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Acyl-CoA:cholesterol O-acyl transferase 2 (ACAT2) promotes cholesterol absorption by the intestine and the secretion of cholesteryl ester-enriched very low density lipoproteins by the liver. Paradoxically, mice lacking ACAT2 also exhibit mild hypertriglyceridemia. The present study addresses the unexpected role of ACAT2 in regulation of hepatic triglyceride (TG) metabolism. Mouse models of either complete genetic deficiency or pharmacological inhibition of ACAT2 were fed low fat diets containing various amounts of cholesterol to induce hepatic steatosis. Mice genetically lacking ACAT2 in both the intestine and the liver were dramatically protected against hepatic neutral lipid (TG and cholesteryl ester) accumulation, with the greatest differences occurring in situations where dietary cholesterol was elevated. Further studies demonstrated that liver-specific depletion of ACAT2 with antisense oligonucleotides prevents dietary cholesterol-associated hepatic steatosis both in an inbred mouse model of non-alcoholic fatty liver disease (SJL/J) and in a humanized hyperlipidemic mouse model (LDLr(-/-), apoB(100/100)). All mouse models of diminished ACAT2 function showed lowered hepatic triglyceride concentrations and higher plasma triglycerides secondary to increased hepatic secretion of TG into nascent very low density lipoproteins. This work demonstrates that inhibition of hepatic ACAT2 can prevent dietary cholesterol-driven hepatic steatosis in mice. These data provide the first evidence to suggest that ACAT2-specific inhibitors may hold unexpected therapeutic potential to treat both atherosclerosis and non-alcoholic fatty liver disease.

Loss of ABCA1 activity in Tangier disease (TD) is associated with abnormal apoB lipoprotein (Lp) metabolism in addition to the complete absence of high density lipoprotein (HDL). We used hepatocyte-specific ABCA1 knock-out (HSKO) mice to test the hypothesis that hepatic ABCA1 plays dual roles in regulating Lp metabolism and nascent HDL formation. HSKO mice recapitulated the TD lipid phenotype with postprandial hypertriglyceridemia, markedly decreased LDL, and near absence of HDL. Triglyceride (TG) secretion was 2-fold higher in HSKO compared with wild type mice, primarily due to secretion of larger TG-enriched VLDL secondary to reduced hepatic phosphatidylinositol 3-kinase signaling. HSKO mice also displayed delayed clearance of postprandial TG and reduced post-heparin plasma lipolytic activity. In addition, hepatic LDLr expression and plasma LDL catabolism were increased 2-fold in HSKO compared with wild type mice. Last, adenoviral repletion of hepatic ABCA1 in HSKO mice normalized plasma VLDL TG and hepatic phosphatidylinositol 3-kinase signaling, with a partial recovery of HDL cholesterol levels, providing evidence that hepatic ABCA1 is involved in the reciprocal regulation of apoB Lp production and HDL formation. These findings suggest that altered apoB Lp metabolism in TD subjects may result from hepatic VLDL TG overproduction and increased hepatic LDLr expression and highlight hepatic ABCA1 as an important regulatory factor for apoB-containing Lp metabolism.

Cholesteryl ester synthesis by the acyl-CoA:cholesterol acyltransferase enzymes ACAT1 and ACAT2 is, in part, a cellular homeostatic mechanism to avoid toxicity associated with high free cholesterol levels. In hepatocytes and enterocytes, cholesteryl esters are secreted as part of apoB lipoproteins, the assembly of which is critically dependent on microsomal triglyceride transfer protein (MTP). Conditional genetic ablation of MTP reduces cholesteryl esters and enhances free cholesterol in the liver and intestine without diminishing ACAT1 and ACAT2 mRNA levels. As expected, increases in hepatic free cholesterol are associated with decreases in 3-hydroxy-3-methylglutaryl-CoA reductase and increases in ATP-binding cassette transporter 1 mRNA levels. Chemical inhibition of MTP also decreases esterification of cholesterol in Caco-2 and HepG2 cells. Conversely, coexpression of MTP and apoB in AC29 cells stably transfected with ACAT1 and ACAT2 increases cholesteryl ester synthesis. Liver and enterocyte microsomes from MTP-deficient animals synthesize lesser amounts of cholesteryl esters in vitro, but addition of purified MTP and low density lipoprotein corrects this deficiency. Enrichment of microsomes with cholesteryl esters also inhibits cholesterol ester synthesis. Thus, MTP enhances cellular cholesterol esterification by removing cholesteryl esters from their site of synthesis and depositing them into nascent apoB lipoproteins. Therefore, MTP plays a novel role in regulating cholesteryl ester biosynthesis in cells that produce lipoproteins. We speculate that non-lipoprotein-producing cells may use different mechanisms to alleviate product inhibition and modulate cholesteryl ester biosynthesis.

Syndecan 1 is the major proteoglycan produced by epithelial cells. It is strategically localized at the plasma membrane to participate in growth factor signaling and cell-cell and cell-matrix interactions. Its expression may modulate the properties of epithelial lineage tumor cells in which it is generally down-regulated compared with nontumor progenitors. The present study examined the regulation of syndecan 1 in prostate epithelial cells by n-3 polyunsaturated fatty acids. In prostate tissue of mice, syndecan 1 immunostaining was demonstrated in epithelial cells throughout each gland. In animals fed an n-3 polyunsaturated fatty acid-enriched diet, syndecan 1 mRNA was increased in all prostate glands. In the human prostate cancer cell line, PC-3, delivery of exogenous n-3 (but not n-6) fatty acids resulted in up-regulation of syndecan 1 expression. This effect was mimicked by a peroxisome proliferator-activated receptor (PPAR) gamma agonist, troglitazone, and inhibited in the presence of a PPARgamma antagonist and in cells transfected with dominant negative PPARgamma cDNA. Using a luciferase gene driven either by a PPAR response element or by a DR-1 site present in the syndecan 1 promoter, reporter activation was increased by n-3 low density lipoprotein, docosahexaenoic acid, and troglitazone, whereas activity of a luciferase gene placed downstream of a mutant DR-1 site was unresponsive. These findings indicate that syndecan 1 is up-regulated by n-3 fatty acids by a transcriptional pathway involving PPARgamma. This mechanism may contribute to the chemopreventive properties of n-3 fatty acids in prostate cancer.

Targeted deletion of acyl-CoA:cholesterol acyltransferase 2 (ACAT2) (A2), especially in the liver, protects hyperlipidemic mice from diet-induced hypercholesterolemia and atherosclerosis, whereas the deletion of ACAT1 (A1) is not as effective, suggesting ACAT2 may be the more appropriate target for treatment of atherosclerosis. Among the numerous ACAT inhibitors known, pyripyropene A (PPPA) is the only compound that has high selectivity (>2000-fold) for inhibition of ACAT2 compared with ACAT1. In the present study we sought to determine the PPPA interaction site of ACAT2. To achieve this goal we made several chimeric proteins where parts of ACAT2 were replaced by the analogous region of ACAT1. Differences in the amino acid sequence and the membrane topology were utilized to design the chimeras. Among chimeras, A2:1-428/A1:444-550 had 50% reduced PPPA selectivity, whereas C-terminal-truncated ACAT2 mutant A2:1-504 (C-terminal last 22 amino acids were deleted) remained selectively inhibited, indicating the PPPA-sensitive site is located within a region between amino acids 440 and 504. Three additional chimeras within this region helped narrow down the PPPA-sensitive site to a region containing amino acids 480-504, representing the fifth putative transmembrane domain of ACAT2. Subsequently, for this region we made single amino acid mutants where each amino acid in ACAT2 was individually changed to its ACAT1 counterpart. Mutation of Q492L, V493L, S494A resulted in only 30, 50, and 70% inhibition of the activity by PPPA, respectively (as opposed to greater than 95% with the wild type enzyme), suggesting these three residues are responsible for the selective inhibition by PPPA of ACAT2. Additionally, we found that PPPA non-covalently interacts with ACAT2 apparently without altering the oligomeric structure of the protein. The present study provides the first evidence for a unique motif in ACAT2 that can be utilized for making an ACAT2-specific drug.

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.

Although NPC1L1 is required for intestinal cholesterol absorption, data demonstrating mechanisms by which this protein facilitates the process are few. In this study, a hepatoma cell line stably expressing human NPC1L1 was established, and cholesterol uptake was studied. A relationship between NPC1L1 intracellular trafficking and cholesterol uptake was apparent. At steady state, NPC1L1 proteins localized predominantly to the transferrin-positive endocytic recycling compartment, where free cholesterol also accumulated as revealed by filipin staining. Interestingly, acute cholesterol depletion induced with methyl-beta-cyclodextrin stimulated relocation of NPC1L1 to the plasma membrane, preferentially to a newly formed "apical-like" subdomain. This translocation was associated with a remarkable increase in cellular cholesterol uptake, which in turn was dose-dependently inhibited by ezetimibe, a novel cholesterol absorption inhibitor that specifically binds to NPC1L1. These findings define a cholesterol-regulated endocytic recycling of NPC1L1 as a novel mechanism regulating cellular cholesterol uptake.

The capacity of acyl-CoA:cholesterol O-acyltransferase (ACAT) 2 to differentiate cholesterol from the plant sterol, sitosterol, was compared with that of the sterol esterifying enzymes, ACAT1 and lecithin:cholesterol acyltransferase (LCAT). Cholesterol-loaded microsomes from transfected cells containing either ACAT1 or ACAT2 exhibited significantly more ACAT activity than their sitosterol-loaded counterparts. In sitosterol-loaded microsomes, both ACAT1 and ACAT2 were able to esterify sitosterol albeit with lower efficiencies than cholesterol. The mass ratios of cholesterol ester to sitosterol ester formed by ACAT1 and ACAT2 were 1.6 and 7.2, respectively. Compared with ACAT1, ACAT2 selectively esterified cholesterol even when sitosterol was loaded into the microsomes. To further characterize the difference in sterol specificity, ACAT1 and ACAT2 were compared in intact cells loaded with either cholesterol or sitosterol. Despite a lower level of ACAT activity, the ACAT1-expressing cells esterified 4-fold more sitosterol than the ACAT2 cells. The data showed that compared with ACAT1, ACAT2 displayed significantly greater selectively for cholesterol compared with sitosterol. The plasma cholesterol esterification enzyme lecithin:cholesterol acyltransferase was also compared. With recombinant high density lipoprotein particles, the esterification rate of cholesterol by LCAT was only 15% greater than for sitosterol. Thus, LCAT was able to efficiently esterify both cholesterol and sitosterol. In contrast, ACAT2 demonstrated a strong preference for cholesterol rather than sitosterol. This sterol selectivity by ACAT2 may reflect a role in the sorting of dietary sterols during their absorption by the intestine in vivo.

The role of liver acyl-CoA:cholesterol acyltransferase 2 (ACAT2), earlier shown to be the principal ACAT enzyme within primate hepatocytes, as a regulator of the hypercholesterolemia induced by dietary cholesterol was studied. At the end of low and high cholesterol diet periods, liver biopsies were taken from cynomolgus monkeys, a species highly responsive to dietary cholesterol, and less responsive African green monkeys. Liver cholesterol and cholesteryl ester concentrations were highest in cynomolgus monkeys fed cholesterol, despite the fact that in order to induce equivalent hypercholesterolemia, dietary cholesterol levels were 50% lower than was fed to green monkeys. Hepatic cholesteryl oleate secretion rate, measured during liver perfusion as an indicator of ACAT activity, was significantly higher in cynomolgus monkeys. Liver microsomal ACAT activity was 2-3-fold higher in cynomolgus monkeys than in green monkeys. The responses of ACAT2 were compared with those of ACAT1 that is found primarily in Kupffer cells. ACAT2 protein mass was significantly correlated to microsomal total ACAT activity in both species; ACAT1 mass was less well correlated. Dietary cholesterol induced a significant 3-fold increase of ACAT2 protein mass in cynomolgus monkeys, a much greater increase than was found for mRNA abundance; neither ACAT2 mRNA nor protein was diet-responsive in green monkeys. In cynomolgus monkeys but not in green monkeys, liver free cholesterol concentrations were elevated when cholesterol was fed and were correlated with ACAT2 protein levels. The data suggest a mechanism whereby the elevation of hepatic free cholesterol concentrations by dietary cholesterol, seen only in cynomolgus monkeys, resulted in higher ACAT2 protein levels in hepatocytes, either through increased production or stabilization of the protein. Regulation of ACAT2 gene transcription was not a factor.

The present study demonstrates that two different forms of the intracellular cholesterol esterification enzyme acyl-CoA:cholesterol acyltransferase (ACAT) are present in the nonhuman primate hepatocyte; one is similar to that originally cloned from human genomic DNA, here termed ACAT1, while a second gene product, termed ACAT2, is reported here. The primate ACAT2 gene product was cloned from an African green monkey liver cDNA library. Sequence analysis of an isolated, full-length clone of ACAT2 cDNA identified an open reading frame encoding a 526-amino acid protein with essentially no sequence similarity to the ACAT1 cDNA over the N-terminal 101 amino acids but with 57% identity predicted over the remaining 425 amino acids. Transfection of the cloned ACAT2 cDNA into two different mammalian cell types resulted in the production of abundant ACAT activity which was sensitive to ACAT inhibitors. Northern blot analysis showed that the ACAT2 mRNA was expressed primarily in liver and intestine in monkeys. In contrast, ACAT1 mRNA was expressed in almost all tissues examined. Topologic predictions from the amino acid sequence of ACAT2 indicates that it has seven trans-membrane domains in a configuration that places the putative active site of the enzyme in the lumen of the endoplasmic reticulum. This orientation of ACAT2 in the endoplasmic reticulum membrane, in addition to its expression only in liver and intestine, suggests that this enzyme may have as a primary function, the secretion of cholesteryl esters into apoB-containing lipoproteins.

Lecithin-cholesterol acyltransferase (LCAT) catalyzes the intravascular synthesis of lipoprotein cholesteryl esters by converting cholesterol and lecithin to cholesteryl ester and lysolecithin. LCAT is unique in that it catalyzes sequential reactions within a single polypeptide sequence, a phospholipase A2 reaction followed by a transacylation reaction. In this report we find that LCAT mediates a partial reverse reaction, the transacylation of lipoprotein cholesteryl oleate, in whole plasma and in a purified, reconstituted system. As a result of the reverse transacylation reaction, a linear accumulation of [3H]cholesterol occurred during incubations of plasma containing high density lipoprotein labeled with [3H]cholesteryl oleate. When high density lipoprotein labeled with cholesteryl [14C]oleate was also included in the incubation the labeled fatty acyl moiety remained in the cholesteryl [14C]oleate pool showing that the formation of labeled cholesterol did not result from hydrolysis of the doubly labeled cholesteryl esters. The rate of release of [3H]cholesterol was only about 10% of the forward rate of esterification of cholesterol using partially purified human LCAT and was approximately 7% in whole monkey plasma. Therefore, net production of cholesterol via the reverse LCAT reaction would not occur. [3H]Cholesterol production from [3H]cholesteryl oleate was almost completely inhibited by a final concentration of 1.4 mM 5,5'-dithiobis(nitrobenzoic acid) during incubation with either purified LCAT or whole plasma. Addition of excess lysolecithin to the incubation system did not result in the formation of [14C]oleate-labeled lecithin, showing that the reverse reaction found here for LCAT was limited to the last step of the reaction. To explain these results we hypothesize that LCAT forms a [14C]oleate enzyme thioester intermediate after its attack on the cholesteryl oleate molecule. Formation of this intermediate allows [3H]cholesterol to be liberated from the enzyme by exchange with unlabeled cholesterol of plasma lipoproteins. The liberated [3H]cholesterol thereby becomes available for reesterification by LCAT as indicated by its appearance as newly synthesized cholesteryl linoleate.

Differential scanning calorimetry (DSC) and x-ray diffraction studies were performed on chylomicrons and very low density lipoproteins (VLDL), from nonhuman primates fed saturated fat, isolated from lymph at two different temperatures (15 and 39 degrees C). When heated from -10 leads to 60 degrees C, chylomicrons and VLDL isolated at 15 degrees C had two endothermic transitions resulting from the melting of triglycerides (TG). Cooling resulted in the onset of crystallization of the TG core at 16-19 degrees C; 50% of the TG in the particles remained fluid at 4 degrees C. The initial DSC pattern was reproduced not on immediate reheating, but by storing samples at 4 degrees C overnight. Chylomicrons isolated at 39 degrees C contained a metastable (undercooled) TG core until cooled to the onset of TG crystallization at 16-19 degrees C. Reheating the particles with crystalline TG to body temperature (39 degrees C) resulted in a partially crystalline TG core, not an undercooled liquid. To ascertain the effect of particle structure on the TG physical properties, intact particles were compared with heat denatured particles and extracted lipids. The onset of crystallization was lower and the time necessary for isothermal crystallization was much greater for intact versus denatured particles or lipids. Only minor differences in physical properties as a function of particle size were found. It was concluded that: 1) the physical state of chylomicrons and VLDL TG core can be modified by isolation temperature; 2) particle structure affects crystallization but not melting of chylomicrons and VLDL TG; and 3) chylomicrons particle size does not markedly influence the physical properties of the TG core.

A sensitive DNA-excess solution hybridization assay was used to quantitate apo-E mRNA in the liver and peripheral tissues of two nonhuman primates, Macaca fascicularis and Cercopithecus aethiops. When expressed on the basis of total RNA, apo-E mRNA values for M. fascicularis adrenal, brain, testis, and spleen ranged from 17-52% of the liver value. Apo-E mRNA values for mesenteric lymph node, kidney, thymus, and skeletal muscle were 1-5% of the liver value. When expressed on a cellular basis, apo-E mRNA was most abundant in the liver at approximately 1200 molecules/cell. Peripheral tissues showed a continuous range of apo-E mRNA from 1.5 molecules/cell in the thymus up to 350 molecules/cell in the brain. Similar results were obtained with peripheral tissues from C. aethiops in which case apo-E mRNA also was found in skin, lung, skeletal muscle, small intestine, and vascular tissues such as heart, aorta, and brachial artery. Calculation of the total apo-E mRNA/organ showed that most of the apo-E mRNA was present in the liver. However, summation of apo-E mRNA in peripheral tissues indicated that 20-40% of total body apo-E mRNA was extrahepatic. This results indicates that apo-E made in peripheral tissues may play a quantitatively important role in cholesterol metabolism since peripheral tissues have the potential to contribute a significant fraction of plasma apo-E.

Little is known about the mechanism and control of lipoprotein particle fusion, although apoproteins are presumed to be important in maintenance of particle structure. This study characterizes the interaction of apo-B-containing low density lipoproteins (LDL) with cholesterol ester microemulsions (CEME) in the presence and absence of apo-A-I to determine if a role for these apoproteins in particle integrity could be ascertained. CEME are an apoprotein-free analog of LDL formed by sonication of radiolabeled phospholipid (surface) and cholesterol ester (core). Incubation of CEME with LDL followed by precipitation of LDL with MnCl2 resulted in coprecipitation of CEME with LDL that was time-, temperature-, and concentration-, but not pH (pH 6-9)-, dependent and occurred over a wide range of CEME and LDL particle compositions. Particles from the incubation were larger than the unincubated particles and intermediate in density and electrophoretic mobility between the starting LDL and CEME. Differential scanning calorimetry experiments suggested that CEME surface and core lipids had mixed with those of LDL. When particles from incubations were exposed to an anti-apo-B column, radiolabeled surface and core molecules originating from the CEME particles bound to the column. Particles eluted at low pH from the anti-apo-B column were irregularly shaped and had excess surface material as judged by electron microscopy. Incubation of CEME with LDL in the presence of 3 M KBr or 4% bovine serum albumin did not alter the interaction of the particles. However, incubation of CEME with LDL in the presence of apo-A-I (2:1 CEME cholesterol-to-apo-A-I mass ratio) greatly reduced the interaction of the LDL and CEME particles. We conclude that the incubation of CEME with isolated LDL resulted in particle fusion that was prevented by apo-A-I.

Earlier studies have shown that African green monkeys develop a more modest hypercholesterolemia, higher high density lipoprotein (HDL) concentrations, and less atherosclerosis than cynomolgus monkeys fed diets with the same cholesterol content. In the present study, cynomolgus monkeys were fed less cholesterol than was fed to African green monkeys to induce equivalent hypercholesterolemia in both species. African green monkeys still had 2-fold higher plasma HDL cholesterol concentrations and 2.7-fold higher plasma apolipoprotein (apo) A-I concentrations. Therefore, the higher HDL concentration in African green monkeys appears to result from factors that act independently of dietary cholesterol intake or total plasma cholesterol concentration. Two aspects of HDL production were examined to determine the metabolic basis of the species difference in HDL concentration. The rate of hepatic apoA-I secretion, as estimated by the accumulation of apoA-I in the medium during recirculating liver perfusion, was 5-fold higher in livers of African green monkeys. In addition, the concentration of apoA-I mRNA was 2-fold higher in the liver and 3.7-fold higher in the intestine of African green monkeys. Taken together, these findings indicate that differences in apoA-I production in the liver and small intestine are large enough to be responsible for the differences in the plasma concentrations of HDL and apoA-I between these species. Factors which regulate apoA-I secretion, including modulation of tissue apoA-I mRNA concentrations, are important determinants of plasma HDL concentrations and may contribute to the relative resistance of African green monkeys to dietary cholesterol-induced hypercholesterolemia and atherosclerosis. ApoA-I mRNA was also detected at low levels in the kidney and testis of African green and cynomolgus monkeys but not in the adrenal or brain. The tissue distribution and abundance of apoA-I mRNA in peripheral tissues was very different than that seen for apoE mRNA. Kidney and testis apoA-I mRNAs were the same size as liver apoA-I mRNA when examined by Northern blot analysis. Testis apoA-I mRNA appeared to be functionally active as judged by its presence in cytoplasmic polyribosomes. The low levels of apoA-I expression in kidney and testis are unlikely to contribute significantly to the plasma apoA-I pool but might function in some aspect of local lipid metabolism within these tissues.

The tissue distribution of apolipoprotein (apo) E synthesis in the cynomolgus monkey, Macaca fascicularis, was determined via short-term organ culture with radiolabeled amino acid. Tissue extracts were reacted with antiserum to apo-E, and immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Newly synthesized apo-E was detected in liver, adrenal, testis, lung, spleen, mesenteric lymph node, and kidney. Peripheral and hepatic apo-E showed the same electrophoretic mobility. High resolution two-dimensional gel analysis showed that newly synthesized apo-E exists in two major isoforms in each tissue examined. Comparison of isoform patterns and mixing experiments showed that newly synthesized apo-E isoforms have identical charge properties in each tissue examined. These data indicate that numerous peripheral tissues synthesize apo-E that is indistinguishable from liver apo-E by the criteria tested. Measurements of relative synthetic capacities illustrate that apo-E is a moderately abundant protein product of a variety of peripheral tissues although quantitative differences in apo-E synthesis occur. Apo-E mRNA from cynomolgus monkey liver and human Hep G2 cells co-migrated with an electrophoretic mobility corresponding to approximately 1200 nucleotides. Apo-E mRNA from liver, brain, thymus, kidney, testis, lymph node, and spleen was the same size. Primer extension analysis yielded a cDNA product representing complete copying of the 5' untranslated region of human Hep G2 apo-E mRNA. A cDNA of identical size was produced with cynomolgus monkey apo-E mRNA from liver, spleen, brain, lymph node, kidney, lung, and thymus. These data suggest that transcription of the apo-E gene is initiated at or near the same site in each tissue examined.

The size of low density lipoproteins (LDL) is strongly correlated with LDL cholesteryl ester (CE) content and coronary artery atherosclerosis in monkeys fed cholesterol and saturated fat. African green monkeys fed 11% (weight) fish oil diets have smaller LDL and less CE per LDL particle than lard-fed animals. We hypothesized that this might be due to a lower plasma lecithin:cholesterol acyltransferase (LCAT) activity in fish oil-fed animals. Using recombinant particles made of egg yolk lecithin-[14C]cholesterol-apoA-I as exogenous substrate, we found no difference in plasma LCAT activity (27 versus 28 nmol CE formed per h/ml) of fish oil- versus lard-fed animals, respectively; furthermore, no diet-induced difference in immunodetectable LCAT was found. However, plasma phospholipids from fish oil-fed animals were over 4-fold enriched in n-3 fatty acids in the sn-2 position compared to those of lard-fed animals. Additionally, the proportion of n-3 fatty acid-containing CE products formed by LCAT, relative to the available n-3 fatty acid in the sn-2 position of phospholipids, was less than one-tenth of that for linoleic acid. The overall rate of LCAT-catalyzed CE formation with phospholipid substrates from fish oil-fed animals was lower (5-50%) than with phospholipid substrates from lard-fed animals. These data show that n-3 fatty acids in phospholipids are not readily utilized by LCAT for formation of CE; rather, LCAT preferentially utilizes linoleic acid for CE formation. The amount of linoleic acid in the sn-2 position of plasma phospholipids is reduced and replaced with n-3 fatty acids in fish oil-fed animals. As a result, LCAT-catalyzed plasma CE formation in vivo is likely reduced in fish oil-fed animals contributing to the decreased cholesteryl ester content and smaller size of LDL particles in the animals of this diet group.

African green monkeys were fed diets containing low and moderate cholesterol concentrations with either polyunsaturated or unsaturated fat as 40% of calories. Plasma total cholesterol, low density lipoprotein (LDL) cholesterol, and apoB concentrations generally were higher in animals fed (a) the higher dietary cholesterol concentration and (b) saturated fat. At necropsy, liver and intestine were removed, and measurement of mRNAs for LDL receptors (liver) and for apolipoprotein B (liver and intestine) was done. Monkey small intestine mucosa made exclusively apoB48 while the liver made only apoB100, although apoB mRNA in both tissues was the same size (14 kilobases). No dietary cholesterol or fat effects were found for apoB mRNA abundance in the liver, while the animals fed the higher dietary cholesterol level had 50% lower levels of hepatic LDL receptor mRNA. In a separate group of animals, livers were perfused and the rate of apoB secretion was measured. No dietary fat effect on apoB secretion rate was found, and no relationship between plasma LDL cholesterol concentration and the rate of hepatic apoB production existed. These findings support the idea that the dietary factors that increase LDL concentrations act by reducing clearance of apoB-containing particles rather than by increasing production of these lipoproteins. Hepatic LDL receptor mRNA was similar in abundance in polyunsaturated fat and saturated fat-fed animals, suggesting that the difference in plasma cholesterol concentration between these groups is not mediated via effects on LDL receptor mRNA abundance. The level of intestinal apoB mRNA was about 30% higher in animals fed the moderate dietary cholesterol concentration. Earlier studies have shown that more cholesterol is transported in chylomicrons from the intestine when dietary cholesterol levels are higher, and the increased intestinal apoB mRNA abundance may reflect increased intestinal cholesterol transport and chylomicron apoB48 production.