, and GPR109A. To explore the role of these receptors in hypercholesterolemia and CVD, we examined atherosclerosis progression and lipid metabolism in Gpr41-/-, Gpr43-/-, and Gpr109a-/- mice using a proprotein convertase subtilisin/kexin type 9 adeno-associated virus (PCSK9-AAV) model of hypercholesterolemia. Deficiency of any single SCFA receptor did not significantly affect atherosclerotic plaque burden compared with wild-type (WT) littermates. However, male Gpr41-/- mice exhibited decreased gonadal fat, plasma triacylglycerol, and low-density lipoprotein cholesterol levels compared to their WT littermates. GPR41 deficiency in males was also associated with increased cecal propionate levels, reduced ileal expression of nutrient transporters such as Npc1l1 and a trend toward increased gut motility. In addition, male Gpr41-/- mice displayed altered gut microbiota composition and lower levels of microbially generated bile acids relative to their WT counterparts. Together, these findings highlight GPR41 as a key intestinal chemosensor regulating nutrient uptake, lipid storage, and microbiota composition.

Reduced estrogen action is associated with obesity and insulin resistance. However, the cell and tissue-specific actions of estradiol in maintaining metabolic health remain inadequately understood, especially in men. We observed that skeletal muscle ESR1/Esr1 (encodes estrogen receptor α [ERα]) is positively correlated with insulin sensitivity and metabolic health in humans and mice. Because skeletal muscle is a primary tissue involved in oxidative metabolism and insulin sensitivity, we generated muscle-selective Esr1 loss- and gain-of-expression mouse models. We determined that Esr1 links mitochondrial DNA replication and cristae-nucleoid architecture with metabolic function and insulin action in the skeletal muscle of male mice. Overexpression of human ERα in muscle protected male mice from diet-induced disruption of metabolic health and enhanced mitochondrial adaptation to exercise training intervention. Our findings indicate that muscle expression of Esr1 is critical for the maintenance of mitochondrial function and metabolic health in males and that tissue-selective activation of ERα can be leveraged to combat metabolic-related diseases in both sexes.

The lactating mammary gland strongly induces de novo lipogenesis (DNL) to support the synthesis of fatty acids, triglycerides, and cholesterol found within milk. In monogastric species, glucose is a major substrate utilized for DNL within the lactating mammary gland and must be efficiently taken up and processed to supply cytosolic acetyl-CoA for DNL. Along with the enzymes of the DNL pathway, the glycolytic enzyme, Aldolase C (Aldoc), is transcriptionally upregulated and is highly expressed during lactation in the mammary gland, suggesting a role for Aldoc in lactation. Aldoc is also a transcriptional target of the sterol regulatory element binding proteins 1 and 2 (Srebp1 and Srebp2), which transcriptionally regulate enzymes within the DNL pathway and has recently been shown to regulate plasma cholesterol and triglycerides. Here, we investigate the role of Aldoc in lactation, by utilizing a whole-body Aldoc knockout mouse. Our results demonstrate that Aldoc has a significant impact on lactation, whereby pups nursing from Aldoc-/- dams have reduced body weight. Biochemical analysis of milk identified that milk from Aldoc-/- dams have significantly higher galactose, lower lactose, and cholesterol content. Mass spectrometry analysis of milk lipids from Aldoc-/- dams revealed significantly lower quantities of medium and long chain fatty acid containing triglycerides, which has direct implications on lactation as these are the predominant triglycerides synthesized from glucose in human mammary gland. Overall, our results provide functional evidence for the contribution of Aldoc in mammary gland lactose and lipid synthesis during lactation.

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 genomewide association studies locus for human plasma cholesterol levels, our studies establish Aldoc as a causal gene within this locus.(jlr) Through our work, we develop a framework for leveraging mouse genome-wide liver datasets for identifying and validating genes involved in cholesterol metabolism.

Agpat5 (1-acylglycerol-3-phosphate O-acyltransferase 5) is a broadly expressed lipid regulatory enzyme involved in glycerophospholipid metabolism. Multiple genetic studies in mice and humans have identified that Agpat5 is associated with plasma insulin, cholesterol, and alanine aminotransferase levels. Despite the strong genetic evidence on Agpat5, no study has investigated its liver-specific role in physiology. Here, we conducted a series of metabolic studies under four distinct dietary conditions to assess the impact of liver-specific Agpat5 deletion on plasma insulin levels, glucose tolerance, plasma cholesterol levels, and hepatic steatosis. Liver-specific deletion of Agpat5 did not affect plasma insulin levels, glucose tolerance, plasma cholesterol levels, or hepatic steatosis in mice fed a chow diet, high-fat diet, or Western diet. However, when mice consumed a chow diet combined with liquid sucrose, liver-specific deletion of Agpat5 resulted in significantly decreased plasma insulin levels and improved glucose tolerance without alterations in body weight or fat mass. Using global lipidomics, we identified that Agpat5 specifically modulated levels of phosphatidylglycerol and cardiolipin within the livers of mice consuming liquid sucrose. Overall, our findings indicate a liver-specific role of Agpat5 in contributing to hyperinsulinemia and glucose tolerance in the absence of body weight changes when consuming liquid sucrose.

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/β-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.

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.

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.

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.

Nearly 40 percent of adult Americans suffer from high blood cholesterol levels, which is directly associated with cardiovascular disease (CVD) risk. Genetics and environmental factors play an impor...

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.

Hyperinsulinemia in association with obesity is a key contributor to type 2 diabetes. Using a mouse genetic reference population, we identified Agpat5, 1-acylglycerol-3-phosphate O-acyltransferase 5, as a gene associated with plasma insulin levels after high fat feeding. Using a genetic interaction analysis, we also uncovered a relationship between Agpat5 and Foxo1, Forkhead Box O1. Agpat5 is an acyltransferase that produces diverse phospholipids by esterifying lysophospholipids. Foxo1 is an insulin responsive transcription factor. To biologically validate the role of Agpat5 in obesity associated hyperinsulinemia, we treated obese high fat fed mice with an antisense oligonucleotide (ASO) that targets Agpat5 to inhibit its gene expression. We show that Agpat5 ASO treated obese mice have significantly lower fed and fasting plasma insulin, as well as improved glucose tolerance and reduced Foxo1 protein. Analysis of insulin secretion and clearance indicates that Agpat5 ASO treated mice clear insulin more efficiently, as assessed by c-peptide to insulin ratio. To understand the tissue-specific roles of Agpat5 in obesity, we generated liver- and adipose- specific knockout mice. We show that hepatic Agpat5 promotes hepatic insulin resistance and hyperinsulinemia through a mechanism involving hepatic Foxo1. Furthermore, we show that adipose Agpat5 promotes hyperinsulinemia and adiposity. Our studies suggest that Agpat5 promotes hyperinsulinemia in obesity through regulation of Foxo1 signaling and insulin clearance. Disclosure S.L. St. Clair: None. S.L. Belisle: None. S. Bruggeman: None. F.B. Leyva Jaimes: None. B.A. Burgess: None. Z. Li: None. B. Parks: None.

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.

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.

There is increasing need to understand the molecular mechanisms contributing to obesity and type 2 diabetes. Agpat5, 1-acylglycerol-3-phosphate O-acyltransferase 5, is a lipid acyltransferase that esterifies the SN2 position of lysophospholipids to produce phospholipids. We previously identified Agpat5 within a genome-wide significant quantitative trait locus (QTL) associated with plasma insulin levels after high fat feeding in a mouse genetic reference population. We validated the effect of Agpat5, showing in multiple mouse models that treatment with Agpat5 antisense oligonucleotide (ASO) reduces plasma insulin after high-fat feeding. To investigate the hepatic role of Agpat5, we developed a liver-specific Agpat5 knockout mouse model. After 12 weeks of high-fat feeding, liver-specific Agpat5 knockout mice have significantly reduced fasting plasma insulin relative to control mice. Additionally, liver-specific Agpat5 knockout mice have significantly reduced hepatic triglycerides and are protected from hepatic steatosis. Investigating the effect of Agpat5 on hepatic insulin resistance, we found that loss of Agpat5 improves insulin signaling and reduces Foxo1 protein levels. Our studies suggest that hepatic Agpat5 may serve as a link between lipid metabolism and insulin signaling to regulate plasma insulin in obesity. Thus, drug therapies targeting Agpat5 could be developed to treat type 2 diabetes in obese populations. Disclosure S.L. St. Clair: None. S.L. Belisle: None. F.B. Leyva Jaimes: None. Z. Li: None. B. Parks: None. Funding National Institutes of Health

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.”

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.

Genetic factors play an important role in contributing to variation in plasma lipid levels across the human population. Large genome-wide association studies (GWAS) in humans have identified more than 100 loci significantly associated with plasma levels of low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, total cholesterol (TC), and triglycerides. To prioritize genes that are involved in cholesterol metabolism, we developed a systematic approach to leverage genome-wide liver transcriptomic and proteomic data from multiple mouse reference populations along with human lipid GWAS data. We constructed global co-expression networks from twelve distinct mouse liver datasets, encompassing more than 800 unique mice and identified a conserved module of genes highly enriched for cholesterol biosynthesis. Based on replication across datasets and presence in transcript and protein, we prioritized 112 unique genes. Intersection of these 112 prioritized genes with human GWAS data for LDL, HDL, TC, and triglycerides identified 54 genes to be within 100 kilobases of a significant or suggestive significant single nucleotide polymorphism (SNP). Out of the 54 identified genes that overlap with human GWAS data, 29 have well documented biological roles in cholesterol metabolism, such as LDLR , PCSK9 , and INSIG1 . With the 25 identified genes with no described role in cholesterol metabolism, we tested for transcriptional regulation to cholesterol and performed a functional screen by siRNA knockdown. From this analysis, we identified twelve genes that show transcriptional regulation to cholesterol levels and nine genes that are able to modulate cholesterol metabolism when targeted in vitro with siRNA. Five genes out of the 25 prioritized genes show both transcriptional regulation to cholesterol and ability to modulate cholesterol metabolism in vitro. One of these genes, Sestrin1 , we validate in vivo and in vitro as a modulator of cholesterol metabolism. Collectively, through a systematic approach we have identified 25 highly prioritized genes that have no documented role in cholesterol metabolism, five genes are transcriptionally regulated by cholesterol and influence cholesterol metabolism.