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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.

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 Abca1 flox/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.

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.

Background— Stearoyl-CoA desaturase 1 (SCD1) is a critical regulator of energy metabolism and inflammation. We have previously reported that inhibition of SCD1 in hyperlipidemic mice fed a saturated fatty acid (SFA)-enriched diet prevented development of the metabolic syndrome, yet surprisingly promoted severe atherosclerosis. In this study we tested whether dietary fish oil supplementation could prevent the accelerated atherosclerosis caused by SCD1 inhibition. Methods and Results— LDLr −/− , ApoB 100/100 mice were fed diets enriched in saturated fat or fish oil in conjunction with antisense oligonucleotide (ASO) treatment to inhibit SCD1. As previously reported, in SFA-fed mice, SCD1 inhibition dramatically protected against development of the metabolic syndrome, yet promoted atherosclerosis. In contrast, in mice fed fish oil, SCD1 inhibition did not result in augmented macrophage inflammatory response or severe atherosclerosis. In fact, the combined therapy of dietary fish oil and SCD1 ASO treatment effectively prevented both the metabolic syndrome and atherosclerosis. Conclusions— SCD1 ASO treatment in conjunction with dietary fish oil supplementation is an effective combination therapy to comprehensively combat the metabolic syndrome and atherosclerosis in mice.

Objectives— The enzyme acyl-coenzymeA (CoA):cholesterol O-acyl trans ferase 2 (ACAT2) in the liver synthesizes cholesteryl esters (CE) from cholesterol and fatty acyl-CoA, which get incorporated into apoB-containing lipoproteins that are secreted into the bloodstream. Dietary fatty acid composition influences the amount and fatty acid composition of CE within apoB-containing lipoproteins. We hypothesized that when ACAT2 activity is removed by gene deletion, hepatic CE synthesis and secretion would be minimal and, as a result, dietary fat-related differences in atherosclerosis would be eliminated. Methods and Results— Groups of female apoB100 only, LDLr −/− mice with and without ACAT2 were fed diets enriched in either ω-3 or ω-6 polyunsaturated fat, saturated fat, and cis or trans monounsaturated fat. After 20 weeks on diet, mice fed diets enriched in monounsaturated or saturated fat exhibited significantly higher amounts of plasma cholesterol, larger LDL particles enriched in monounsaturated CE, and more atherosclerosis than mice fed polyunsaturated fat. The dietary fat-induced shifts in plasma cholesterol, LDL size, LDL CE composition, and atherosclerosis were not observed in ACAT2 −/− mice. Regardless of the diet fed, the ACAT2 −/− mice were protected from atherosclerosis. Conclusions— The results indicate that in apoB100 only, LDLr −/− mice, ACAT2 plays an essential role in facilitating dietary fat type–specific atherosclerosis through its various effects on plasma lipoprotein concentration and composition.

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.

Abstract Atherogenic diets enriched in saturated, n-6 polyunsaturated, and monounsaturated fatty acids were fed to African green monkeys for 5 years to define effects on plasma lipoproteins and coronary artery atherosclerosis. The monkeys fed polyunsaturated and monounsaturated fat had similar plasma concentrations of LDL cholesterol, and these values were significantly lower than for LDL in the animals fed saturated fat. Plasma HDL cholesterol concentrations were comparable in animals fed saturated and monounsaturated fat and were significantly higher than in animals fed polyunsaturated fat. Thus, the monounsaturated fat group had the lowest LDL/HDL ratio. LDL particle size was largest in the saturated and monounsaturated fat groups, significantly larger than in the polyunsaturated fat group. LDL particle enrichment with cholesteryl oleate was the greatest in the animals fed monounsaturated fat, next greatest in the saturated fat–fed animals, and was least in the polyunsaturated fat–fed animals. Coronary artery atherosclerosis as measured by intimal area was less in the polyunsaturated fat compared with the saturated fat groups, was less in the animals fed polyunsaturated fat compared with the monounsaturated fat–fed animals, but did not differ between the monounsaturated and saturated fat groups. Cholesteryl ester, particularly cholesteryl oleate, accumulation in the coronary arteries was also similar between groups fed monounsaturated and saturated fat but was minimal in the animals fed polyunsaturated fat. In sum, the monkeys fed monounsaturated fat developed equivalent amounts of coronary artery atherosclerosis as those fed saturated fat, but monkeys fed polyunsaturated fat developed less. The beneficial effects of the lower LDL and higher HDL in the animals fed monounsaturated fat apparently were offset by the atherogenic shifts in LDL particle composition. Dietary polyunsaturated fat appears to result in the least amount of coronary artery atherosclerosis because it prevents cholesteryl oleate accumulation in LDL and the coronary arteries in these primates.

The hypothesis tested was that juvenile African green monkeys consuming diets enriched with n-6 polyunsaturated fat from birth until young adulthood would have significantly less coronary artery atherosclerosis than comparable animals consuming diets enriched with saturated fat. African green monkeys (Cercopithecus aethiops, n = 108) of both sexes were fed atherogenic diets (0.8 mg cholesterol/kcal) throughout their lives so that death at 16, 32, or 60 months of age permitted quantification of atherosclerosis. In the coronary arteries, the average intimal area increased significantly with age (P = .02), showing increases of 28-fold and sevenfold between 32 and 60 months in the saturated fat- and polyunsaturated fat-fed groups, respectively. Young adult male animals at 60 months of age were found to have significantly (P = .03) more coronary artery atherosclerosis than female animals. Animals fed polyunsaturated fat had significantly (P < or = .01) less coronary artery atherosclerosis. By 60 months of age in the animals consuming polyunsaturated fat, the average coronary artery intimal area was one fourth and the average size of the largest coronary intimal lesion was one fifth that in monkeys fed saturated fat. Low-density lipoprotein (LDL) cholesterol and LDL particle size were each found to be positively correlated with coronary artery atherosclerosis end points in both diet groups. In addition to the coronary arteries, atherosclerosis in the abdominal and thoracic aorta and carotid arteries was also evaluated; the coronary arteries were the only arterial system with significantly less atherosclerosis in the polyunsaturated fat group as measured by intimal area. However, evaluation of histological sections of abdominal aorta showed relatively more sterol clefts in the saturated fat-fed group, and more free cholesterol was measured, suggesting that lesions were more complicated in this group. These results show that dietary intervention early in life with n-6 polyunsaturated fat can be effective in decreasing the development of atherosclerosis, particularly in the coronary arteries of primates. This outcome supports the concept that dietary intervention beginning early in childhood can have beneficial effects on the coronary heart disease of later life.