The etiology of non-alcoholic fatty liver disease (NAFLD), the most common form of chronic liver disease, is poorly understood. To understand the causal mechanisms underlying NAFLD, we conducted a multi-omics, multi-tissue integrative study using the Hybrid Mouse Diversity Panel, consisting of ∼100 strains of mice with various degrees of NAFLD. We identified both tissue-specific biological processes and processes that were shared between adipose and liver tissues. We then used gene network modeling to predict candidate regulatory genes of these NAFLD processes, including Fasn, Thrsp, Pklr, and Chchd6. In vivo knockdown experiments of the candidate genes improved both steatosis and insulin resistance. Further in vitro testing demonstrated that downregulation of both Pklr and Chchd6 lowered mitochondrial respiration and led to a shift toward glycolytic metabolism, thus highlighting mitochondria dysfunction as a key mechanistic driver of NAFLD.

Highlights d Sex differences in phenotype and gene expression depend upon genetic background d The gene Lypla1 impacts obesity in a sex-specific manner d Functional analyses reveal sex differences for adipose tissue beiging d Adipose mitochondrial function depends upon gene-by-sex interactions

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.

We report the genetic analysis of a "humanized" hyperlipidemic mouse model for progressive nonalcoholic steatohepatitis (NASH) and fibrosis. Mice carrying transgenes for human apolipoprotein E*3-Leiden and cholesteryl ester transfer protein and fed a "Western" diet were studied on the genetic backgrounds of over 100 inbred mouse strains. The mice developed hepatic inflammation and fibrosis that was highly dependent on genetic background, with vast differences in the degree of fibrosis. Histological analysis showed features characteristic of human NASH, including macrovesicular steatosis, hepatocellular ballooning, inflammatory foci, and pericellular collagen deposition. Time course experiments indicated that while hepatic triglyceride levels increased steadily on the diet, hepatic fibrosis occurred at about 12 weeks. We found that the genetic variation predisposing to NASH and fibrosis differs markedly from that predisposing to simple steatosis, consistent with a multistep model in which distinct genetic factors are involved. Moreover, genome-wide association identified distinct genetic loci contributing to steatosis and NASH. Finally, we used hepatic expression data from the mouse panel and from 68 bariatric surgery patients with normal liver, steatosis, or NASH to identify enriched biological pathways. Conclusion: The pathways showed substantial overlap between our mouse model and the human disease.

Aberrant lipid metabolism promotes the development of skeletal muscle insulin resistance, but the exact identity of lipid-mediated mechanisms relevant to human obesity remains unclear. A comprehensive lipidomic analysis of primary myocytes from individuals that are insulin-sensitive and lean (LN) or insulin-resistant with obesity (OB) revealed several species of lysophospholipids (lyso-PL) that were differentially-abundant. These changes coincided with greater expression of lysophosphatidylcholine acyltransferase 3 (LPCAT3), an enzyme involved in phospholipid transacylation (Lands cycle). Strikingly, mice with skeletal muscle-specific knockout of LPCAT3 (LPCAT3-MKO) exhibited greater muscle lyso-PC/PC, concomitant with improved skeletal muscle insulin sensitivity. Conversely, skeletal muscle-specific overexpression of LPCAT3 (LPCAT3-MKI) promoted glucose intolerance. The absence of LPCAT3 reduced phospholipid packing of cellular membranes and increased plasma membrane lipid clustering, suggesting that LPCAT3 affects insulin receptor phosphorylation by modulating plasma membrane lipid organization. In conclusion, obesity accelerates the skeletal muscle Lands cycle, whose consequence might induce the disruption of plasma membrane organization that suppresses muscle insulin action.

Sex differences in body fat distribution, glucose homeostasis, insulin signaling, ectopic fat accumulation, and lipid metabolism during normal growth and in response to hormonal or nutritional imbalance are mediated partly through sex hormones and the sex chromosome complement. Most of these differences are mediated in a tissue-specific manner. Important future goals are to better understand the interactions between genetic variation and sex differences, and to bring an understanding of sex differences into clinical practice.

The etiology of non-alcoholic fatty liver disease (NAFLD), the most common form of chronic liver disease, is poorly understood. To understand the causal mechanisms underlying NAFLD, we conducted a multi-omics, multi-tissue integrative study using the Hybrid Mouse Diversity Panel (HMDP), consisting of ~100 strains of mice with various degrees of NAFLD. We identified both tissue-specific biological processes as well as processes that were shared between adipose and liver tissues. We then used gene network modeling to predict candidate regulatory genes of these NAFLD processes, including Fasn, Thrsp, Pklr, and Chchd6. In vivo knockdown experiments of the candidate genes improved both steatosis and insulin resistance. Further in vitro testing demonstrated that down regulation of both Pklr and Chchd6 lowered mitochondrial respiration and

ObjectiveLipocalin-2 (LCN2) is a secreted protein involved in innate immunity and has also been associated with several cardiometabolic traits in both mouse and human studies. However, the causal relationship of LCN2 to these traits is unclear, and most studies have examined only males.MethodsUsing adeno-associated viral vectors we expressed LCN2 in either adipose or liver in a tissue specific manner on the background of a whole-body Lcn2 knockout or wildtype mice. Metabolic phenotypes including body weight, body composition, plasma and liver lipids, glucose homeostasis, insulin resistance, mitochondrial phenotyping, and metabolic cage studies were monitored.ResultsWe studied the genetics of LCN2 expression and associated clinical traits in both males and females in a panel of 100 inbred strains of mice (HMDP). The natural variation in Lcn2 expression across the HMDP exhibits high heritability, and genetic mapping suggests that it is regulated in part by Lipin1 gene variation. The correlation analyses revealed striking tissue dependent sex differences in obesity, insulin resistance, hepatic steatosis, and dyslipidemia. To understand the causal relationships, we examined the effects of expression of LCN2 selectively in liver or adipose. On a Lcn2-null background, LCN2 expression in white adipose promoted metabolic disturbances in females but not males. It acted in an autocrine/paracrine manner, resulting in mitochondrial dysfunction and an upregulation of inflammatory and fibrotic genes. On the other hand, on a null background, expression of LCN2 in liver had no discernible impact on the traits examined despite increasing the levels of circulating LCN2 more than adipose LCN2 expression. The mechanisms underlying the sex-specific action of LCN2 are unclear, but our results indicate that adipose LCN2 negatively regulates its receptor, LRP2 (or megalin), and its repressor, ERα, in a female-specific manner and that the effects of LCN2 on metabolic traits are mediated in part by LRP2.ConclusionsFollowing up on our population-based studies, we demonstrate that LCN2 acts in a highly sex- and tissue-specific manner in mice. Our results have important implications for human studies, emphasizing the importance of sex and the tissue source of LCN2.