Thomas Q. de Aguiar Vallim

Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation.

Circulating trimethylamine-N-oxide (TMAO) levels are strongly associated with atherosclerosis. We now examine genetic, dietary, and hormonal factors regulating TMAO levels. We demonstrate that two flavin mono-oxygenase family members, FMO1 and FMO3, oxidize trimethylamine (TMA), derived from gut flora metabolism of choline, to TMAO. Further, we show that FMO3 exhibits 10-fold higher specific activity than FMO1. FMO3 overexpression in mice significantly increases plasma TMAO levels while silencing FMO3 decreases TMAO levels. In both humans and mice, hepatic FMO3 expression is reduced in males compared to females. In mice, this reduction in FMO3 expression is due primarily to downregulation by androgens. FMO3 expression is induced by dietary bile acids by a mechanism that involves the farnesoid X receptor (FXR), a bile acid-activated nuclear receptor. Analysis of natural genetic variation among inbred strains of mice indicates that FMO3 and TMAO are significantly correlated, and TMAO levels explain 11% of the variation in atherosclerosis.

Pleiotropic Roles of Bile Acids in Metabolism

Enzymatic oxidation of cholesterol generates numerous distinct bile acids that function both as detergents that facilitate digestion and absorption of dietary lipids, and as hormones that activate four distinct receptors. Activation of these receptors alters gene expression in multiple tissues, leading to changes not only in bile acid metabolism but also in glucose homeostasis, lipid and lipoprotein metabolism, energy expenditure, intestinal motility and bacterial growth, inflammation, liver regeneration, and hepatocarcinogenesis. This review covers the roles of specific bile acids, synthetic agonists, and their cognate receptors in controlling these diverse functions, as well as their current use in treating human diseases.

The TMAO-Generating Enzyme Flavin Monooxygenase 3 Is a Central Regulator of Cholesterol Balance

Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here, we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT) and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic endoplasmic reticulum (ER) stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation.

Role of ABC transporters in lipid transport and human disease

Almost half of the 48 human ATP-binding cassette (ABC) transporter proteins are thought to facilitate the ATP-dependent translocation of lipids or lipid-related compounds. Such substrates include cholesterol, plant sterols, bile acids, phospholipids, and sphingolipids. Mutations in a substantial number of the 48 human ABC transporters have been linked to human disease. Indeed the finding that 12 diseases have been associated with abnormal lipid transport and/or homeostasis demonstrates the importance of this family of transporters in cell physiology. This review highlights the role of ABC transporters in lipid transport and movement, in addition to discussing their roles in cellular homeostasis and inherited disorders.

MicroRNA-144 regulates hepatic ATP binding cassette transporter A1 and plasma high-density lipoprotein after activation of the nuclear receptor farnesoid X receptor.

Rationale: The bile acid receptor farnesoid X receptor (FXR) regulates many aspects of lipid metabolism by variouscomplex and incompletely understood molecular mechanisms. We set out to investigate the molecular mechanisms for FXR-dependent regulation of lipid and lipoprotein metabolism. Objective: To identify FXR-regulated microRNAs that were subsequently involved in regulating lipid metabolism. Methods and Results: ATP binding cassette transporter A1 (ABCA1) is a major determinant of plasma high-density lipoprotein (HDL)-cholesterol levels. Here, we show that activation of the nuclear receptor FXR in vivo increases hepatic levels of miR-144, which in turn lowers hepatic ABCA1 and plasma HDL levels. We identified 2 complementary sequences to miR-144 in the 3′ untranslated region of ABCA1 mRNA that are necessary for miR-144–dependent regulation. Overexpression of miR-144 in vitro decreased both cellular ABCA1 protein and cholesterol efflux to lipid-poor apolipoprotein A-I protein, whereas overexpression in vivo reduced hepatic ABCA1 protein and plasma HDL-cholesterol. Conversely, silencing miR-144 in mice increased hepatic ABCA1 protein and HDL-cholesterol. In addition, we used tissue-specific FXR-deficient mice to show that induction of miR-144 and FXR-dependent hypolipidemia requires hepatic, but not intestinal, FXR. Finally, we identified functional FXR response elements upstream of the miR-144 locus, consistent with direct FXR regulation. Conclusions: We have identified a novel pathway involving FXR, miR-144, and ABCA1 that together regulate plasma HDL-cholesterol.Rationale: The bile acid receptor Farnesoid-X-Receptor (FXR) regulates many aspects of lipid metabolism by various complex and not fully understood molecular mechanisms. We set out to investigate the molecular mechanisms for FXR-dependent regulation of lipid and lipoprotein metabolism. Objective: To identify FXR-regulated microRNAs that were subsequently involved in regulating lipid metabolism. Methods and Results: ATP binding cassette transporter A1 (ABCA1) is a major determinant of plasma High Density Lipoprotein (HDL)-cholesterol levels. Here we show that activation of the nuclear receptor FXR in vivo increases hepatic levels of miR-144, which in turn lower hepatic ABCA1 and plasma HDL levels. We identified two complementary sequences to miR-144 in the 39 untranslated region (UTR) of ABCA1 mRNA that are necessary for miR-144-dependent regulation. Overexpression of miR-144 in vitro decreased both cellular ABCA1 protein and cholesterol efflux to lipid-poor apolipoprotein A-I (ApoA-I) protein, whilst overexpression in vivo reduced hepatic ABCA1 protein and plasma HDL-cholesterol. Conversely, silencing miR-144 in mice increased hepatic ABCA1 protein and HDL-cholesterol. In addition, we utilized tissue-specific FXR deficient mice to show that induction of miR-144 and FXR-dependent hypolipidemia requires hepatic, but not intestinal FXR. Finally, we identified functional FXR response elements (FXREs) upstream of the miR-144 locus, consistent with direct FXR regulation. Conclusions: We have identified a novel pathway involving FXR, miR-144 and ABCA1 that together regulate plasma HDL cholesterol.

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.

Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity

The nuclear receptor, farnesoid X receptor (FXR, NR1H4), is known to regulate cholesterol, bile acid, lipoprotein, and glucose metabolism. In the current study, we provide evidence to support a role for FXR in hepatoprotection from acetaminophen (APAP)-induced toxicity. Pharmacological activation of FXR induces the expression of several genes involved in phase II and phase III xenobiotic metabolism in wild-type, but not Fxr(-/-) mice. We used chromatin immunoprecipitation-based genome-wide response element analyses coupled with luciferase reporter assays to identify functional FXR response elements within promoters, introns, or intragenic regions of these genes. Consistent with the observed transcriptional changes, FXR gene dosage is positively correlated with the degree of protection from APAP-induced hepatotoxicity in vivo. Further, we demonstrate that pretreatment of wild-type mice with an FXR-specific agonist provides significant protection from APAP-induced hepatotoxicity. Based on these findings, we propose that FXR plays a role in hepatic xenobiotic metabolism and, when activated, provides hepatoprotection against toxins such as APAP.

Feedback modulation of cholesterol metabolism by the lipid-responsive non-coding RNA LeXis

The liver X receptors (LXRs) are transcriptional regulators of cellular and systemic cholesterol homeostasis. In the setting of cholesterol excess, LXR activation induces the expression of a battery of genes involved in cholesterol efflux 1, facilities cholesterol esterification by promoting fatty acid synthesis 2, and inhibits cholesterol uptake by the low-density lipoprotein receptor (LDLR)3. The fact that sterol content is maintained in a narrow range in most cell types and in the organism as a whole suggests that extensive crosstalk between regulatory pathways must exist. However, the molecular mechanisms that integrate LXRs with other lipid metabolic pathways, are incompletely understood. Here we show that ligand activation of LXRs in liver not only promotes cholesterol efflux, but also simultaneously inhibits cholesterol biosynthesis. We further identify the long non-coding RNA LeXis as one mediator of this effect. Hepatic LeXis expression is robustly induced in response to western diet feeding or pharmacologic LXR activation. Raising or lowering the levels of LeXis in liver affects the expression of cholesterol biosynthetic genes, and the levels of cholesterol in the liver and plasma. LeXis interacts with and affects the DNA interactions of Raly, a heterogeneous ribonucleoprotein that is required for the maximal expression of cholesterologenic genes in mouse liver. These studies outline a regulatory role for a non-coding RNA in lipid metabolism and advance our understanding of the mechanisms orchestrating sterol homeostasis.

Skeletal muscle action of estrogen receptor α is critical for the maintenance of mitochondrial function and metabolic homeostasis in females

ERα action in skeletal muscle is involved in the preservation of mitochondrial health and insulin sensitivity in female mice and can serve as a defense against metabolic disease in women. Postmenopausal muscle and mitochondrial mayhem Menopause ushers in a host of changes that range from unpleasant to undesirable. One undesirable shift is a loss of protection against insulin resistance, which brings with it a constellation of consequences in the form of chronic disease associated with metabolic dysfunction. Now, Ribas et al. investigate the mechanism underlying the postmenopausal chinks in a woman’s energy homeostasis armor. The estrogen receptor (ER) is known to participate in the preservation of mitochondrial health and insulin sensitivity in mice, but the precise tissue-specific mechanisms remain unclear. Because skeletal muscle is a main tissue responsible for insulin-stimulated glucose disposal, the authors first showed that ERα expression in muscle correlated with metabolic health in human females. They then created a muscle-specific ERα knockout (MERKO) mouse and found that glucose homeostasis was disrupted, fat accumulation increased, and mitochondrial function impaired. These findings imply that ERα action in skeletal muscle helps maintain mitochondrial function and metabolic homeostasis in females. Impaired estrogen receptor α (ERα) action promotes obesity and metabolic dysfunction in humans and mice; however, the mechanisms underlying these phenotypes remain unknown. Considering that skeletal muscle is a primary tissue responsible for glucose disposal and oxidative metabolism, we established that reduced ERα expression in muscle is associated with glucose intolerance and adiposity in women and female mice. To test this relationship, we generated muscle-specific ERα knockout (MERKO) mice. Impaired glucose homeostasis and increased adiposity were paralleled by diminished muscle oxidative metabolism and bioactive lipid accumulation in MERKO mice. Aberrant mitochondrial morphology, overproduction of reactive oxygen species, and impairment in basal and stress-induced mitochondrial fission dynamics, driven by imbalanced protein kinase A–regulator of calcineurin 1–calcineurin signaling through dynamin-related protein 1, tracked with reduced oxidative metabolism in MERKO muscle. Although muscle mitochondrial DNA (mtDNA) abundance was similar between the genotypes, ERα deficiency diminished mtDNA turnover by a balanced reduction in mtDNA replication and degradation. Our findings indicate the retention of dysfunctional mitochondria in MERKO muscle and implicate ERα in the preservation of mitochondrial health and insulin sensitivity as a defense against metabolic disease in women.

The chromosome 3p21.3-encoded gene, LIMD1, is a critical tumor suppressor involved in human lung cancer development

Loss of heterozygosity (LOH) and homozygous deletions at chromosome 3p21.3 are common in both small and nonsmall cell lung cancers, indicating the likely presence of tumor suppressor genes (TSGs). Although genetic and epigenetic changes within this region have been identified, the functional significance of these changes has not been explored. Concurrent protein expression and genetic analyses of human lung tumors coupled with functional studies have not been done. Here, we show that expression of the 3p21.3 gene, LIMD1, is frequently down-regulated in human lung tumors. Loss of LIMD1 expression occurs through a combination of gene deletion, LOH, and epigenetic silencing of transcription without evidence for coding region mutations. Experimentally, LIMD1 is a bona fide TSG. Limd1−/− mice are predisposed to chemical-induced lung adenocarcinoma and genetic inactivation of Limd1 in mice heterozygous for oncogenic K-RasG12D markedly increased tumor initiation, promotion, and mortality. Thus, we conclude that LIMD1 is a validated chromosome 3p21.3 tumor-suppressor gene involved in human lung cancer development. LIMD1 is a LIM domain containing adapter protein that localizes to E-cadherin cell–cell adhesive junctions, yet also translocates to the nucleus where it has been shown to function as an RB corepressor. As such, LIMD1 has the potential to communicate cell extrinsic or environmental cues with nuclear responses.