BACKGROUNDPreclinical studies suggest that cholesterol accumulation leads to insulin resistance. We previously reported that alterations in a monocyte cholesterol metabolism transcriptional network (CMTN) - suggestive of cellular cholesterol accumulation - were cross-sectionally associated with obesity and type 2 diabetes (T2D). Here, we sought to determine whether the CMTN alterations independently predict incident prediabetes/T2D risk, and correlate with cellular cholesterol accumulation.METHODSMonocyte mRNA expression of 11 CMTN genes was quantified among 934 Multi-Ethnic Study of Atherosclerosis (MESA) participants free of prediabetes/T2D; cellular cholesterol was measured in a subset of 24 monocyte samples.RESULTSDuring a median 6-year follow-up, lower expression of 3 highly correlated LXR target genes - ABCG1 and ABCA1 (cholesterol efflux) and MYLIP (cholesterol uptake suppression) - and not other CMTN genes, was significantly associated with higher risk of incident prediabetes/T2D. Lower expression of the LXR target genes correlated with higher cellular cholesterol levels (e.g., 47% of variance in cellular total cholesterol explained by ABCG1 expression). Further, adding the LXR target genes to overweight/obesity and other known predictors significantly improved prediction of incident prediabetes/T2D.CONCLUSIONThese data suggest that the aberrant LXR/ABCG1-ABCA1-MYLIP pathway (LAAMP) is a major T2D risk factor and support a potential role for aberrant LAAMP and cellular cholesterol accumulation in diabetogenesis.FUNDINGThe MESA Epigenomics and Transcriptomics Studies were funded by NIH grants 1R01HL101250, 1RF1AG054474, R01HL126477, R01DK101921, and R01HL135009. This work was supported by funding from NIDDK R01DK103531 and NHLBI R01HL119962.

Lawrence (Larry) Lee Rudel, PhD, died on August 29, 2019, in Winston-Salem, North Carolina, at the age of 77, after an extended illness. Larry was an internationally-renowned lipid scientist and biochemist, whose more than 50 years of scientific discovery focused on cholesterol metabolism and cardiovascular disease. His long and productive career was marked by seminal scientific discoveries, rigorous training of graduate students and postdoctoral fellows, and service to his institution, Wake Forest School of Medicine, as well as the National Institutes of Health (NIH), the American Heart Association (AHA), and the Kern Lipid Conference. Larry also served on the Editorial Board of the Journal of Lipid Research for 36 years. Larry Rudel was born on September 19, 1941, in Salt Lake City, Utah, to Lloyd and Grace Tussey Rudel. He spent his early years in Chappell, Nebraska, and Loveland, Colorado, and graduated from Loveland High School in 1959. He attended college at Colorado State University, where he earned a BS in Physical Sciences in 1963, and graduate school at the University of Arkansas Medical Center, where he received an MS (1965) and a PhD (1969) in Biochemistry under the guidance of Manford D. Morris. Subsequently, he completed postdoctoral training with James Felts at the Banting and Best Institute in Toronto, Ontario, Canada (1970–1971) and the Cardiovascular Research Institute of the University of California, San Francisco (1972–1973). Larry joined the faculty at Bowman Gray School of Medicine, later renamed Wake Forest School of Medicine, in 1973 and rose rapidly to the rank of Professor of Pathology-Section on Lipid Sciences in 1982, with a cross-appointment in the Department of Biochemistry. During his distinguished career at Wake Forest School of Medicine, Larry was Section Head of Lipid Sciences in the Department of Pathology (2005–2014) and was awarded the Established Investigator in Basic Sciences in 2001 for his outstanding research achievements. He retired in April 2018 as Professor of Internal Medicine-Section on Molecular Medicine and Biochemistry. Larry's research and training activities left Wake Forest School of Medicine with an exemplary and, in many ways, unprecedented legacy in the field of lipid sciences and cardiometabolic disease research. Larry's early studies as a graduate student and postdoctoral fellow foreshadowed his illustrious career. He was keenly interested in how dietary cholesterol was absorbed by the intestine and assimilated in the body. His 1972 Journal of Clinical Investigation publication demonstrated, using a rabbit model, that dietary cholesterol was preferentially esterified during absorption and assembly into intestinal chylomicrons and very low density lipoproteins and suggested that a mechanism exists to maintain free and esterified exogenous cholesterol in a chemically distinct pool from endogenous biliary cholesterol during assembly of intestinal lipoproteins. These early studies ignited a passion in Larry and a lifelong quest to understand cholesteryl ester metabolism and its role in the development of coronary heart disease, which was responsible for the early death of his father. Larry made two additional contributions as a graduate student and postdoctoral fellow. The first was a reliable and simple colorimetric assay to measure cholesterol accurately and precisely. This discovery, which predated the enzymatic cholesterol assays used commonly now, was key to his progress and that of others at the time. Larry also pioneered a rapid and gentle method for preparative plasma lipoprotein isolation using gel filtration chromatography, demonstrating that his method was a viable and improved alternative to sequential ultracentrifugation, which was the only preparative method available in the 1970s. This approach has since become the method of choice for plasma lipoprotein fractionation. As a new faculty member at Wake Forest University, Larry began investigating the role of dietary cholesterol on atherosclerosis progression using nonhuman primates. His early interests in dietary cholesterol absorption led to the development of a lymph duct cannulation procedure that allowed compositional and metabolic studies using nascent intestinal lipoproteins not previously exposed to plasma. However, Larry's investigative attention refocused on the liver with the discovery that precursors of atherogenic LDLs in plasma were secreted by the liver, not the intestine. Larry subsequently pioneered studies demonstrating that nonhuman primates consuming a Western-type diet and who developed atherosclerosis had larger saturated and monounsaturated cholesteryl ester-enriched LDL. He and his collaborators went on to show that the cholesteryl ester content of these large circulating LDL particles was due to the activity of hepatic acyl CoA-cholesterol acyltransferase 2 (ACAT2), which is exclusively expressed in hepatocytes and intestinal epithelial cells. Using molecular and cellular biology studies as well as genetically modified mouse models, Larry and his team were able to establish a direct relationship between hepatic ACAT2 activity and atherosclerosis. In the latter years of his career, Larry demonstrated the efficacy of a small molecule inhibitor of ACAT2 as a treatment for coronary heart disease in preclinical models. During his career, Larry was an insightful and highly productive scientist. He published 243 peer-reviewed manuscripts, 19 book chapters, and delivered 164 scientific presentations. Larry's research program was continuously funded by the NIH from 1974 to 2014, including his 15 years as Project Leader of an NHLBI-supported Specialized Center of Research in Arteriosclerosis and 20 years as Program Director of an NHLBI program project. His leadership of the program project and the Section on Lipid Sciences resulted in the assembly of what became an internationally recognized and respected team of lipid scientists. Larry's scientific legacy also lives on through his trainees. He directly mentored seventeen PhD students, three MS students, and twenty-nine postdoctoral fellows, and indirectly impacted all trainees and faculty in the Lipid Sciences Program by fostering an extremely rich learning environment. Whether it was informal gatherings at Larry's house, spirited scientific discussions in the weekly program project meetings, or personalized mentoring sessions, Larry was inspiring in the development and support of research careers. Indeed, many of his trainees have gone on to prominent positions in academia and industry, and have made and continue to make critical discoveries in cardiometabolic disease etiology and treatment research. In recognition of his mentoring, Larry received the AHA Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB) Women's Leadership Committee Mentor of Women Award in 2013. Larry also served the greater scientific community. He was an active reviewer for the NIH and served as a member and then Chair of the Metabolism Study Section from 1986−1990. Larry was also very active in the AHA, serving on many committees from 1979−2012, culminating with the position of Chair of the ATVB council (2008−2010). He was recognized by the AHA with the ATVB Special Recognition Award in Arteriosclerosis (2004), the KinMet Robert I. Levy Award (2007), and the Distinguished Achievement Award (2012). He was conference chair for the Aspen Bile Acid/Cholesterol Conference (1993), the Gordon Research Conference on Lipid Metabolism (1996), and the Kern Aspen Lipid Conference (2003). In addition to his longstanding service to the Journal of Lipid Research, Larry was also an Associate Editor (1991−1999) and Editorial Board member (1999−2019) for the ATVB journal. Larry was a committed family man who enjoyed golfing with his sons, traveling with his family, hosting family and friends at his vacation home at Lake Lure, North Carolina, and preparing holiday turkey dinners. He was proud of the creativity of his sons and of the accomplishments of his family. He enjoyed close friendships with many members of the scientific community. He was preceded in death by his parents and his sister Francis Rudel Hadsall. He is survived by his wife, Katherine Bouwman Rudel of Winston-Salem, North Carolina; three sons, Brian (Sheri) Rudel of Tobaccoville, North Carolina, John (Cindee) Rudel of Norfolk, Virginia, and David Rudel of Charlotte, North Carolina; 5 grandchildren, Connor Rudel, Mason Rudel, Rawley Rudel, Ryan Rudel, Ruby Rudel, and a niece, Traci Hadsall, of Acworth, Georgia. Larry will be remembered for his sense of humor and fun-loving nature, his charming but direct personality, his keen scientific insights, and the consistent and effective leadership he provided at Wake Forest University and beyond. Larry's lasting legacy is embodied in his life's work and shared experiences with family, friends, and colleagues. We and others who have been positively influenced by his love of scientific research and his rigorous quest to understand lipid and sterol metabolism and its links to the etiology of atherosclerotic cardiovascular disease will miss him greatly.

Objective: The role of hepatocyte Abca1 (ATP binding cassette transporter A1) in trafficking hepatic free cholesterol (FC) into plasma versus bile for reverse cholesterol transport (RCT) is poorly understood. We hypothesized that hepatocyte Abca1 recycles plasma HDL-C (high-density lipoprotein cholesterol) taken up by the liver back into plasma, maintaining the plasma HDL-C pool, and decreasing HDL-mediated RCT into feces. Approach and Results: Chow-fed hepatocyte-specific Abca1 knockout (HSKO) and control mice were injected with human HDL radiolabeled with 125 I-tyramine cellobiose ( 125 I-TC; protein) and 3 H-cholesteryl oleate ( 3 H-CO). 125 I-TC and 3 H-CO plasma decay, plasma HDL 3 H-CO selective clearance (ie, 3 H- 125 I fractional catabolic rate), liver radiolabel uptake, and fecal 3 H-sterol were significantly greater in HSKO versus control mice, supporting increased plasma HDL RCT. Twenty-four hours after 3 H-CO-HDL injection, HSKO mice had reduced total hepatic 3 H-FC (ie, 3 H-CO hydrolyzed to 3 H-FC in liver) resecretion into plasma, demonstrating Abca1 recycled HDL-derived hepatic 3 H-FC back into plasma. Despite similar liver LDLr (low-density lipoprotein receptor) expression between genotypes, HSKO mice treated with LDLr-targeting versus control antisense oligonucleotide had slower plasma 3 H-CO-HDL decay, reduced selective 3 H-CO clearance, and decreased fecal 3 H-sterol excretion that was indistinguishable from control mice. Increased RCT in HSKO mice was selective for 3 H-CO-HDL, since macrophage RCT was similar between genotypes. Conclusions: Hepatocyte Abca1 deletion unmasks a novel and selective FC trafficking pathway that requires LDLr expression, accelerating plasma HDL-selective CE uptake by the liver and promoting HDL RCT into feces, consequently reducing HDL-derived hepatic FC recycling into plasma.

Although epidemiological data and results from rodent studies support an inverse relationship between nicotine consumption and body weight, the molecular mechanisms are poorly understood. CD-1 mice were fed a basal diet or a basal diet containing low or high dose smokeless tobacco blend or high dose nicotine tartrate for 14 weeks. High dose tobacco blend and nicotine tartrate diets vs. basal diet reduced mouse body weight (16.3% and 19.7%, respectively), epididymal (67.6% and 72.5%, respectively) and brown adipose weight (42% and 38%, respectively), epididymal adipocyte size (46.4% and 41.4%, respectively), and brown adipose tissue lipid droplet abundance, with no elevation of adipose tissue inflammation. High dose tobacco blend and nicotine diets also increased mouse physical activity and decreased respiratory exchange ratio, suggesting that high dose nicotine intake induces adipose tissue triglyceride lipolysis to provide fatty acids as an energy source. Both low and high dose tobacco blend and nicotine diet feeding vs. basal diet increased plasma insulin levels (2.9, 3.6 and 4.3-fold, respectively) and improved blood glucose disposal without affecting insulin sensitivity. Feeding of the high dose tobacco blend or nicotine feeding in mice induces body weight loss likely by increasing physical activity and stimulating adipose tissue triglyceride lipolysis.

OBJECTIVE:Adipose tissue cholesterol increases with adipocyte triglyceride content and size during development of obesity. However, how adipocyte cholesterol affects adipocyte function is poorly understood. The aim of this study was to evaluate the role of the cellular cholesterol exporter, Abca1 (ATP-binding cassette transporter A1), on adipose tissue function during diet-induced obesity.APPROACH AND RESULTS:Adiponectin Cre recombinase transgenic mice were crossed with Abca1flox/flox mice to generate ASKO (adipocyte-specific Abca1 knockout) mice. Control and ASKO mice were then fed a high-fat, high-cholesterol (45% calories as fat and 0.2% cholesterol) diet for 16 weeks. Compared with control mice, ASKO mice had a 2-fold increase in adipocyte plasma membrane cholesterol content and significantly lower body weight, epididymal fat pad weight, and adipocyte size. ASKO versus control adipose tissue had decreased PPARγ (peroxisome proliferator-activated receptor γ) and CCAAT/enhancer-binding protein expression, nuclear SREBP1 (sterol regulatory element-binding protein 1) protein, lipogenesis, and triglyceride accretion but similar Akt activation after acute insulin stimulation. Acute siRNA-mediated Abca1 silencing during 3T3L1 adipocyte differentiation reduced adipocyte Abca1 and PPARγ protein expression and triglyceride content. Systemic stimulated triglyceride lipolysis and glucose homeostasis were similar between control and ASKO mice.CONCLUSIONS:Adipocyte Abca1 is a key regulator of adipocyte lipogenesis and lipid accretion, likely because of increased adipose tissue membrane cholesterol, resulting in decreased activation of lipogenic transcription factors PPARγ and SREBP1.

G protein-coupled receptor (GPR)120/FFA receptor (FFAR)4 (GPR120/FFAR4) activation by n-3 PUFAs attenuates inflammation, but its impact on atherosclerosis is unknown. We determined whether in vivo activation of leukocyte GPR120/FFAR4 by n-3 versus n-6 PUFAs is atheroprotective. Leukocyte GPR120/FFAR4 WT or KO mice in the LDL receptor KO background were generated by bone marrow transplantation. Mice were fed one of the four atherogenic diets containing 0.2% cholesterol and 10% calories as palm oil (PO) + 10% calories as: 1) PO, 2) fish oil (FO; 20:5 n-3 and 22:6 n-3 enriched), 3) echium oil (EO; 18:4 n-3 enriched), or 4) borage oil (BO; 18:3 n-6 enriched) for 16 weeks. Compared with PO, mice fed BO, EO, and FO had significantly reduced plasma cholesterol, TG, VLDL cholesterol, hepatic neutral lipid, and atherosclerosis that were equivalent for WT and KO mice. In BO-, EO-, and FO-fed mice, but not PO-fed mice, lack of leukocyte GPR120/FFAR4 resulted in neutrophilia, pro-inflammatory Ly6Chi monocytosis, increased aortic root monocyte recruitment, and increased hepatic inflammatory gene expression. In conclusion, leukocyte GPR120 expression has minimal effects on dietary PUFA-induced plasma lipid/lipoprotein reduction and atheroprotection, and there is no distinction between n-3 versus n-6 PUFAs in activating anti-inflammatory effects of leukocyte GPR120/FFAR4 in vivo.

Objective— To test the hypothesis that the attenuation of cholesterol oleate packaging into apoB-containing lipoproteins will arrest progression of pre-existing atherosclerotic lesions. Approach and Results— Atherosclerosis was induced in apoB-100 only, LDLr –/– mice by feeding a diet enriched in cis -monounsaturated fatty acids for 24 weeks. A subset of mice was then euthanized to quantify the extent of atherosclerosis. The remaining mice were continued on the same diet (controls) or assigned to the following treatments for 16 weeks: (1) a diet enriched in n-3 polyunsaturated fatty acids, (2) the cis -monounsaturated fatty acid diet plus biweekly injections of an antisense oligonucleotide specific to hepatic sterol-O-acyltransferase 2 (SOAT2); or (3) the cis -monounsaturated fatty acid diet and biweekly injections of a nontargeting hepatic antisense oligonucleotide. Extent of atherosclerotic lesions in the aorta was monitored morphometrically in vivo with magnetic resonance imaging and ex vivo histologically and immunochemically. Hepatic knockdown of SOAT2 via antisense oligonucleotide treatment arrested lesion growth and stabilized lesions. Conclusions— Hepatic knockdown of SOAT2 in apoB100-only, LDLr –/– mice resulted in remodeling of aortic atherosclerotic lesions into a stable phenotype, suggesting SOAT2 is a viable target for the treatment of atherosclerosis.

Background: We recently reported that hepatic apolipoprotein A-IV (apoA-IV) gene expression is increased in mouse models of steatosis and is positively correlated with liver triglyceride (TG) content. The most common human apoA-IV polymorphism T347S (rs675) decreases its lipid affinity and is associated with increased adiposity in population studies. Hypothesis: 347S allele carriers will exhibit increased hepatic TG content in conditions predisposing to hepatic steatosis. Methods: We examined the impact of the 347S allele on liver TG content in a cohort of very obese humans. Total hepatic mRNA was extracted from snap frozen tissue obtained from 40 obese subjects (11 male, 29 female) at the time of bariatric surgery. ApoA-IV abundance was quantitated by RT-PCR; copy numbers were normalized to GAPDH. Presence of the T347S polymorphism and the next most common apoA-IV allele, S127N (rs5104), was determined by cDNA sequencing. Liver total TG was measured in solvent extracts by GLC and normalized as mg TG/g protein. Results: Mean BMI in the cohort was 43.8 ± 1.5 kg/m 2 ; T347S allele frequency was 0.38 and S127N allele frequency was 0.23; 16/40 (40%) had severe steatosis (hepatic TG content > 400 mg TG/g protein). There was no difference in total hepatic apoA-IV mRNA abundance among carriers of the wild type and variant alleles; mean liver TG content was highest in subjects carrying a 347S allele; a significantly higher percentage of subjects carrying a 347S allele, but not a 127N allele, had severe steatosis (P=0.024). Conclusions: These data establish that the apoA-IV 347S allele is associated with severe hepatic steatosis in a cohort of obese subjects, and suggest that apoA-IV genotype plays a role in hepatic lipid metabolism. As apoA-IV facilitates hepatic TG export by enabling secretion of larger VLDL particles, the impact of the 347S allele on hepatic TG content in the absence of altered gene expression suggests that its reduced lipid affinity may impair VLDL particle expansion.

Dietary n‐9 monounsaturated fatty acids (FA) decrease metabolic syndrome (MetS) risk factors; however, whether consumption of novel vegetable oils high in MUFA affect LDL binding characteristics has not been determined. We utilized the Canola Oil Multi‐Centre Intervention Trial (COMIT), a human intervention study consisting of five dietary treatments differing in qualitative fat content, each treatment fed for 29 days and separated by a 4 week washout period. We evaluated the efficacy of three vegetable oil treatments from COMIT including corn/safflower oil (control), high oleic canola oil, and high oleic canola oil with DHA on plasma FA profiles, LDL cholesterol ester FA composition and in vitro LDL proteoglycan binding affinity. A multi‐centre, double blind, randomized, 3‐period crossover, controlled feeding study was conducted where subjects with MetS criteria (n=50) were fed isocaloric diets (50% CHO, 15% PRO, 35% FAT) containing three of the five treatments, each for 29 days. Plasma FA profiles at day 29 reflected the FA composition of each diet, suggesting high compliance to diet. Similarly, LDL cholesterol ester FA distribution mirrored the FA composition of the dietary fat at day 29, but not day 1, on each treatment. However, proteoglycan binding affinity values of LDL particles did not differ significantly across day 1 to day 29 for corn/safflower (‐2.46+3.63%) (mean+SEM), high oleic canola (‐6.47+1.95%) or oleic canola oil with DHA (‐0.52+2.85%). No difference was observed in binding affinity assessed at day 29 as a function of diet treatment. These data suggest that, despite the demonstration of diet induced shifts in LDL cholesterol ester FA patterns, n‐9 FA rich canola oil diets does not induce any change in LDL binding affinity compared with a n‐6 FA rich diet.Grant Funding Source: Supported by Canola Council of Canada/Agr Agr Food Canada and Canada Research Chairs program

Rationale: Cholesterol esters (CE), especially cholesterol oleate, generated by hepatic and intestinal sterol O-acyltransferase 2 (SOAT2) play a critical role in cholesterol homeostasis. However, it is unknown whether the contribution of intestine-derived CE from SOAT2 would have similar effects in promoting atherosclerosis progression as for liver-derived CE.Objective: To test whether, in low-density lipoprotein receptor null (LDLr-/-) mice, the conditional knockout of intestinal SOAT2 (SOAT2(SI-/SI-)) or hepatic SOAT2 (SOAT2(L-/L-)) would equally limit atherosclerosis development compared with the global deletion of SOAT2 (SOAT2(-/-) ).Methods and Results: SOAT2 conditional knockout mice were bred with LDLr-/- mice creating LDLr-/- mice with each of the specific SOAT2 gene deletions. All mice then were fed an atherogenic diet for 16 weeks. SOAT2(SI-/SI-)LDLr(-/-) and SOAT2(-/-)LDLr(-/-) mice had significantly lower levels of intestinal cholesterol absorption, more fecal sterol excretion, and lower biliary cholesterol levels. Analysis of plasma LDL showed that all mice with SOAT2 gene deletions had LDL CE with reduced percentages of cholesterol palmitate and cholesterol oleate. Each of the LDLr-/- mice with SOAT2 gene deletions had lower accumulations of total cholesterol and CE in the liver compared with control mice. Finally, aortic atherosclerosis development was significantly lower in all mice with global or tissue-restricted SOAT2 gene deletions. Nevertheless, SOAT2(-/-)LDLr(-/-) and SOAT2(L-/L-)LDLr(-/-) mice had less aortic CE accumulation and smaller aortic lesions than SOAT2(SI-/SI-)LDLr(-/-) mice.Conclusions: SOAT2-derived CE from both the intestine and liver significantly contribute to the development of atherosclerosis, although the CE from the hepatic enzyme appeared to promote more atherosclerosis development.

Rationale: Signal initiation by the high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain. The C-terminal transmembrane domain uniquely interacts with plasma membrane (PM) cholesterol. Objective: The molecular basis and functional significance of SR-BI interaction with PM cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a PM cholesterol sensor. Methods and Results: In studies performed in COS-M6 cells, mutation of a highly conserved C-terminal transmembrane domain glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-β-cyclodextrin also was prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or methyl-β-cyclodextrin in cultured enterocytes, and it is required for HDL activation of endothelial NO synthase and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. Conclusions: Through interaction with PM cholesterol, SR-BI serves as a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.

Several studies in humans and animals suggest that LDL particle core enrichment in cholesteryl oleate (CO) is associated with increased atherosclerosis. Diet enrichment with MUFAs enhances LDL CO content. Steroyl O-acyltransferase 2 (SOAT2) is the enzyme that catalyzes the synthesis of much of the CO found in LDL, and gene deletion of SOAT2 minimizes CO in LDL and protects against atherosclerosis. The purpose of this study was to test the hypothesis that the increased atherosclerosis associated with LDL core enrichment in CO results from an increased affinity of the LDL particle for arterial proteoglycans. ApoB-100-only Ldlr−/− mice with and without Soat2 gene deletions were fed diets enriched in either cis-MUFA or n-3 PUFA, and LDL particles were isolated. LDL:proteogylcan binding was measured using surface plasmon resonance. Particles with higher CO content consistently bound with higher affinity to human biglycan and the amount of binding was shown to be proportional to the extent of atherosclerosis of the LDL donor mice. The data strongly support the thesis that atherosclerosis was induced through enhanced proteoglycan binding of LDL resulting from LDL core CO enrichment.

Reverse cholesterol transport (RCT) can proceed through the classic hepatobiliary route or through the nonbiliary transintestinal cholesterol efflux (TICE) pathway. Scavenger receptor class B type I (SR-BI) plays a critical role in the classic hepatobiliary route of RCT. However, the role of SR-BI in TICE has not been studied. To examine the role of intestinal SR-BI in TICE, sterol balance was measured in control mice and mice transgenically overexpressing SR-BI in the proximal small intestine (SR-BIhApoCIII-ApoAIV-Tg). SR-BIhApoCIII-ApoAIV-Tg mice had significantly lower plasma cholesterol levels compared with wild-type controls, yet SR-BIhApoCIII-ApoAIV-Tg mice had normal fractional cholesterol absorption and fecal neutral sterol excretion. Both in the absence or presence of ezetimibe, intestinal SR-BI overexpression had no impact on the amount of cholesterol excreted in the feces. To specifically study effects of intestinal SR-BI on TICE we crossed SR-BIhApoCIII-ApoAIV-Tg mice into a mouse model that preferentially utilized the TICE pathway for RCT (Niemann-Pick C1-like 1 liver transgenic), and likewise found no alterations in cholesterol absorption or fecal sterol excretion. Finally, mice lacking SR-BI in all tissues also exhibited normal cholesterol absorption and fecal cholesterol disposal. Collectively, these results suggest that SR-BI is not rate limiting for intestinal cholesterol absorption or for fecal neutral sterol loss through the TICE pathway.

The serine hydrolase α/β hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs) to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6's role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.

Acyl-CoA:cholesterol acyltransferase 2 (ACAT2) is exclusively expressed in the small intestine and liver. ACAT2 facilitates the movement of cholesterol among tissues by generating cholesteryl ester (CE) for packaging into newly synthesized chylomicrons and very low-density lipoproteins (VLDL). In these studies we investigated whether CE derived from either the intestine or liver would differentially affect hepatic and plasma cholesterol homeostasis. For this purpose, we generated both liver-specific (ACAT2L-/L-) and intestine-specific (ACAT2SI-/SI-) ACAT2 knockout mice, and studied dietary cholesterol-induced hepatic lipid accumulation and hypercholesterolemia. Interestingly, diet-induced accumulation of hepatic CE was similarly decreased in both ACAT2L-/L- and ACAT2SI-/SI- mice, and free cholesterol did not build up in the liver. Compared with control mice, both ACAT2L-/L- and ACAT2SI-/SI- mice had lower levels of plasma VLDL-cholesterol but higher plasma triglycerides. ACAT2SI-/SI- but not ACAT2L-/L- mice had blunted cholesterol absorption. Collectively, both ACAT2L-/L- and ACAT2SI-/SI- mice were equally protected from diet-induced hepatic CE accumulation and hypercholesterolemia. These results suggest that inhibition of either intestinal or hepatic ACAT2 improves atherogenic hyperlipidemia and limits hepatic CE accumulation in mice, indicating that inhibition of ACAT2 expression in either tissue likely would be beneficial for atheroprotection.

INTRODUCTION: Accumulation of lipids in the artery wall, particularly cholesteryl esters (CE), is a classic feature of atherosclerosis. Apo B-containing lipoprotein particles are the primary vehicles by which CEs are delivered across the endothelial barrier into the intima and once present in the subendothelial space these particles are subject to sequestration by native proteoglycans. Several studies in both non-human primate and murine models of atherosclerosis strongly suggest that core enrichment in cholesteryl oleate of low-density lipoprotein (LDL) particles play a significant role in determination of the extent of atherosclerosis. It has also been shown that Acyl-CoA:cholesterol O-acyltransferase 2 (ACAT2) is the enzyme responsible for cholesteryl oleate enrichment of apo B-containing lipoproteins and gene deletion of ACAT2 in animal models is protective against the development of atherosclerosis. Thus, we hypothesized that the selective accumulation of LDL within the intima is the result of LDL particle core enrichment of cholesteryl oleate that results in modification of key surface characteristics of ApoB promoting interaction with resident proteoglycans. METHODS: Apo B-100 only, LDLr -/- mice (W/T) and Apo B-100 only, LDLr-/-, ACAT2 -/- (KO) mice were fed diets enriched in either cis-monounsaturated fatty acids (cis-MUFA) or n-3 polyunsaturated fatty acids (n-3 PUFA) for 16 weeks. Blood and plasma was harvested and LDL particles were isolated by size exclusion chromatography. The major lipid constituents of the LDL particle were measured along with the fatty acids of the cholesteryl ester fraction of the particle core. LDL affinity to arterial proteoglycans was determined using an immunocapture surface plasmon resonance (SPR) technique we developed. Atherosclerosis was quantified by measuring the cholesterol content of the aorta. RESULTS: W/T mice fed a cis-MUFA diet displayed the highest degree of cholesteryl oleate packaging into the particle core and the highest propensity to bind to arterial proteoglycans. Feeding a diet enriched in n-3 PUFA and/or knocking out ACAT2 successfully inhibited the packaging of cholesteryl oleate into the LDL particles. Accompanying this decrease in cholesteryl oleate content was a significant decrease in binding to arterial proteoglycans and development of atherosclerosis. CONCLUSION: Elimination of cholesteryl oleate from the LDL particle core results in significantly less binding to arterial wall proteoglycans and in turn, less development of atherosclerosis.

Acyl-CoA:cholesterol acyltransferase 2 (ACAT2) generates cholesterol esters (CE) for packaging into newly synthesized lipoproteins and thus is a major determinant of blood cholesterol levels. ACAT2 is expressed exclusively in the small intestine and liver, but the relative contributions of ACAT2 expression in these tissues to systemic cholesterol metabolism is unknown. We investigated whether CE derived from the intestine or liver would differentially affect hepatic and plasma cholesterol homeostasis. We generated liver-specific (ACAT2L−/L−) and intestine-specific (ACAT2SI−/SI−) ACAT2 knockout mice and studied dietary cholesterol-induced hepatic lipid accumulation and hypercholesterolemia. ACAT2SI−/SI− mice, in contrast to ACAT2L−/L− mice, had blunted cholesterol absorption. However, specific deletion of ACAT2 in the intestine generated essentially a phenocopy of the conditional knockout of ACAT2 in the liver, with reduced levels of plasma very low-density lipoprotein and hepatic CE, yet hepatic-free cholesterol does not build up after high cholesterol intake. ACAT2L−/L− and ACAT2SI−/SI− mice were equally protected from diet-induced hepatic CE accumulation and hypercholesterolemia. These results suggest that inhibition of intestinal or hepatic ACAT2 improves atherogenic hyperlipidemia and limits hepatic CE accumulation in mice and that depletion of intestinal ACAT2 is sufficient for most of the beneficial effects on cholesterol metabolism. Inhibitors of ACAT2 targeting either tissue likely would be beneficial for atheroprotection.