Control of ACAT2 liver expression by HNF4{alpha}: lesson from MODY1 patients.

OBJECTIVE: ACAT2 is thought to be responsible for cholesteryl ester production in chylomicron and VLDL assembly. Recently, we identified HNF1alpha as an important regulator of the human ACAT2 promoter. Thus, we hypothesized that MODY3 (HNF1alpha gene mutations) and possibly MODY1 (HNF4alpha, upstream regulator of HNF1alpha, gene mutations) subjects may have lower VLDL esterified cholesterol. METHODS AND RESULTS: Serum analysis and lipoprotein separation using size-exclusion chromatography were performed in controls and MODY1 and MODY3 subjects. In vitro analyses included mutagenesis and cotransfections in HuH7 cells. Finally, the relevance in vivo of these findings was tested by ChIP assays in human liver. Whereas patients with MODY3 had normal lipoprotein composition, those with MODY1 had lower levels of VLDL and LDL esterified cholesterol, as well as of VLDL triglyceride. Mutagenesis revealed one important HNF4 binding site in the human ACAT2 promoter. ChIP assays and protein-to-protein interaction studies showed that HNF4alpha, directly or indirectly (via HNF1alpha), can bind to the ACAT2 promoter. CONCLUSIONS: We identified HNF4alpha as an important regulator of the hepatocyte-specific expression of the human ACAT2 promoter. Our results suggest that the lower levels of esterified cholesterol in VLDL- and LDL-particles in patients with MODY1 may-at least in part-be attributable to lower ACAT2 activity in these patients.

Influence of dietary fatty acid composition on the relationship between CETP activity and plasma lipoproteins in monkeys.

CETP activity, measured as transfer of cholesteryl ester from exogenous HDL to exogenous VLDL and LDL, reflecting CETP mass as determined by ELISA, was documented in three groups of St. Kitts vervet monkeys fed diets enriched in saturated (Sat), monounsaturated (Mono), or n-6 polyunsaturated (Poly) fatty acids. CETP activity was not different when comparing the three dietary fats. However, CETP activity was significantly higher when cholesterol was added to each of the diets. Significant positive associations between CETP activity and VLDL and LDL cholesterol concentrations were found whereas significant negative associations were seen between CETP activity and HDL cholesterol in each of the diet groups. The strength of these associations was highest in the Sat group. Cholesteryl ester (CE) fatty acid composition of lipoproteins varied widely among diet groups, with the more polyunsaturated CE of the Poly group being associated with a higher rate of CE transfer to endogenous acceptor apolipoprotein B-containing lipoproteins. Finally, only the Sat diet group showed significant positive correlations of CETP activity with LDL particle diameter (r = 0.76), cholesteryl ester percentage (r = 0.67), and a strong negative correlation (r = -0.86) with LDL receptor function, estimated as the difference between native and methylated LDL turnover rates. We speculate that strong associations between CETP and LDL metabolism may explain, at least in part, the increased atherogenicity of dietary saturated fat.

Caloric restriction lowers plasma lipoprotein (a) in male but not female rhesus monkeys.

Many age-associated pathophysiological changes are retarded by caloric restriction (CR). The present study has investigated the effect of CR on plasma lipoprotein (a) [Lp(a)], an independent risk factor for the age-associated process of atherosclerosis. Rhesus monkeys were fed a control diet (n=19 males, 12 females) or subjected to CR (n=20 males, 11 females fed 30% less calories) for >2 years. All female animals were premenopausal. Plasma Lp(a) levels in control animals were almost two fold higher for males than females (47+/-9 vs 25+/-5mg/dl mean+/-SEM, p=0.05). CR resulted in a reduction in circulating Lp(a) in males to levels similar to those measured in calorie-restricted females, (27+/-5 vs 24+/-4 mg/dl mean+/-SEM). For all animals, plasma Lp(a) was correlated with total cholesterol (r=0.27, p=0.03) and LDL cholesterol (r=0.50, p=0.0001) whether unadjusted or after adjustment for treatment, gender or group. These studies introduce a new mechanism whereby CR may have a beneficial effect on risk factors for the development of atherosclerosis in primates.

Dietary n-3 polyunsaturated fat increases the fractional catabolic rate of medium-sized HDL particles in African green monkeys.

We have previously described a novel pathway for the metabolism of HDL subfractions in which small [2 apolipoprotein (apoA-I) molecules per particle] HDL particles are converted in a unidirectional manner outside the plasma compartment to medium (3 apoA-I molecules per particle) or large (4 apoA-I molecules per particle) HDL particles, which are subsequently removed from the circulation by the liver (Colvin et al. 1999. J. Lipid Res. 40: 1782;-1792; Huggins et al. 2000. J. Lipid Res. 41: 384;-394). The purpose of the present study was to determine whether the reduction in concentration of medium HDL in African green monkeys consuming n-3 polyunsaturated versus saturated fat diets resulted from decreased in vivo production or increased catabolism. Tracer small LpA-I (HDL containing only apoA-I) were isolated, without ultracentrifugation, by gel filtration and immunoaffinity chromatography and radiolabeled. After injection, the specific activity of apoA-I in small, medium, and large HDL was determined, and the kinetic data were analyzed using our previously published multicompartmental model for HDL subfraction metabolism. We found a significant reduction of apoA-I concentration in medium HDL in the animals fed n-3 polyunsaturated fat (31.2 +/- 0.7 mg/dl) compared with animals fed saturated fat (85.4 +/- 11.9 mg/dl; P = 0.002). The production rates of apoA-I in small, medium, and large HDL were similar in both diet groups; however, there was a significant increase in the fractional catabolic rate of apoA-I in medium HDL in the animals fed n-3 polyunsaturated fat (2.188 +/- 0.501 pools/day) compared with animals fed saturated fat (0.714 +/- 0.191 pools/day; P = 0.02). We conclude that n-3 polyunsaturated fat reduces HDL cholesterol concentration by increasing the fractional catabolic rate of medium-sized HDL particles in African green monkeys.

Isoprostane levels in lipids extracted from atherosclerotic arteries of nonhuman primates.

Nonhuman primates used in these studies had been fed for 5 years diets enriched with cholesterol and one of three classes of fatty acids: saturated, monounsaturated, or polyunsaturated fatty acids. Atherosclerotic iliac artery lipid extracts were quantitatively analyzed for cholesterol, cholesteryl esters, fatty acid composition, and a marker of lipid oxidation, the F(2)-isoprostanes. There was no significant difference in the mean accumulation of F(2)-isoprostanes among the different diet groups. To account for the small, individual variation in the arachidonate concentration the F(2)-isoprostane mass from each sample was normalized by dividing by arachidonate mass: F(2)-isoprostane mass/(mass arachidonate). At lower levels of cholesterol accumulation, the F(2)-isoprostane mass/(mass arachidonate) ratio was greater in lipids from POLY arteries compared to SAT arteries, but the reverse was true at high levels of cholesterol. F(2)-isoprostane/(mass arachidonate) increased with mole fraction linoleate for the SAT group, but decreased for the POLY group. In summary, these studies demonstrated that there is no simple explanation of how F(2)-isoprostane accumulation did not depend on the concentration of oxidizable lipids that promote free-radical lipid oxidation.

Is the oxidation of high-density lipoprotein lipids different than the oxidation of low-density lipoprotein lipids?

This article gives detailed insight into the kinetics of high-density lipoprotein (HDL) oxidation catalyzed by azobis(2-amidinopropane).dihydrochloride (ABAP) or by copper. ABAP initialized oxidation of human HDL 3-4 times faster than non-human primate HDL with a similar composition. The oxidizability of non-human primate HDL was 1000 times lower than the oxidizability calculated from rate constants derived from liposome oxidation, suggesting that there is a slow step in HDL oxidation not present in liposomes. Saturable binding of copper to HDL was a significant feature of copper-catalyzed oxidation. Binding constants (K(m)) for non-human primate HDL were 2-3-fold lower than those for human HDL. Copper-catalyzed oxidation of non-human primate HDL was slower than that of human HDL, but human HDL(2) and HDL(3) oxidized at about the same rate. Overall, the kinetics describing the oxidation of HDL were mechanistically similar to those reported for LDL, suggesting that HDL lipids were as easily oxidized as LDL lipids and that HDL will be easily oxidized in vivo when exposed to agents that oxidize LDL.

Biochemical analysis of cell-derived apoE3 particles active in stimulating neurite outgrowth.

Susceptibility to the development of late-onset Alzheimer's disease is increased for individuals harboring one or more apolipoprotein E4 (apoE4) alleles. Although several isoform-specific effects of apoE have been identified, the relationship between biochemical function and risk factor assessment is unknown. Our previous studies showed that a physiologically relevant cell-derived apoE3 particle stimulates neurite outgrowth in an isoform-specific manner. In an attempt to delineate the biochemical mechanism responsible for the stimulatory effects of apoE3 on neurite outgrowth, we performed a detailed physical characterization of cell-derived apoE3 and apoE4 particles. Immunoaffinity chromatography followed by SDS-PAGE illustrated homogeneity in protein content (apoE >95%). The affinity-purified particles contained phospholipid and 1 mol of cholesterol per mole of apoE but no core lipids. Nondenaturing gradient gel electrophoresis identified two major particle populations with hydrated diameters of 8.0 and 9.2 nm. Neurite outgrowth assays performed with the affinity-purified particles resulted in similar isoform-specific differences as seen previously, apoE3 stimulatory and apoE4 neutral. Interestingly, we did not observe a reduction in apoE medium concentrations over the duration of the neurite outgrowth assays, suggesting little or no endocytic uptake. Ligand blot analysis demonstrated that the affinity-purified apoE particles bind to several Neuro-2a membrane proteins. Western blots of the Neuro-2a membrane proteins indicated that the LDL receptor, gp330, and LR8B might be involved in the apoE-binding event. These results discriminate against the lipid delivery hypothesis and suggest that the biological activity of the phospholipid apoE3 particles may be due to cell surface signaling.

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.

Defining the atherogenicity of large and small lipoproteins containing apolipoprotein B100.

Apo-E-deficient apo-B100-only mice (APOE:(-/-)APOB:(100/100)) and LDL receptor-deficient apo-B100-only mice (LDLR:(-/-)APOB:(100/100)) have similar total plasma cholesterol levels, but nearly all of the plasma cholesterol in the former animals is packaged in VLDL particles, whereas, in the latter, plasma cholesterol is found in smaller LDL particles. We compared the apo-B100-containing lipoprotein populations in these mice to determine their relation to susceptibility to atherosclerosis. The median size of the apo-B100-containing lipoprotein particles in APOE:(-/-)APOB:(100/100) plasma was 53.4 nm versus only 22.1 nm in LDLR:(-/-)APOB:(100/100) plasma. The plasma levels of apo-B100 were three- to fourfold higher in LDLR:(-/-)APOB:(100/100) mice than in APOE:(-/-)APOB:(100/100) mice. After 40 weeks on a chow diet, the LDLR:(-/-)APOB:(100/100) mice had more extensive atherosclerotic lesions than APOE:(-/-)APOB:(100/100) mice. The aortic DNA synthesis rate and the aortic free and esterified cholesterol contents were also higher in the LDLR:(-/-)APOB:(100/100) mice. These findings challenge the notion that all non-HDL lipoproteins are equally atherogenic and suggest that at a given cholesterol level, large numbers of small apo-B100-containing lipoproteins are more atherogenic than lower numbers of large apo-B100-containing lipoproteins.

Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates.

Two closely related enzymes with more than 50% sequence identity have been identified that catalyze the esterification of cholesterol using acyl-CoA substrates, namely acyl-CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2. Both are membrane-spanning proteins believed to reside in the endoplasmic reticulum of cells. ACAT2 has been hypothesized to be associated with lipoprotein particle secretion whereas ACAT1 is ubiquitous and may serve a more general role in cellular cholesterol homeostasis. We have prepared and affinity purified rabbit polyclonal antibodies unique to either ACAT enzyme to identify their cellular localization in liver and intestine, the two main lipoprotein-secreting tissues of the body, and for comparison, kidney and adrenal. In the liver, ACAT2 was identified in the rough endoplasmic reticulum of essentially all hepatocytes whereas ACAT1 was confined to cells lining the intercellular spaces among hepatocytes in a pattern typical of Kupffer cells. In the intestine, ACAT2 signal was strongly present in the apical third of the mucosal cells, whereas ACAT1 staining was diffuse throughout the mucosal cell, but with strong signal in goblet cells, Paneth cells, and villus macrophages. In the kidney, ACAT1 immunostaining was specific for the distal tubules and podocytes within the glomerulus. In the adrenal, ACAT1 signal was strongly present in the cells of the cortex, and absent from other adrenal cell types. No ACAT2 signal was identified in the kidney or adrenal. We conclude that only the cells of the liver and intestine that secrete apolipoprotein B-containing lipoproteins contain ACAT2, whereas ACAT1 is present in numerous other cell types. The data clearly suggest separate functions for these two closely related enzymes, with ACAT2 being most closely associated with plasma cholesterol levels.

ACAT1 and ACAT2 membrane topology segregates a serine residue essential for activity to opposite sides of the endoplasmic reticulum membrane.

A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determined based on glycosylation site use and accessibility to exogenous protease. The accessibility of the C-terminal FLAG epitope in constructs was determined by immunofluorescence microscopy of permeabilized transfected cells. Both ACAT1 and ACAT2 span the membrane five times with their N termini in the cytosol and C termini in the ER lumen. The fourth transmembrane domain is located in a different region for each protein, placing the putative active site ACAT1 serine (Ser(269)) in the cytosol and the analogous residue in ACAT2 (Ser(249)) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to lipoprotein particle assembly and secretion, whereas ACAT1 may function primarily to maintain the balance of free and esterified cholesterol intracellularly.

Low levels of extrahepatic nonmacrophage ApoE inhibit atherosclerosis without correcting hypercholesterolemia in ApoE-deficient mice.

The prevention of atherosclerosis by apolipoprotein E (apoE) is generally attributed to the removal of plasma lipoprotein remnant particles. We developed transgenic apoE-knockout mice expressing apoE specifically in the adrenal gland and found that only 3% of the wild-type plasma level of apoE was sufficient to normalize plasma cholesterol levels in the apoE-deficient mouse. As expected, mice expressing apoE at levels that correct hypercholesterolemia had almost no cholesteryl ester deposition in their aortas. In contrast, their nontransgenic siblings had significant atherosclerosis. Unexpectedly, we found that atherosclerosis was also reduced in 2 transgenic lines expressing too little apoE (<1% to 2% of wild type) to correct their hypercholesterolemia. Gel exclusion chromatographic profiles of plasma lipoproteins and the size distributions of lipoproteins with density <1.063 (low density and very low density lipoproteins), as determined by dynamic laser light scattering, were the same in mice expressing <2 microg/mL plasma apoE and their nontransgenic littermates. We conclude that the antiatherogenic action of low levels of plasma apoE is not due to the clearance of remnant lipoproteins. Thus, low levels of apoE provided systemically, but not made in the liver or in macrophages, can block atherogenesis in the vascular wall independently of normalizing the plasma concentration of atherogenic remnant lipoprotein particles.

In vitro oxidation of low-density lipoprotein in two species of nonhuman primates subjected to caloric restriction.

Caloric restriction (CR), which increases longevity and retards age-associated diseases in laboratory rodents, is being evaluated in nonhuman primate trials. CR reduces oxidative stress in rodents and appears to improve risk factors for cardiovascular disease in nonhuman primates. We tested the hypothesis that low-density lipoprotein (LDL) oxidizability is reduced in two monkey species (rhesus and cynomolgus) subjected to chronic CR. In both species, no significant differences occurred between CR and control animals on total, LDL, or high-density lipoprotein (HDL) cholesterol. In rhesus monkeys, triglycerides were higher in controls than CR (139 +/- 23 vs 66 +/- 8 mg/dl,p < .01, respectively). LDL from CR rhesus monkeys was reduced in triglyceride content and molecular weight compared to controls, whereas LDL composition in cynomolgus monkeys was similar in CR and control animals. In keeping with minor deviations in lipids, antioxidants, and LDL composition, no consistent differences in in vitro LDL oxidizability were apparent between CR and controls in either species.

Consumption of soy protein reduces cholesterol absorption compared to casein protein alone or supplemented with an isoflavone extract or conjugated equine estrogen in ovariectomized cynomolgus monkeys.

Dietary intake of soy protein is associated with reductions in plasma cholesterol. Isoflavones are thought to be active components of soy and responsible for the beneficial effects because of their structural similarities to estrogen. The purposes of this study were to determine if i) soy protein or a semipurified soy extract, rich in isoflavones, is responsible for improving the lipid profile and ii) altered intestinal cholesterol metabolism is one mechanism for hypocholesterolemic effects. Ovariectomized adult female cynomolgus monkeys (40) were assigned to groups fed diets containing i) casein-lactalbumin (CAS) ii) intact soy protein (SOY), iii) CAS plus an isoflavone-rich semipurified soy extract similar in isoflavone content as SOY (ISO) or iv) CAS plus conjugated equine estrogen (CEE) for 20 wk. Cholesterol absorption was determined using the fecal isotope ratio method. Bile acid excretion was measured using the 3alpha-hydroxysteroid dehydrogenase assay. The SOY group had significantly lower total- and VLDL + LDL-cholesterol compared to the other three groups and significantly higher HDL-cholesterol compared to the CAS and CEE groups. Cholesterol absorption was significantly lower in the SOY group compared to the other groups, but bile acid excretion was not significantly affected. The hypocholesterolemic effect of soy protein appears to be mediated in part by decreased cholesterol absorption. The semipurified soy extract, rich in isoflavones, added to casein protein did not have lipid-lowering effects. Other components of soy such as saponins, phytic acid or the amino acid composition may be involved in the hypocholesterolemic effects seen in this study.

Determination of the tissue sites responsible for the catabolism of large high density lipoprotein in the African green monkey.

In vivo multicompartmental modeling of the turnover of HDL subfractions has suggested that HDL containing four molecules of apoA-I per particle and no other apolipoproteins (large LpA-I) are terminal particles in plasma. We hypothesized that these terminal particles were the end product of HDL metabolism and, as such, would be cleared preferentially by the liver. Thus, the purpose of this study was to determine: 1) the tissue sites of catabolism of large LpA-I in African green monkeys, and 2) whether saturated versus n;-6 polyunsaturated dietary fat affected tissue accumulation. Large LpA-I were isolated, without ultracentrifugation, by size exclusion and immunoaffinity chromatography and radiolabeled with either the residualizing compound, (125)I-labeled tyramine cellobiose (TC), or with (131)I. After injection into recipient animals, the plasma die-away of the radiolabels was followed for 12 or 24 h, after which the animals were killed and tissues were collected for determining radiolabel sites of catabolism. The plasma die-away of the (125)I-labeled TC-LpA-I and (131)I-labeled LpA-I doses was similar suggesting that the TC radiolabeling did not modify the metabolism of the large LpA-I dose. The liver, adrenal, kidney, and spleen had the greatest accumulation of large LpA-I degradation products on a per gram tissue basis. On a whole organ basis, the liver was the major site of large LpA-I degradation in both the 12-h (15.4 +/- 0.3% of injected dose) and 24-h (9.1 +/- 0.6% of injected dose) catabolic studies. The kidney, compared to the liver, had less uptake of large LpA-I radioactivity in either study (1.3 +/- 0.4% and 1.2 +/- 0.3% of injected dose). There was no apparent influence of dietary fat type on the tissue accumulation of large LpA-I. We conclude that the liver is the primary site of catabolism of large LpA-I in the African green monkey.

Validity of animal models for the cholesterol-raising effects of coffee diterpenes in human subjects.

Cafestol and kahweol, coffee lipids present in unfiltered coffee brews, potently increase LDL-cholesterol concentration in human subjects. We searched for an animal species in which cafestol similarly increases LDL-cholesterol. Such an animal model could be used subsequently as a model to study the mechanism of action of cafestol and kahweol. Cafestol and kahweol increased serum lipids in African green monkeys (Cercopithecus aethiops), cebus (Cebus apella) and rhesus (Macaca mulatta) monkeys, hamsters, rats and gerbils differently from the increase in human subjects. In African green monkeys, the rise in total cholesterol was less pronounced than that in human subjects. In addition, the increase in total cholesterol was predominantly due to a rise in HDL-cholesterol rather than LDL-cholesterol. Thus, the rise in plasma lipids might illustrate the mechanism in these monkeys rather than the mechanism in human subjects. In other animal species, cafestol and kahweol did not raise cholesterol consistently. The variability in effects on serum lipids could not be explained by the mode of administration or dose of diterpenes, nor by the amount of cholesterol in the diet. In conclusion, we did not find an animal model in which cafestol and kahweol elevate plasma lipoproteins to the same extent as in human subjects. For the time being, therefore, studies on the mechanism of action should be done preferably in human subjects.