ACAT Activity Research Articles

Su2010 Chronic and Selective Inhibition of Na-K-ATPase Uniquely Regulates Brush Border Na Dependent Nutrient Absorption in Intestinal Epithelial Cells

cells were added to the basolateral medium of intestinal Caco-2/15 cells grown on Transwell filter plates. Results: An evident decrease was noted in LDLr in the whole-cell extracts and basolateral cell-surface while exogenous WTand D374Y-PCSK9 were accumulated in cells. Clearly, co-immunoprecipitation studies indicated an association of PCSK9 and LDLr. Both forms of exogenous PCSK9 caused a significant enhancement in cholesterol uptake also evidenced by a raised protein expression of cholesterol transporters NPC1L1 and CD36, but SR-B-I, ABCA1, ABGC5 and ABCG8 expressions remained unchanged. Moreover, PCSK9 altered the activity of HMG-CoA reductase and ACAT, two key proteins involved in cholesterol synthesis and esterification, respectively. Finally, exogenous PCSK9 was able to enhance triglyceride-rich lipoproteins by positively modulating lipid synthesis, Sar1GTPase expression as well as apolipoprotein B-48 biogenesis. Conclusion: These data suggest that, in addition to its modulatory action on LDLr, PCSK9 significantly influences intracellular cholesterol homeostasis and the machinery governing lipid transport in the gut. Acknowledgment: This study was supported by the Canadian Institutes of Health Research (EL, NS) and the J.A. DeSeve Research Chair in Nutrition (EL).

Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation

The hypothesis tested in this study was that cholesterol esterification by ACAT2 would increase cholesterol absorption efficiency by providing cholesteryl ester (CE) for incorporation into chylomicrons. The assumption was that absorption would be proportional to Acat2 gene dosage. Male ACAT2⁺/⁺, ACAT2⁺/⁻, and ACAT2⁻/⁻ mice were fed a diet containing 20% of energy as palm oil with 0.2% (w/w) cholesterol. Cholesterol absorption efficiency was measured by fecal dual-isotope and thoracic lymph duct cannulation (TLDC) methods using [³H]sitosterol and [¹⁴C]cholesterol tracers. Excellent agreement among individual mice was found for cholesterol absorption measured by both techniques. Cholesterol absorption efficiency in ACAT2⁻/⁻ mice was 16% compared with 46-47% in ACAT2⁺/⁺ and ACAT2⁺/⁻ mice. Chylomicrons from ACAT2⁺/⁺ and ACAT2⁺/⁻ mice carried ∼80% of total sterol mass as CE, whereas ACAT2⁻/⁻ chylomicrons carried >90% of sterol mass in the unesterified form. The total percentage of chylomicron mass as CE was reduced from 12% in the presence of ACAT2 to ∼1% in ACAT2⁻/⁻ mice. Altogether, the data demonstrate that ACAT2 increases cholesterol absorption efficiency by providing CE for chylomicron transport, but one copy of the Acat2 gene, providing ∼50% of ACAT2 mRNA and enzyme activity, was as effective as two copies in promoting cholesterol absorption.

Subcellular localization and regulation of StarD4 protein in macrophages and fibroblasts

StarD4 is a member of the StarD4 subfamily of START domain proteins with a characteristic lipid binding pocket specific for cholesterol. The objective of this study was to define StarD4 subcellular localization, regulation, and function. Immunobloting showed that StarD4 is highly expressed in the mouse fibroblast cell line 3T3-L1, in human THP-1 macrophages, Kupffer cells (liver macrophages), and hepatocytes. In 3T3-L1 cells and THP-1 macrophages, StarD4 protein appeared localized to the cytoplasm and the endoplasmic reticulum (ER). More specifically, in THP-1 macrophages StarD4 co-localized to areas of the ER enriched in Acyl-CoA:cholesterol acyltransferase-1 (ACAT-1), and was closely associated with budding lipid droplets. The addition of purified StarD4 recombinant protein to an in vitro assay increased ACAT activity 2-fold, indicating that StarD4 serves as a rate-limiting step in cholesteryl ester formation by delivering cholesterol to ACAT-1-enriched ER. In addition, StarD4 protein was found to be highly regulated and to redistribute in response to sterol levels. In summary, these observations, together with our previous findings demonstrating the ability of increased StarD4 expression to increase bile acid synthesis and cholesteryl ester formation, provide strong evidence for StarD4 as a highly regulated, non-vesicular, directional, intracellular transporter of cholesterol which plays a key role in the maintenance of intracellular cholesterol homeostasis.

Urotensin II differentially regulates macrophage and hepatic cholesterol homeostasis

Urotensin II (UII) is a vasoactive peptide with pleotropic activity. Interestingly, UII levels are elevated in hyperlipidemic patients, and UII induces lipase activity in some species. However, the exact role UII plays in cholesterol homeostasis remains to be elucidated. UII knockout (UII KO) mice were generated and a plasma lipoprotein profile, and hepatocytes and macrophages cholesterol uptake, storage and synthesis was determined. UII KO had a decreased LDL cholesterol profile and liver steatosis compared to wildtype mice (WT). UII KO macrophages demonstrated enhanced ACAT activity and LDL uptake in the short term (up to 4 h), of which more LDL-delivered exogenously derived cholesterol was incorporated into cholesteryl ester (CE) than the WT macrophages. UII KO macrophages generated more than two times the amount of de novo endogenously synthesized cholesterol, and of this cholesterol more than two times the relative amount was esterified to CE. In comparison, results in hepatocytes demonstrated that far more exogenously derived cholesterol was incorporated into CE in the WT cells, generating almost ten times the amount of CE than UII KO. WT cells synthesize de novo almost ten times the amount of cholesterol than UIIKO, and of that cholesterol, almost two times the amount of CE in WT than UII KO hepatocytes. In addition, more ApoB lipoproteins were secreted from WT than UII KO hepatocytes. These results demonstrate a fundamental difference between macrophages and hepatocytes in terms of cholesterol homeostasis, and suggest an important role for UII in modulating cholesterol regulation.

Autophosphorylation Activates Dictyostelium Myosin II Heavy Chain Kinase A by Providing a Ligand for an Allosteric Binding Site in the α-Kinase Domain

Dictyostelium discoideum myosin II heavy chain kinase A (MHCK A), a member of the atypical α-kinase family, phosphorylates sites in the myosin II tail that block filament assembly. Here we show that the catalytic activity of A-CAT, the α-kinase domain of MHCK A (residues 552-841), is severely inhibited by the removal of a disordered C-terminal tail sequence (C-tail; residues 806-841). The key residue in the C-tail was identified as Thr(825), which was found to be constitutively autophosphorylated. Dephosphorylation of Thr(825) using shrimp alkaline phosphatase decreased A-CAT activity. The activity of a truncated A-CAT lacking Thr(825) could be rescued by P(i), phosphothreonine, and a phosphorylated peptide, but not by threonine, glutamic acid, aspartic acid, or an unphosphorylated peptide. These results focused attention on a P(i)-binding pocket located in the C-terminal lobe of A-CAT. Mutational analysis demonstrated that the P(i)-pocket was essential for A-CAT activity. Based on these results, it is proposed that autophosphorylation of Thr(825) activates ACAT by providing a covalently tethered ligand for the P(i)-pocket. Ab initio modeling studies using the Rosetta FloppyTail and FlexPepDock protocols showed that it is feasible for the phosphorylated Thr(825) to dock intramolecularly into the P(i)-pocket. Allosteric activation is predicted to involve a conformational change in Arg(734), which bridges the bound P(i) to Asp(762) in a key active site loop. Sequence alignments indicate that a comparable regulatory mechanism is likely to be conserved in Dictyostelium MHCK B-D and metazoan eukaryotic elongation factor-2 kinases.

Abolished synthesis of cholic acid reduces atherosclerotic development in apolipoprotein E knockout mice

To investigate the effects of abolished cholic acid (CA) synthesis in the ApoE knockout model [apolipoprotein E (apoE) KO],a double-knockout (DKO) mouse model was created by crossbreeding Cyp8b1 knockout mice (Cyp8b1 KO), unable to synthesize the primary bile acid CA, with apoE KO mice. After 5 months of cholesterol feeding, the development of atherosclerotic plaques in the proximal aorta was 50% less in the DKO mice compared with the apoE KO mice. This effect was associated with reduced intestinal cholesterol absorption, decreased levels of apoB-containing lipoproteins in the plasma, enhanced bile acid synthesis, reduced hepatic cholesteryl esters, and decreased hepatic activity of ACAT2. The upregulation of Cyp7a1 in DKO mice seemed primarily caused by reduced expression of the intestinal peptide FGF15. Treatment of DKO mice with the farnesoid X receptor (FXR) agonist GW4064 did not alter the intestinal cholesterol absorption, suggesting that the action of CA in this process is confined mainly to formation of intraluminal micelles and less to its ability to activate the nuclear receptor FXR. Inhibition of CA synthesis may offer a therapeutic strategy for the treatment of hyperlipidemic conditions that lead to atherosclerosis.

Cholesterol loading in macrophages stimulates formation of ER-derived vesicles with elevated ACAT1 activity

Cholesterol loading in macrophages stimulates formation of ER-derived vesicles with elevated ACAT1 activity

High ACAT1 expression in estrogen receptor negative basal-like breast cancer cells is associated with LDL-induced proliferation

The specific role of dietary fat in breast cancer progression is unclear, although a low-fat diet was associated with decreased recurrence of estrogen receptor alpha negative (ER(-)) breast cancer. ER(-) basal-like MDA-MB-231 and MDA-MB-436 breast cancer cell lines contained a greater number of cytoplasmic lipid droplets compared to luminal ER(+) MCF-7 cells. Therefore, we studied lipid storage functions in these cells. Both triacylglycerol and cholesteryl ester (CE) concentrations were higher in the ER(-) cells, but the ability to synthesize CE distinguished the two types of breast cancer cells. Higher baseline, oleic acid- and LDL-stimulated CE concentrations were found in ER(-) compared to ER(+) cells. The differences corresponded to greater mRNA and protein levels of acyl-CoA:cholesterol acyltransferase 1 (ACAT1), higher ACAT activity, higher caveolin-1 protein levels, greater LDL uptake, and lower de novo cholesterol synthesis in ER(-) cells. Human LDL stimulated proliferation of ER(-) MDA-MB-231 cells, but had little effect on proliferation of ER(+) MCF-7 cells. The functional significance of these findings was demonstrated by the observation that the ACAT inhibitor CP-113,818 reduced proliferation of breast cancer cells, and specifically reduced LDL-induced proliferation of ER(-) cells. Taken together, our studies show that a greater ability to take up, store and utilize exogenous cholesterol confers a proliferative advantage to basal-like ER(-) breast cancer cells. Differences in lipid uptake and storage capability may at least partially explain the differential effect of a low-fat diet on human breast cancer recurrence.

Leptin modulates ACAT1 expression and cholesterol efflux from human macrophages

Leptin is an adipose tissue-derived hormone implicated in atherosclerosis and macrophage foam cell formation. The current study was conducted to examine the effect of leptin on cholesteryl ester accumulation in human monocytes/macrophages. Exogenously added leptin at 5 nM during differentiation of monocytes into macrophages for 7 days accelerated acetylated LDL (acetyl-LDL)-induced cholesteryl ester accumulation by 30-50%. Leptin did not affect endocytic uptake of acetyl-LDL; however, it increased ACAT activity 1.8-fold and ACAT-1 protein expression 1.9-fold. Among the four ACAT-1 mRNA transcripts, two shorter transcripts (2.8 and 3.6 kb) were upregulated approximately 1.7-fold upon leptin treatment. The enhanced expression of ACAT-1 protein by leptin was suppressed by inhibitors of Janus-activated kinase2 (JAK2) and phosphatidylinositol 3-kinase (PI3K). HDL-mediated cholesterol efflux was suppressed by leptin, which was canceled by K-604, an ACAT-1 inhibitor. Expression of long form of leptin receptor was upregulated during monocytic differentiation into macrophages and sustained after differentiation. Thus, the results suggest that leptin accelerates cholesteryl ester accumulation in human monocyte-derived macrophages by increasing ACAT-1 expression via JAK2 and PI3K, thereby suppressing cholesterol efflux.

Control of ACAT2 Liver Expression by HNF4α

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. 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. 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.

Synthesis and structure–activity relationships of N-(4-amino-2,6-diisopropylphenyl)- N’-(1,4-diarylpiperidine-4-yl)methylureas as anti-hyperlipidemic agents

Based on 1,4-diarylpiperidine-4-methylureas, a new class of ACAT inhibitors, we examined in the study the SAR of a series of compounds prepared by replacing the substituent at the three aromatic parts. Introduction of long alkoxy group onto the phenyl moiety at the B-part was effective in improving both the inhibitory activity for ACAT and the up-regulatory activity for LDL-R expression. Particularly, 3-hydroxypropoxy group ( 43) on the phenyl moiety of B-part led to improved solubility, while keeping both biological activities. Compound 43 inhibited ACAT activity with an IC 50 value of 18 nM, which is superior to that of a known ACAT inhibitor, CI-1011. In addition, compound 43 revealed an LDL-R up-regulatory activity comparable to that of SMP-797. We therefore expect this compound to be a novel ACAT inhibitor.

ACAT2 and human hepatic cholesterol metabolism: Identification of important gender-related differences in normolipidemic, non-obese Chinese patients

Objective ACAT2 is a major cholesterol esterification enzyme specifically expressed in hepatocytes and may control the amount of hepatic free (unesterified) cholesterol available for secretion into bile or into HDL. This study aims to further elucidate physiologic roles of ACAT2 in human hepatic cholesterol metabolism. Methods and results Liver biopsies from 40 normolipidemic, non-obese gallstone patients including some gallstone-free patients (female/male, 18/22) were collected and analyzed for microsomal ACAT2 activity, protein and mRNA expression. Plasma HDL-cholesterol (HDL-C) was significantly higher in females than in males, while triglycerides were significantly lower. ACAT2 activity in females was significantly lower than observed in males, regardless of the presence of gallstone disease. Moreover, the activity of ACAT2 correlated negatively with plasma levels of HDL-C ( r = −0.57, P < 0.05) and with Apo AI ( r = −0.49, P < 0.05). Conclusion This is the first description of a gender-related difference in hepatic ACAT2 activity in normolipidemic non-obese Chinese patients suggesting a possible role for ACAT2 in the regulation of cholesterol metabolism in humans. The negative correlation between ACAT2 activity and HDL-C or Apo AI may reflect this regulation. Since ACAT2 activity generally has been found to be pro-atherogenic in animal models, the observed sex-related difference may contribute to female protection from complications of coronary heart disease (CHD).

Are side-chain oxidized oxysterols regulators also in vivo?

Oxsterols are oxygenated metabolites of cholesterol that are short-lived intermediates or end products in cholesterol excretion pathways. They are present in very low concentrations in mammalian systems, always accompanied by a high excess of cholesterol. According to current concepts, side-chain oxidized oxysterols may be mediators of many cholesterol-induced regulatory effects. When added to cultured cells in vitro, side-chain oxidized oxysterols limit intracellular cholesterol levels by at least three different mechanisms: 1) binding to Insig with subsequent block of the sterol regulatory element-binding proteins (SREBP)-mediated mechanism for regulation of sterol sensitive genes; 2) increasing degradation of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, eventually by a mechanism involving binding of Insig to the enzyme; 3) activation of LXR-mediated stimulation of cholesterol transporters and cholesterol metabolism. Addition of pure unesterified oxysterols to cell cultures is however highly unphysiological, and the in vivo relevance of such experiments is questionable. Transgenic mouse models with markedly reduced or increased concentration of some specific oxysterols do not present marked disturbances in cholesterol turnover and homeostasis. Oxysterol-binding proteins such as LXR have been conclusively shown to be of importance for cholesterol turnover in vivo, but their physiological ligands have not yet been defined with certainty. During the last few years, new experimental data has accumulated supporting the contention that side-chain oxysterols are involved in some LXR-mediated regulation in vivo, at least in some biological systems. The new findings will be critically reviewed here.

The Molecular Target of Rubimaillin in the Inhibition of Lipid Droplet Accumulation in Macrophages

The naphthohydroquinone rubimaillin, which has an angular-type three cyclic skeleton and was isolated from the Chinese medical plant Rubia cordifola, was found to inhibit lipid droplet accumulation in mouse macrophages and to selectively inhibit cholesteryl ester synthesis (IC(50): 18 microM). The metabolism of cholesterol from lysosomes to lipid droplets was inhibited by the compound with a similar IC(50) (45 microM). Moreover, rubimaillin inhibited acyl-CoA:cholesterol acyltransferase (ACAT1) activity in ACAT1-expressing cells (IC(50): 80 microM). Thus, these data strongly suggest that rubimaillin inhibits macrophage ACAT activity in order to decrease cholesteryl ester (CE) synthesis, leading to a reduction in the number of lipid droplets. Furthermore, rubimaillin was found to inhibit the ACAT2 isozyme in ACAT2-expressing cells (IC(50): 22 microM). We concluded that rubimaillin is a dual inhibitor of ACAT1 and ACAT2, but is more selective for the ACAT2 isozyme.

Modulation of intestinal cholesterol absorption by high glucose levels: impact on cholesterol transporters, regulatory enzymes, and transcription factors

Growing evidence suggests that the small intestine may contribute to excessive postprandial lipemia, which is highly prevalent in insulin-resistant/Type 2 diabetic individuals and substantially increases the risk of cardiovascular disease. The aim of the present study was to determine the role of high glucose levels on intestinal cholesterol absorption, cholesterol transporter expression, enzymes controlling cholesterol homeostasis, and the status of transcription factors. To this end, we employed highly differentiated and polarized cells (20 days of culture), plated on permeable polycarbonate filters. In the presence of [(14)C]cholesterol, glucose at 25 mM stimulated cholesterol uptake compared with Caco-2/15 cells supplemented with 5 mM glucose (P < 0.04). Because combination of 5 mM glucose with 20 mM of the structurally related mannitol or sorbitol did not change cholesterol uptake, we conclude that extracellular glucose concentration is uniquely involved in the regulation of intestinal cholesterol transport. The high concentration of glucose enhanced the protein expression of the critical cholesterol transporter NPC1L1 and that of CD36 (P < 0.02) and concomitantly decreased SR-BI protein mass (P < 0.02). No significant changes were observed in the protein expression of ABCA1 and ABCG8, which act as efflux pumps favoring cholesterol export out of absorptive cells. At the same time, 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity was decreased (P < 0.007), whereas ACAT activity remained unchanged. Finally, increases were noted in the transcription factors LXR-alpha, LXR-beta, PPAR-beta, and PPAR-gamma along with a drop in the protein expression of SREBP-2. Collectively, our data indicate that glucose at high concentrations may regulate intestinal cholesterol transport and metabolism in Caco-2/15 cells, thus suggesting a potential influence on the cholesterol absorption process in Type 2 diabetes.

Hormone-sensitive lipase is involved in hepatic cholesteryl ester hydrolysis

Hormone-sensitive lipase (HSL) regulates the hydrolysis of acylglycerol and cholesteryl ester (CE) in various organs, including adipose tissues. However, the hepatic expression level of HSL has been reported to be almost negligible. In the present study, we found that mice lacking both leptin and HSL (Lep(ob/ob)/HSL(-/-)) showed massive accumulation of CE in the liver compared with Lep(ob/ob)/HSL(+/+) mice, while triacylglycerol (TG) accumulation was modest. Similarly, feeding with a high-cholesterol diet induced hepatic CE accumulation in HSL(-/-) mice. Supporting these observations, we detected significant expression of protein as well as mRNA of HSL in the liver. HSL(-/-) mice showed reduced activity of CE hydrolase, but not of TG lipase, in the liver compared with wild-type mice. Furthermore, we confirmed the expression of HSL in viable parenchymal cells isolated from wild-type mice. The hepatocytes from HSL(-/-) mice showed reduced activity of CE hydrolase and contained more CE than those from HSL(+/+) mice even without the incubation with lipoproteins. Incubation with LDL further augmented the accumulation of CE in the HSL-deficient hepatocytes. From these results, we conclude that HSL is involved in the hydrolysis of CE in hepatocyes.

Identification of putative active site residues of ACAT enzymes

In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

A New Twist in the Cholesterol Metabolism-Based Enterohepatic Connection

A New Twist in the Cholesterol Metabolism-Based Enterohepatic Connection

S1582 Start Domain Protein Stard4 Is Able to Bind Free Cholesterol and Increases Cholesterol Ester Formation and Microsomal Acat Activity

S1582 Start Domain Protein Stard4 Is Able to Bind Free Cholesterol and Increases Cholesterol Ester Formation and Microsomal Acat Activity

Cholesterol Synthesis Inhibition Elicits an Integrated Molecular Response in Human Livers Including Decreased ACAT2

The purpose of this study was to identify how different degrees of cholesterol synthesis inhibition affect human hepatic cholesterol metabolism. Thirty-seven normocholesterolemic gallstone patients randomized to treatment with placebo, 20 mg/d fluvastatin, or 80 mg/d atorvastatin for 4 weeks were studied. Based on serum lathosterol determinations, cholesterol synthesis was reduced by 42% and 70% in the 2 groups receiving statins. VLDL cholesterol was reduced by 20% and 55%. During gallstone surgery, a liver biopsy was obtained and hepatic protein and mRNA expression of rate-limiting steps in cholesterol metabolism were assayed and related to serum lipoproteins. A marked induction of LDL receptors and 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase was positively related to the degree of cholesterol synthesis inhibition (ChSI). The activity, protein, and mRNA for ACAT2 were all reduced during ChSI, as was apoE mRNA. The lowering of HDL cholesterol in response to high ChSI could not be explained by altered expression of the HDL receptor CLA-1, ABCA1, or apoA-I. Statin treatment reduces ACAT2 activity in human liver and this effect, in combination with a reduced Apo E expression, may contribute to the favorable lowering of VLDL cholesterol seen in addition to the LDL lowering during statin treatment.