Richard G. Lee

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.

Antisense Oligonucleotide Inhibition of Apolipoprotein C-III Reduces Plasma Triglycerides in Rodents, Nonhuman Primates, and Humans

Rationale: Elevated plasma triglyceride levels have been recognized as a risk factor for the development of coronary heart disease. Apolipoprotein C-III (apoC-III) represents both an independent risk factor and a key regulatory factor of plasma triglyceride concentrations. Furthermore, elevated apoC-III levels have been associated with metabolic syndrome and type 2 diabetes mellitus. To date, no selective apoC-III therapeutic agent has been evaluated in the clinic. Objective: To test the hypothesis that selective inhibition of apoC-III with antisense drugs in preclinical models and in healthy volunteers would reduce plasma apoC-III and triglyceride levels. Methods and Results: Rodent- and human-specific second-generation antisense oligonucleotides were identified and evaluated in preclinical models, including rats, mice, human apoC-III transgenic mice, and nonhuman primates. We demonstrated the selective reduction of both apoC-III and triglyceride in all preclinical pharmacological evaluations. We also showed that inhibition of apoC-III was well tolerated and not associated with increased liver triglyceride deposition or hepatotoxicity. A double-blind, placebo-controlled, phase I clinical study was performed in healthy subjects. Administration of the human apoC-III antisense drug resulted in dose-dependent reductions in plasma apoC-III, concomitant lowering of triglyceride levels, and produced no clinically meaningful signals in the safety evaluations. Conclusions: Antisense inhibition of apoC-III in preclinical models and in a phase I clinical trial with healthy subjects produced potent, selective reductions in plasma apoC-III and triglyceride, 2 known risk factors for cardiovascular disease. This compelling pharmacological profile supports further clinical investigations in hypertriglyceridemic subjects.

Acyl coenzyme A: cholesterol acyltransferase types 1 and 2: structure and function in atherosclerosis.

Two enzymes are responsible for cholesterol ester formation in tissues, acyl coenzyme A : cholesterol acyltransferase types 1 and 2 (ACAT1 and ACAT2). The available evidence suggests different cell locations, membrane orientations, and metabolic functions for each enzyme. ACAT1 and ACAT2 gene disruption experiments in mice have shown complementary results, with ACAT1 being responsible for cholesterol homeostasis in the brain, skin, adrenal, and macrophages. ACAT1 −/− mice have less atherosclerosis than their ACAT1 +/+ counterparts, presumably because of the decreased ACAT activity in the macrophages. By contrast, ACAT2 −/− mice have limited cholesterol absorption in the intestine, and decreased cholesterol ester content in the liver and plasma lipoproteins. Almost no cholesterol esterification was found when liver and intestinal microsomes from ACAT2 −/− mice were assayed. Studies in non-human primates have shown the presence of ACAT1 primarily in the Kupffer cells of the liver, in non-mucosal cell types in the intestine, and in kidney and adrenal cortical cells, whereas ACAT2 is present only in hepatocytes and in intestinal mucosal cells. The membrane topology for ACAT1 and ACAT2 is also apparently different, with ACAT1 having a serine essential for activity on the cytoplasmic side of the endoplasmic reticulum membrane, whereas the analogous serine is present on the lumenal side of the endoplasmic reticulum for ACAT2. Taken together, the data suggest that cholesterol ester formation by ACAT1 supports separate functions compared with cholesterol esterification by ACAT2. The latter enzyme appears to be responsible for cholesterol ester formation and secretion in lipoproteins, whereas ACAT1 appears to function to maintain appropriate cholesterol availability in cell membranes.

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.

CGI-58 knockdown in mice causes hepatic steatosis, but prevents diet-induced obesity and glucose intolerance

Mutations of Comparative Gene Identification-58 (CGI-58) in humans cause triglyceride (TG) accumulation in multiple tissues. Mice genetically lacking CGI-58 die shortly after birth due to a skin barrier defect. To study the role of CGI-58 in integrated lipid and energy metabolism, we utilized antisense oligonucleotides (ASOs) to inhibit CGI-58 expression in adult mice. Treatment with two distinct CGI-58-targeting ASOs resulted in ∼80–95% knockdown of CGI-58 protein expression in both liver and white adipose tissue. In chow-fed mice, ASO-mediated depletion of CGI-58 did not alter weight gain, plasma TG, or plasma glucose, yet raised hepatic TG levels ∼4-fold. When challenged with a high-fat diet (HFD), CGI-58 ASO-treated mice were protected against diet-induced obesity, but their hepatic contents of TG, diacylglycerols, and ceramides were all elevated, and intriguingly, their hepatic phosphatidylglycerol content was increased by 10-fold. These hepatic lipid alterations were associated with significant decreases in hepatic TG hydrolase activity, hepatic lipoprotein-TG secretion, and plasma concentrations of ketones, nonesterified fatty acids, and insulin. Additionally, HFD-fed CGI-58 ASO-treated mice were more glucose tolerant and insulin sensitive. Collectively, this work demonstrates that CGI-58 plays a critical role in limiting hepatic steatosis and maintaining hepatic glycerophospholipid homeostasis and has unmasked an unexpected role for CGI-58 in promoting HFD-induced obesity and insulin resistance.

Plasma Cholesteryl Esters Provided by Lecithin:Cholesterol Acyltransferase and Acyl-Coenzyme A:Cholesterol Acyltransferase 2 Have Opposite Atherosclerotic Potential

Evidence suggests that ACAT2 is a proatherogenic enzyme that contributes cholesteryl esters (CEs) to apoB-containing lipoproteins, whereas LCAT is an antiatherogenic enzyme that facilitates reverse cholesterol transport by esterifying free cholesterol on HDL particles. We hypothesized that deletion of LCAT and ACAT2 would lead to absence of plasma CEs and reduced atherosclerosis. To test this hypothesis, ACAT2−/− LCAT−/− LDLr−/−, ACAT2−/− LDLr−/−, and LCAT−/− LDLr−/− mice were fed a 0.15% cholesterol diet for 20 weeks. In comparison to LDLr−/− mice, the total plasma cholesterol (TPC) of ACAT2−/− LCAT−/− LDLr−/− mice was 67% lower because of the complete absence of plasma CEs, leading to 94% less CE accumulation in the aorta. In the LCAT−/− LDLr−/− mice, TPC and atherosclerosis were significantly higher because of increased accumulations of ACAT2-derived CE. In ACAT2−/− LDLr−/− mice, again compared with LDLr−/− mice, TPC was 19% lower, whereas atherosclerosis was 88% lower. Therefore, the absence of ACAT2 led to a significant reduction in TPC although benefits in reduction of atherosclerosis were much more pronounced. Overall, the data suggest that ACAT2-derived CE is the predominant atherogenic lipid in blood, and that an important goal for prevention of atherosclerosis is to limit ACAT2-derived CE accumulation in lipoproteins.

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.

Targeted Depletion of Hepatic ACAT2-driven Cholesterol Esterification Reveals a Non-biliary Route for Fecal Neutral Sterol Loss

Deletion of acyl-CoA:cholesterol O-acyltransferase 2 (ACAT2) in mice results in resistance to diet-induced hypercholesterolemia and protection against atherosclerosis. Recently, our group has shown that liver-specific inhibition of ACAT2 via antisense oligonucleotide (ASO)-mediated targeting likewise limits atherosclerosis. However, whether this atheroprotective effect was mediated by: 1) prevention of packaging of cholesterol into apoB-containing lipoproteins, 2) augmentation of nascent HDL cholesterol secretion, or 3) increased hepatobiliary sterol secretion was not examined. Therefore, the purpose of these studies was to determine whether hepatic ACAT2 is rate-limiting in all three of these important routes of cholesterol homeostasis. Liver-specific depletion of ACAT2 resulted in reduced packaging of cholesterol into apoB-containing lipoproteins (very low density lipoprotein, intermediate density lipoprotein, and low density lipoprotein), whereas high density lipoprotein cholesterol levels remained unchanged. In the liver of ACAT2 ASO-treated mice, cholesterol ester accumulation was dramatically reduced, yet there was no reciprocal accumulation of unesterified cholesterol. Paradoxically, ASO-mediated depletion of hepatic ACAT2 promoted fecal neutral sterol excretion without altering biliary sterol secretion. Interestingly, during isolated liver perfusion, ACAT2 ASO-treated livers had augmented secretion rates of unesterified cholesterol and phospholipid. Furthermore, we demonstrate that liver-derived cholesterol from ACAT2 ASO-treated mice is preferentially delivered to the proximal small intestine as a precursor to fecal excretion. Collectively, these studies provide the first insight into the hepatic itinerary of cholesterol when cholesterol esterification is inhibited only in the liver, and provide evidence for a novel non-biliary route of fecal sterol loss.

Antisense oligonucleotide lowers plasma levels of apolipoprotein (a) and lipoprotein (a) in transgenic mice.

OBJECTIVES This study sought to assess whether an antisense oligonucleotide (ASO) directed to apolipoprotein (a) [apo(a)] reduces apo(a) and lipoprotein (a) [Lp(a)] levels in transgenic mouse models. BACKGROUND Elevated Lp(a) is a causal, independent, genetic risk factor for cardiovascular disease and myocardial infarction. Effective therapies to specifically lower plasma Lp(a) levels are lacking. METHODS Three transgenic mouse models were utilized: 8K-apo(a) mice expressing 8 kringle IV (KIV) repeats with a single copy of KIV-2; 8K-Lp(a) mice expressing both the 8K apo(a) plus human apolipoprotein B-100; and 12K-apo(a) mice expressing a 12K apo(a) with 3 KIV-2 repeats. The mice were treated intraperitoneally with saline, a control ASO, or ASO 144367 directed to KIV-2 for 4 to 6 weeks. Apo(a), Lp(a), and oxidized phospholipids present on human apoB (OxPL/h-apoB) or apo(a) [OxPL/apo(a)] were measured at baseline and on and off therapy. RESULTS ASO 144367 significantly reduced Lp(a) by 24.8% in 8K-Lp(a) mice, and reduced apo(a) levels by 19.2% in 8K-Lp(a) mice, 30.0% in 8K-apo(a) mice, and 86% in 12K-apo(a) mice; ASO 144367 also significantly reduced OxPL/apoB 22.4% in 8K-Lp(a) mice, and OxPL/apo(a) levels by 19.9% in 8K-Lp(a) mice, 22.1% in 8K-apo(a) mice, and 92.5% in 12K-apo(a) mice (p < 0.004, or less, for all). No significant changes occurred in Lp(a), apo(a), OxPL/apoB, or OxPL/apo(a) levels with control ASO or saline. CONCLUSIONS This study documents the first specific therapy, to our knowledge, for lowering apo(a)/Lp(a) levels and their associated OxPL. A more potent effect was documented in mice expressing apo(a) with multiple KIV-2 repeats. Targeting liver expression of apo(a) with ASOs directed to KIV-2 repeats may provide an effective approach to lower elevated Lp(a) levels in humans.

ACAT2 Is a Target for Treatment of Coronary Heart Disease Associated With Hypercholesterolemia

The inhibition of intracellular cholesterol esterification as a means to prevent atherosclerosis has been considered to have potential for many years. Two different ACAT enzymes were discovered about 7 years ago, and it has become clear that the two enzymes provide separate physiologic functions. Much has been learned from mice with gene deletions for either ACAT1 or ACAT2. Deletion of ACAT2 has consistently been atheroprotective whereas deletion of ACAT1 has been varyingly problematic. ACAT1 functions in converting cellular cholesterol into cholesteryl ester in response to cholesterol abundance inside the cells. In atherosclerotic lesions, where macrophages ingest excess cholesterol, the ability to esterify the newly-acquired cholesterol seems important for cell survival. Inhibition of ACAT1 may bring undesired consequences with destabilization of cellular membrane function upon cholesterol accumulation leading to macrophage cell death. In contrast, ACAT2 is expressed only in hepatocytes and enterocytes, where ACAT1 is silent, and appears to provide cholesteryl esters for transport in lipoproteins. These two cell types have an abundance of additional mechanisms for disposing of cholesterol so that depletion of ACAT2 does not signal apoptosis. At the present time, the bulk of the available data suggest that the strategy seeming to bear the most potential for treatment of coronary heart disease associated with hypercholesterolemia would be to specifically inhibit ACAT2.