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Lawrence L Rudel - One of the best experts on this subject based on the ideXlab platform.
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Intestine-specific MTP and global ACAT2 deficiency lowers acute cholesterol absorption with chylomicrons and HDLs
Journal of lipid research, 2014Co-Authors: Jahangir Iqbal, Lawrence L Rudel, Mohamed Boutjdir, M. Mahmood HussainAbstract:Intestinal cholesterol absorption involves the chylomicron and HDL pathways and is dependent on microsomal triglyceride transfer protein (MTP) and ABCA1, respectively. Chylomicrons transport free and esterified cholesterol, whereas HDLs transport free cholesterol. ACAT2 esterifies cholesterol for secretion with chylomicrons. We hypothesized that free cholesterol accumulated during ACAT2 deficiency may be secreted with HDLs when chylomicron assembly is blocked. To test this, we studied cholesterol absorption in mice deficient in intestinal MTP, global ACAT2, and both intestinal MTP and global ACAT2. Intestinal MTP ablation significantly increased intestinal triglyceride and cholesterol levels and reduced their transport with chylomicrons. In contrast, global ACAT2 deficiency had no effect on triglyceride absorption but significantly reduced cholesterol absorption with chylomicrons and increased cellular free cholesterol. Their combined deficiency reduced cholesterol secretion with both chylomicrons and HDLs. Thus, contrary to our hypothesis, free cholesterol accumulated in the absence of MTP and ACAT2 is unavailable for secretion with HDLs. Global ACAT2 deficiency causes mild hypertriglyceridemia and reduces hepatosteatosis in mice fed high cholesterol diets by increasing hepatic lipoprotein production by unknown mechanisms. We show that this phenotype is preserved in the absence of intestinal MTP in global ACAT2-deficient mice fed a Western diet. Further, we observed increases in hepatic MTP activity in these mice. Thus, ACAT2 deficiency might increase MTP expression to avoid hepatosteatosis in cholesterol-fed animals. Therefore, ACAT2 inhibition might avert hepatosteatosis associated with high cholesterol diets by increasing hepatic MTP expression and lipoprotein production.
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Synthesis and structure-activity relationship of pyripyropene A derivatives as potent and selective acyl-CoA:cholesterol acyltransferase 2 (ACAT2) inhibitors: part 1.
Bioorganic & medicinal chemistry letters, 2013Co-Authors: Masaki Ohtawa, Lawrence L Rudel, Satoshi Ohte, Taichi Ohshiro, Daisuke Matsuda, Hiroshi Tomoda, Satoshi Ōmura, Hiroyuki Yamazaki, Tohru NagamitsuAbstract:Abstract In an effort to develop potent and selective inhibitors toward ACAT2, structure–activity relationship studies were carried out using derivatives based on pyripyropene A (PPPA, 1). We have successfully developed novel PPPA derivatives with a 7-O-substituted benzoyl substituent that significantly exhibit more potent ACAT2 inhibitory activity and higher ACAT2 isozyme selectivity than 1.
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ACAT2 and abcg5 g8 are both required for efficient cholesterol absorption in mice evidence from thoracic lymph duct cannulation
Journal of Lipid Research, 2012Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L RudelAbstract:Beginning in 1974, the identification of sitosterolemia (1), a rare recessive disorder characterized by elevated plasma and tissue concentrations of phytosterols, has focused attention on the basic molecular processes that govern how the body normally absorbs animal-derived dietary cholesterol while excluding all other similarly structured plant-derived sterols, generally called phytosterols. Phytosterols, including campesterol, stigmasterol, and sitosterol, differ from cholesterol mainly in possessing one or two additional carbons in side chains at C24. The average North American diet contains about equal amounts of cholesterol and phytosterols (∼150–400 mg/day) (2–5), yet <5% of phytosterols are absorbed compared with ∼50% of cholesterol (4, 6). Thus in healthy individuals, sensitive mechanisms exist that allow the body to distinguish among modestly different sterol structures. In general, different species of sterols have different absorption efficiencies; the closer the structural similarity to the cholesterol molecule, the higher the percentage absorption (4, 7). Even mildly hypercholesterolemic patients have serum concentrations of phytosterols that are 500 (campesterol) to 20,000 (sitosterol) times lower than that of cholesterol. Patients with sitosterolemia have increased fractional sterol absorption rates and impaired biliary secretion of neutral sterols, resulting in accumulations of these sterols in the blood and tissues (1, 8), premature atherosclerosis, and tendon/skin xanthomatosis (8, 9). Sitosterolemia has been linked to mutations in either adenosine triphosphate-binding cassette transporter G5 or G8 (ABCG5 or ABCG8), which are expressed almost exclusively in hepatocytes and enterocytes forming a heterodimer (G5G8) typically localized to the plasma membrane (10, 11). Another protein that is expressed exclusively in the same two cell types is acyl CoA:cholesterol acyltransferase type 2 (ACAT2), a cholesterol-esterifying enzyme residing in the endoplasmic reticulum (ER) membrane (12). Mice lacking G5G8 have phenotypes resembling patients with sitosterolemia, including increased fractional absorption of noncholesterol sterols, marked accumulation of sitosterol and campesterol in the blood and liver, reduced levels of biliary cholesterol, and various hemolytic disorders (7, 13). These findings suggested that the physiological role of G5G8 is to limit phytosterol accumulation in the body by limiting its absorption in the intestine and by promoting its secretion from the liver. Because sitosterolemic patients and G5G8 knockout (G5G8−/−) mice can still maintain the same rank order of absorption efficiency (cholesterol > campesterol > sitosterol), it has been suggested that other proteins independent of G5G8 are responsible for the selectivity of cholesterol over phytosterol absorption (13, 14). In the enterocyte, the esterification of a free cholesterol (FC) molecule with a fatty acid from acyl-CoA to synthesize a cholesterol ester (CE) molecule changes the physicochemical state of cholesterol from a relatively membrane-soluble lipid into a more insoluble CE molecule that must be packaged into the neutral lipid core of chylomicron particles for transport (15). Chylomicrons are secreted directly into the lymphatic system where they pool in the cisternae chyli, travel up the thoracic lymph duct, and enter the circulatory system at the subclavian vein (15, 16). Although the cross-talk between G5G8 and ACAT2 is unknown, it is possible that, together, the relative functions of G5G8 and ACAT2 in the enterocyte dictate the fate of newly absorbed cholesterol and phytosterols as they traverse the enterocyte from the gut lumen. The majority (70–92%) of the sterols exported into thoracic duct lymph of various animals, including rats, rabbits, monkeys, and humans, are esterified (15–19). More particularly, the percentage esterification of absorbed cholesterol is constant regardless of the extent of absorption (19). Lee et al. (12) showed that ACAT2, but not ACAT1, is exclusively expressed in hepatocytes and enterocytes. Buhman et al. (20) reported that the loss of ACAT2 activity in the intestine leads to a decrease in cholesterol absorption efficiency despite unchanged cholesterol synthesis. To date, the degree to which ACAT2 handles cholesterol differently than phytosterols remains unclear. In vitro studies using CaCo-2 cells and rabbit intestinal microsomes have concluded that membrane sitosterol does not interfere or compete with cholesterol for esterification, and it does not alter intracellular cholesterol trafficking or CE secretion (21, 22). On the contrary, experiments performed using microsomes isolated from Chinese hamster ovary cells overexpressing either ACAT1 or ACAT2 showed that ACAT2 displayed a greater capacity to differentiate cholesterol from sitosterol, with a preference for esterifying cholesterol over sitosterol (23). These conflicting reports led us to question what actually happens in animal models in vivo. We hypothesized that sterols enter the enterocyte through the brush border membrane via a Niemann-Pick C1-like 1 (NPC1L1)-mediated process but that G5G8 can limit the substrate availability for ACAT2 esterification by subsequently excreting newly absorbed phytosterols and cholesterol out of the cell, in the process possibly decreasing the overall absorption efficiency of both sterols. In addition, sterol esterification by ACAT2 may enhance absorption efficiency by generating sterol esters that get packaged into chylomicron particles for secretion into lymph and, in the process, become unavailable to G5G8. In this study, we investigated the relative contributions of G5G8 and ACAT2 in intestinal cholesterol absorption compared and contrasted with phytosterol absorption. Using the thoracic lymph duct cannulation (TLDC) technique, which allows for quantitative collection of newly absorbed sterols after a meal and characterization of chylomicron particles, this study directly addressed the following question: How does the absence of G5G8 affect cholesterol esterification and absorption efficiency during sterol metabolism in enterocytes of physiologically intact mice?
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ACAT2 and ABCG5/G8 are both required for efficient cholesterol absorption in mice: evidence from thoracic lymph duct cannulation.
Journal of lipid research, 2012Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L RudelAbstract:Beginning in 1974, the identification of sitosterolemia (1), a rare recessive disorder characterized by elevated plasma and tissue concentrations of phytosterols, has focused attention on the basic molecular processes that govern how the body normally absorbs animal-derived dietary cholesterol while excluding all other similarly structured plant-derived sterols, generally called phytosterols. Phytosterols, including campesterol, stigmasterol, and sitosterol, differ from cholesterol mainly in possessing one or two additional carbons in side chains at C24. The average North American diet contains about equal amounts of cholesterol and phytosterols (∼150–400 mg/day) (2–5), yet campesterol > sitosterol), it has been suggested that other proteins independent of G5G8 are responsible for the selectivity of cholesterol over phytosterol absorption (13, 14). In the enterocyte, the esterification of a free cholesterol (FC) molecule with a fatty acid from acyl-CoA to synthesize a cholesterol ester (CE) molecule changes the physicochemical state of cholesterol from a relatively membrane-soluble lipid into a more insoluble CE molecule that must be packaged into the neutral lipid core of chylomicron particles for transport (15). Chylomicrons are secreted directly into the lymphatic system where they pool in the cisternae chyli, travel up the thoracic lymph duct, and enter the circulatory system at the subclavian vein (15, 16). Although the cross-talk between G5G8 and ACAT2 is unknown, it is possible that, together, the relative functions of G5G8 and ACAT2 in the enterocyte dictate the fate of newly absorbed cholesterol and phytosterols as they traverse the enterocyte from the gut lumen. The majority (70–92%) of the sterols exported into thoracic duct lymph of various animals, including rats, rabbits, monkeys, and humans, are esterified (15–19). More particularly, the percentage esterification of absorbed cholesterol is constant regardless of the extent of absorption (19). Lee et al. (12) showed that ACAT2, but not ACAT1, is exclusively expressed in hepatocytes and enterocytes. Buhman et al. (20) reported that the loss of ACAT2 activity in the intestine leads to a decrease in cholesterol absorption efficiency despite unchanged cholesterol synthesis. To date, the degree to which ACAT2 handles cholesterol differently than phytosterols remains unclear. In vitro studies using CaCo-2 cells and rabbit intestinal microsomes have concluded that membrane sitosterol does not interfere or compete with cholesterol for esterification, and it does not alter intracellular cholesterol trafficking or CE secretion (21, 22). On the contrary, experiments performed using microsomes isolated from Chinese hamster ovary cells overexpressing either ACAT1 or ACAT2 showed that ACAT2 displayed a greater capacity to differentiate cholesterol from sitosterol, with a preference for esterifying cholesterol over sitosterol (23). These conflicting reports led us to question what actually happens in animal models in vivo. We hypothesized that sterols enter the enterocyte through the brush border membrane via a Niemann-Pick C1-like 1 (NPC1L1)-mediated process but that G5G8 can limit the substrate availability for ACAT2 esterification by subsequently excreting newly absorbed phytosterols and cholesterol out of the cell, in the process possibly decreasing the overall absorption efficiency of both sterols. In addition, sterol esterification by ACAT2 may enhance absorption efficiency by generating sterol esters that get packaged into chylomicron particles for secretion into lymph and, in the process, become unavailable to G5G8. In this study, we investigated the relative contributions of G5G8 and ACAT2 in intestinal cholesterol absorption compared and contrasted with phytosterol absorption. Using the thoracic lymph duct cannulation (TLDC) technique, which allows for quantitative collection of newly absorbed sterols after a meal and characterization of chylomicron particles, this study directly addressed the following question: How does the absence of G5G8 affect cholesterol esterification and absorption efficiency during sterol metabolism in enterocytes of physiologically intact mice?
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Abstract 129: Liver-Specific and Intestine-Specific ACAT2 Knockout Mice Are Equally Protected from Diet-Induced Hepatic Cholesterol Accumulation
Arteriosclerosis Thrombosis and Vascular Biology, 2012Co-Authors: Jun Zhang, Matthew L. Davis, Janet K. Sawyer, Kathryn Kelley, Stephanie M. Marshall, Martha D. Wilson, Jonathan Mark Brown, Lawrence L RudelAbstract:Acyl-CoA:cholesterol acyltransferase 2 (ACAT2) is exclusively expressed in the small intestine and liver. ACAT2 facilitates the movement of cholesterol among tissues by generating cholesteryl ester (CE) for packaging into newly synthesized chylomicrons and very low-density lipoproteins (VLDL). In these studies we investigated whether CE derived from either the intestine or liver would differentially affect hepatic and plasma cholesterol homeostasis. For this purpose, we generated both liver-specific (ACAT2L-/L-) and intestine-specific (ACAT2SI-/SI-) ACAT2 knockout mice, and studied dietary cholesterol-induced hepatic lipid accumulation and hypercholesterolemia. Interestingly, diet-induced accumulation of hepatic CE was similarly decreased in both ACAT2L-/L- and ACAT2SI-/SI- mice, and free cholesterol did not build up in the liver. Compared with control mice, both ACAT2L-/L- and ACAT2SI-/SI- mice had lower levels of plasma VLDL-cholesterol but higher plasma triglycerides. ACAT2SI-/SI- but not ACAT2L-/L- mice had blunted cholesterol absorption. Collectively, both ACAT2L-/L- and ACAT2SI-/SI- mice were equally protected from diet-induced hepatic CE accumulation and hypercholesterolemia. These results suggest that inhibition of either intestinal or hepatic ACAT2 improves atherogenic hyperlipidemia and limits hepatic CE accumulation in mice, indicating that inhibition of ACAT2 expression in either tissue likely would be beneficial for atheroprotection.
Matthew A. Davis - One of the best experts on this subject based on the ideXlab platform.
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ACAT2 and abcg5 g8 are both required for efficient cholesterol absorption in mice evidence from thoracic lymph duct cannulation
Journal of Lipid Research, 2012Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L RudelAbstract:Beginning in 1974, the identification of sitosterolemia (1), a rare recessive disorder characterized by elevated plasma and tissue concentrations of phytosterols, has focused attention on the basic molecular processes that govern how the body normally absorbs animal-derived dietary cholesterol while excluding all other similarly structured plant-derived sterols, generally called phytosterols. Phytosterols, including campesterol, stigmasterol, and sitosterol, differ from cholesterol mainly in possessing one or two additional carbons in side chains at C24. The average North American diet contains about equal amounts of cholesterol and phytosterols (∼150–400 mg/day) (2–5), yet <5% of phytosterols are absorbed compared with ∼50% of cholesterol (4, 6). Thus in healthy individuals, sensitive mechanisms exist that allow the body to distinguish among modestly different sterol structures. In general, different species of sterols have different absorption efficiencies; the closer the structural similarity to the cholesterol molecule, the higher the percentage absorption (4, 7). Even mildly hypercholesterolemic patients have serum concentrations of phytosterols that are 500 (campesterol) to 20,000 (sitosterol) times lower than that of cholesterol. Patients with sitosterolemia have increased fractional sterol absorption rates and impaired biliary secretion of neutral sterols, resulting in accumulations of these sterols in the blood and tissues (1, 8), premature atherosclerosis, and tendon/skin xanthomatosis (8, 9). Sitosterolemia has been linked to mutations in either adenosine triphosphate-binding cassette transporter G5 or G8 (ABCG5 or ABCG8), which are expressed almost exclusively in hepatocytes and enterocytes forming a heterodimer (G5G8) typically localized to the plasma membrane (10, 11). Another protein that is expressed exclusively in the same two cell types is acyl CoA:cholesterol acyltransferase type 2 (ACAT2), a cholesterol-esterifying enzyme residing in the endoplasmic reticulum (ER) membrane (12). Mice lacking G5G8 have phenotypes resembling patients with sitosterolemia, including increased fractional absorption of noncholesterol sterols, marked accumulation of sitosterol and campesterol in the blood and liver, reduced levels of biliary cholesterol, and various hemolytic disorders (7, 13). These findings suggested that the physiological role of G5G8 is to limit phytosterol accumulation in the body by limiting its absorption in the intestine and by promoting its secretion from the liver. Because sitosterolemic patients and G5G8 knockout (G5G8−/−) mice can still maintain the same rank order of absorption efficiency (cholesterol > campesterol > sitosterol), it has been suggested that other proteins independent of G5G8 are responsible for the selectivity of cholesterol over phytosterol absorption (13, 14). In the enterocyte, the esterification of a free cholesterol (FC) molecule with a fatty acid from acyl-CoA to synthesize a cholesterol ester (CE) molecule changes the physicochemical state of cholesterol from a relatively membrane-soluble lipid into a more insoluble CE molecule that must be packaged into the neutral lipid core of chylomicron particles for transport (15). Chylomicrons are secreted directly into the lymphatic system where they pool in the cisternae chyli, travel up the thoracic lymph duct, and enter the circulatory system at the subclavian vein (15, 16). Although the cross-talk between G5G8 and ACAT2 is unknown, it is possible that, together, the relative functions of G5G8 and ACAT2 in the enterocyte dictate the fate of newly absorbed cholesterol and phytosterols as they traverse the enterocyte from the gut lumen. The majority (70–92%) of the sterols exported into thoracic duct lymph of various animals, including rats, rabbits, monkeys, and humans, are esterified (15–19). More particularly, the percentage esterification of absorbed cholesterol is constant regardless of the extent of absorption (19). Lee et al. (12) showed that ACAT2, but not ACAT1, is exclusively expressed in hepatocytes and enterocytes. Buhman et al. (20) reported that the loss of ACAT2 activity in the intestine leads to a decrease in cholesterol absorption efficiency despite unchanged cholesterol synthesis. To date, the degree to which ACAT2 handles cholesterol differently than phytosterols remains unclear. In vitro studies using CaCo-2 cells and rabbit intestinal microsomes have concluded that membrane sitosterol does not interfere or compete with cholesterol for esterification, and it does not alter intracellular cholesterol trafficking or CE secretion (21, 22). On the contrary, experiments performed using microsomes isolated from Chinese hamster ovary cells overexpressing either ACAT1 or ACAT2 showed that ACAT2 displayed a greater capacity to differentiate cholesterol from sitosterol, with a preference for esterifying cholesterol over sitosterol (23). These conflicting reports led us to question what actually happens in animal models in vivo. We hypothesized that sterols enter the enterocyte through the brush border membrane via a Niemann-Pick C1-like 1 (NPC1L1)-mediated process but that G5G8 can limit the substrate availability for ACAT2 esterification by subsequently excreting newly absorbed phytosterols and cholesterol out of the cell, in the process possibly decreasing the overall absorption efficiency of both sterols. In addition, sterol esterification by ACAT2 may enhance absorption efficiency by generating sterol esters that get packaged into chylomicron particles for secretion into lymph and, in the process, become unavailable to G5G8. In this study, we investigated the relative contributions of G5G8 and ACAT2 in intestinal cholesterol absorption compared and contrasted with phytosterol absorption. Using the thoracic lymph duct cannulation (TLDC) technique, which allows for quantitative collection of newly absorbed sterols after a meal and characterization of chylomicron particles, this study directly addressed the following question: How does the absence of G5G8 affect cholesterol esterification and absorption efficiency during sterol metabolism in enterocytes of physiologically intact mice?
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ACAT2 and ABCG5/G8 are both required for efficient cholesterol absorption in mice: evidence from thoracic lymph duct cannulation.
Journal of lipid research, 2012Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L RudelAbstract:Beginning in 1974, the identification of sitosterolemia (1), a rare recessive disorder characterized by elevated plasma and tissue concentrations of phytosterols, has focused attention on the basic molecular processes that govern how the body normally absorbs animal-derived dietary cholesterol while excluding all other similarly structured plant-derived sterols, generally called phytosterols. Phytosterols, including campesterol, stigmasterol, and sitosterol, differ from cholesterol mainly in possessing one or two additional carbons in side chains at C24. The average North American diet contains about equal amounts of cholesterol and phytosterols (∼150–400 mg/day) (2–5), yet campesterol > sitosterol), it has been suggested that other proteins independent of G5G8 are responsible for the selectivity of cholesterol over phytosterol absorption (13, 14). In the enterocyte, the esterification of a free cholesterol (FC) molecule with a fatty acid from acyl-CoA to synthesize a cholesterol ester (CE) molecule changes the physicochemical state of cholesterol from a relatively membrane-soluble lipid into a more insoluble CE molecule that must be packaged into the neutral lipid core of chylomicron particles for transport (15). Chylomicrons are secreted directly into the lymphatic system where they pool in the cisternae chyli, travel up the thoracic lymph duct, and enter the circulatory system at the subclavian vein (15, 16). Although the cross-talk between G5G8 and ACAT2 is unknown, it is possible that, together, the relative functions of G5G8 and ACAT2 in the enterocyte dictate the fate of newly absorbed cholesterol and phytosterols as they traverse the enterocyte from the gut lumen. The majority (70–92%) of the sterols exported into thoracic duct lymph of various animals, including rats, rabbits, monkeys, and humans, are esterified (15–19). More particularly, the percentage esterification of absorbed cholesterol is constant regardless of the extent of absorption (19). Lee et al. (12) showed that ACAT2, but not ACAT1, is exclusively expressed in hepatocytes and enterocytes. Buhman et al. (20) reported that the loss of ACAT2 activity in the intestine leads to a decrease in cholesterol absorption efficiency despite unchanged cholesterol synthesis. To date, the degree to which ACAT2 handles cholesterol differently than phytosterols remains unclear. In vitro studies using CaCo-2 cells and rabbit intestinal microsomes have concluded that membrane sitosterol does not interfere or compete with cholesterol for esterification, and it does not alter intracellular cholesterol trafficking or CE secretion (21, 22). On the contrary, experiments performed using microsomes isolated from Chinese hamster ovary cells overexpressing either ACAT1 or ACAT2 showed that ACAT2 displayed a greater capacity to differentiate cholesterol from sitosterol, with a preference for esterifying cholesterol over sitosterol (23). These conflicting reports led us to question what actually happens in animal models in vivo. We hypothesized that sterols enter the enterocyte through the brush border membrane via a Niemann-Pick C1-like 1 (NPC1L1)-mediated process but that G5G8 can limit the substrate availability for ACAT2 esterification by subsequently excreting newly absorbed phytosterols and cholesterol out of the cell, in the process possibly decreasing the overall absorption efficiency of both sterols. In addition, sterol esterification by ACAT2 may enhance absorption efficiency by generating sterol esters that get packaged into chylomicron particles for secretion into lymph and, in the process, become unavailable to G5G8. In this study, we investigated the relative contributions of G5G8 and ACAT2 in intestinal cholesterol absorption compared and contrasted with phytosterol absorption. Using the thoracic lymph duct cannulation (TLDC) technique, which allows for quantitative collection of newly absorbed sterols after a meal and characterization of chylomicron particles, this study directly addressed the following question: How does the absence of G5G8 affect cholesterol esterification and absorption efficiency during sterol metabolism in enterocytes of physiologically intact mice?
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Tissue-specific knockouts of ACAT2 reveal that intestinal depletion is sufficient to prevent diet-induced cholesterol accumulation in the liver and blood
Journal of lipid research, 2012Co-Authors: Jun Zhang, Matthew A. Davis, Janet K. Sawyer, Stephanie M. Marshall, Martha D. Wilson, J. Mark Brown, Robert V. Farese, Kathryn L. Kelley, Lawrence L RudelAbstract:Acyl-CoA:cholesterol acyltransferase 2 (ACAT2) generates cholesterol esters (CE) for packaging into newly synthesized lipoproteins and thus is a major determinant of blood cholesterol levels. ACAT2 is expressed exclusively in the small intestine and liver, but the relative contributions of ACAT2 expression in these tissues to systemic cholesterol metabolism is unknown. We investigated whether CE derived from the intestine or liver would differentially affect hepatic and plasma cholesterol homeostasis. We generated liver-specific (ACAT2(L-/L-)) and intestine-specific (ACAT2(SI-/SI-)) ACAT2 knockout mice and studied dietary cholesterol-induced hepatic lipid accumulation and hypercholesterolemia. ACAT2(SI-/SI-) mice, in contrast to ACAT2(L-/L-) mice, had blunted cholesterol absorption. However, specific deletion of ACAT2 in the intestine generated essentially a phenocopy of the conditional knockout of ACAT2 in the liver, with reduced levels of plasma very low-density lipoprotein and hepatic CE, yet hepatic-free cholesterol does not build up after high cholesterol intake. ACAT2(L-/L-) and ACAT2(SI-/SI-) mice were equally protected from diet-induced hepatic CE accumulation and hypercholesterolemia. These results suggest that inhibition of intestinal or hepatic ACAT2 improves atherogenic hyperlipidemia and limits hepatic CE accumulation in mice and that depletion of intestinal ACAT2 is sufficient for most of the beneficial effects on cholesterol metabolism. Inhibitors of ACAT2 targeting either tissue likely would be beneficial for atheroprotection.
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Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation.
Journal of lipid research, 2011Co-Authors: Tam Nguyen, Janet K. Sawyer, Matthew A. Davis, Kathryn L. Kelley, Lawrence L RudelAbstract: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.
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Estrogen decreases atherosclerosis in part by reducing hepatic acyl-CoA:cholesterol acyltransferase 2 (ACAT2) in monkeys.
Arteriosclerosis thrombosis and vascular biology, 2009Co-Authors: Kylie Kavanagh, Lawrence L Rudel, Matthew A. Davis, Martha D. Wilson, Li Zhang, Thomas C. Register, Michael R. Adams, Janice D. WagnerAbstract:Objective— Estrogens decrease atherosclerosis progression, mediated in part through changes in plasma lipids and lipoproteins. This study aimed to determine estrogen-induced changes in hepatic cholesterol metabolism, plasma lipoproteins, and the relationship of these changes to atherosclerosis extent. Methods and Results— Ovariectomized monkeys (n=34) consumed atherogenic diets for 30 months which contained either no hormones (control, n=17) or conjugated equine estrogens (CEE, n=17) at a human dose equivalent of 0.625 mg/d. Hepatic cholesterol content, low-density lipoprotein (LDL) receptor expression, cholesterol 7α-hydroxylase and acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity, and expression levels were determined. CEE treatment resulted in lower plasma concentrations of very-low- and intermediate- density lipoprotein cholesterol (V+IDLC; P =0.01), smaller LDL particles ( P =0.002), and 50% lower hepatic cholesterol content (total, free, and esterified; P P =0.01), explained primarily by reductions in the activity of ACAT2. Estrogen regulation of enzymatic activity was at the protein level as both ACAT1 and 2 protein, but not mRNA levels, were lower ( P =0.02 and Conclusions— Atheroprotective effects of estrogen therapy may be related to reduced hepatic secretion of ACAT2-derived cholesteryl esters in plasma lipoproteins.
Janet K. Sawyer - One of the best experts on this subject based on the ideXlab platform.
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ACAT2 and abcg5 g8 are both required for efficient cholesterol absorption in mice evidence from thoracic lymph duct cannulation
Journal of Lipid Research, 2012Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L RudelAbstract:Beginning in 1974, the identification of sitosterolemia (1), a rare recessive disorder characterized by elevated plasma and tissue concentrations of phytosterols, has focused attention on the basic molecular processes that govern how the body normally absorbs animal-derived dietary cholesterol while excluding all other similarly structured plant-derived sterols, generally called phytosterols. Phytosterols, including campesterol, stigmasterol, and sitosterol, differ from cholesterol mainly in possessing one or two additional carbons in side chains at C24. The average North American diet contains about equal amounts of cholesterol and phytosterols (∼150–400 mg/day) (2–5), yet <5% of phytosterols are absorbed compared with ∼50% of cholesterol (4, 6). Thus in healthy individuals, sensitive mechanisms exist that allow the body to distinguish among modestly different sterol structures. In general, different species of sterols have different absorption efficiencies; the closer the structural similarity to the cholesterol molecule, the higher the percentage absorption (4, 7). Even mildly hypercholesterolemic patients have serum concentrations of phytosterols that are 500 (campesterol) to 20,000 (sitosterol) times lower than that of cholesterol. Patients with sitosterolemia have increased fractional sterol absorption rates and impaired biliary secretion of neutral sterols, resulting in accumulations of these sterols in the blood and tissues (1, 8), premature atherosclerosis, and tendon/skin xanthomatosis (8, 9). Sitosterolemia has been linked to mutations in either adenosine triphosphate-binding cassette transporter G5 or G8 (ABCG5 or ABCG8), which are expressed almost exclusively in hepatocytes and enterocytes forming a heterodimer (G5G8) typically localized to the plasma membrane (10, 11). Another protein that is expressed exclusively in the same two cell types is acyl CoA:cholesterol acyltransferase type 2 (ACAT2), a cholesterol-esterifying enzyme residing in the endoplasmic reticulum (ER) membrane (12). Mice lacking G5G8 have phenotypes resembling patients with sitosterolemia, including increased fractional absorption of noncholesterol sterols, marked accumulation of sitosterol and campesterol in the blood and liver, reduced levels of biliary cholesterol, and various hemolytic disorders (7, 13). These findings suggested that the physiological role of G5G8 is to limit phytosterol accumulation in the body by limiting its absorption in the intestine and by promoting its secretion from the liver. Because sitosterolemic patients and G5G8 knockout (G5G8−/−) mice can still maintain the same rank order of absorption efficiency (cholesterol > campesterol > sitosterol), it has been suggested that other proteins independent of G5G8 are responsible for the selectivity of cholesterol over phytosterol absorption (13, 14). In the enterocyte, the esterification of a free cholesterol (FC) molecule with a fatty acid from acyl-CoA to synthesize a cholesterol ester (CE) molecule changes the physicochemical state of cholesterol from a relatively membrane-soluble lipid into a more insoluble CE molecule that must be packaged into the neutral lipid core of chylomicron particles for transport (15). Chylomicrons are secreted directly into the lymphatic system where they pool in the cisternae chyli, travel up the thoracic lymph duct, and enter the circulatory system at the subclavian vein (15, 16). Although the cross-talk between G5G8 and ACAT2 is unknown, it is possible that, together, the relative functions of G5G8 and ACAT2 in the enterocyte dictate the fate of newly absorbed cholesterol and phytosterols as they traverse the enterocyte from the gut lumen. The majority (70–92%) of the sterols exported into thoracic duct lymph of various animals, including rats, rabbits, monkeys, and humans, are esterified (15–19). More particularly, the percentage esterification of absorbed cholesterol is constant regardless of the extent of absorption (19). Lee et al. (12) showed that ACAT2, but not ACAT1, is exclusively expressed in hepatocytes and enterocytes. Buhman et al. (20) reported that the loss of ACAT2 activity in the intestine leads to a decrease in cholesterol absorption efficiency despite unchanged cholesterol synthesis. To date, the degree to which ACAT2 handles cholesterol differently than phytosterols remains unclear. In vitro studies using CaCo-2 cells and rabbit intestinal microsomes have concluded that membrane sitosterol does not interfere or compete with cholesterol for esterification, and it does not alter intracellular cholesterol trafficking or CE secretion (21, 22). On the contrary, experiments performed using microsomes isolated from Chinese hamster ovary cells overexpressing either ACAT1 or ACAT2 showed that ACAT2 displayed a greater capacity to differentiate cholesterol from sitosterol, with a preference for esterifying cholesterol over sitosterol (23). These conflicting reports led us to question what actually happens in animal models in vivo. We hypothesized that sterols enter the enterocyte through the brush border membrane via a Niemann-Pick C1-like 1 (NPC1L1)-mediated process but that G5G8 can limit the substrate availability for ACAT2 esterification by subsequently excreting newly absorbed phytosterols and cholesterol out of the cell, in the process possibly decreasing the overall absorption efficiency of both sterols. In addition, sterol esterification by ACAT2 may enhance absorption efficiency by generating sterol esters that get packaged into chylomicron particles for secretion into lymph and, in the process, become unavailable to G5G8. In this study, we investigated the relative contributions of G5G8 and ACAT2 in intestinal cholesterol absorption compared and contrasted with phytosterol absorption. Using the thoracic lymph duct cannulation (TLDC) technique, which allows for quantitative collection of newly absorbed sterols after a meal and characterization of chylomicron particles, this study directly addressed the following question: How does the absence of G5G8 affect cholesterol esterification and absorption efficiency during sterol metabolism in enterocytes of physiologically intact mice?
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ACAT2 and ABCG5/G8 are both required for efficient cholesterol absorption in mice: evidence from thoracic lymph duct cannulation.
Journal of lipid research, 2012Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L RudelAbstract:Beginning in 1974, the identification of sitosterolemia (1), a rare recessive disorder characterized by elevated plasma and tissue concentrations of phytosterols, has focused attention on the basic molecular processes that govern how the body normally absorbs animal-derived dietary cholesterol while excluding all other similarly structured plant-derived sterols, generally called phytosterols. Phytosterols, including campesterol, stigmasterol, and sitosterol, differ from cholesterol mainly in possessing one or two additional carbons in side chains at C24. The average North American diet contains about equal amounts of cholesterol and phytosterols (∼150–400 mg/day) (2–5), yet campesterol > sitosterol), it has been suggested that other proteins independent of G5G8 are responsible for the selectivity of cholesterol over phytosterol absorption (13, 14). In the enterocyte, the esterification of a free cholesterol (FC) molecule with a fatty acid from acyl-CoA to synthesize a cholesterol ester (CE) molecule changes the physicochemical state of cholesterol from a relatively membrane-soluble lipid into a more insoluble CE molecule that must be packaged into the neutral lipid core of chylomicron particles for transport (15). Chylomicrons are secreted directly into the lymphatic system where they pool in the cisternae chyli, travel up the thoracic lymph duct, and enter the circulatory system at the subclavian vein (15, 16). Although the cross-talk between G5G8 and ACAT2 is unknown, it is possible that, together, the relative functions of G5G8 and ACAT2 in the enterocyte dictate the fate of newly absorbed cholesterol and phytosterols as they traverse the enterocyte from the gut lumen. The majority (70–92%) of the sterols exported into thoracic duct lymph of various animals, including rats, rabbits, monkeys, and humans, are esterified (15–19). More particularly, the percentage esterification of absorbed cholesterol is constant regardless of the extent of absorption (19). Lee et al. (12) showed that ACAT2, but not ACAT1, is exclusively expressed in hepatocytes and enterocytes. Buhman et al. (20) reported that the loss of ACAT2 activity in the intestine leads to a decrease in cholesterol absorption efficiency despite unchanged cholesterol synthesis. To date, the degree to which ACAT2 handles cholesterol differently than phytosterols remains unclear. In vitro studies using CaCo-2 cells and rabbit intestinal microsomes have concluded that membrane sitosterol does not interfere or compete with cholesterol for esterification, and it does not alter intracellular cholesterol trafficking or CE secretion (21, 22). On the contrary, experiments performed using microsomes isolated from Chinese hamster ovary cells overexpressing either ACAT1 or ACAT2 showed that ACAT2 displayed a greater capacity to differentiate cholesterol from sitosterol, with a preference for esterifying cholesterol over sitosterol (23). These conflicting reports led us to question what actually happens in animal models in vivo. We hypothesized that sterols enter the enterocyte through the brush border membrane via a Niemann-Pick C1-like 1 (NPC1L1)-mediated process but that G5G8 can limit the substrate availability for ACAT2 esterification by subsequently excreting newly absorbed phytosterols and cholesterol out of the cell, in the process possibly decreasing the overall absorption efficiency of both sterols. In addition, sterol esterification by ACAT2 may enhance absorption efficiency by generating sterol esters that get packaged into chylomicron particles for secretion into lymph and, in the process, become unavailable to G5G8. In this study, we investigated the relative contributions of G5G8 and ACAT2 in intestinal cholesterol absorption compared and contrasted with phytosterol absorption. Using the thoracic lymph duct cannulation (TLDC) technique, which allows for quantitative collection of newly absorbed sterols after a meal and characterization of chylomicron particles, this study directly addressed the following question: How does the absence of G5G8 affect cholesterol esterification and absorption efficiency during sterol metabolism in enterocytes of physiologically intact mice?
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Abstract 129: Liver-Specific and Intestine-Specific ACAT2 Knockout Mice Are Equally Protected from Diet-Induced Hepatic Cholesterol Accumulation
Arteriosclerosis Thrombosis and Vascular Biology, 2012Co-Authors: Jun Zhang, Matthew L. Davis, Janet K. Sawyer, Kathryn Kelley, Stephanie M. Marshall, Martha D. Wilson, Jonathan Mark Brown, Lawrence L RudelAbstract:Acyl-CoA:cholesterol acyltransferase 2 (ACAT2) is exclusively expressed in the small intestine and liver. ACAT2 facilitates the movement of cholesterol among tissues by generating cholesteryl ester (CE) for packaging into newly synthesized chylomicrons and very low-density lipoproteins (VLDL). In these studies we investigated whether CE derived from either the intestine or liver would differentially affect hepatic and plasma cholesterol homeostasis. For this purpose, we generated both liver-specific (ACAT2L-/L-) and intestine-specific (ACAT2SI-/SI-) ACAT2 knockout mice, and studied dietary cholesterol-induced hepatic lipid accumulation and hypercholesterolemia. Interestingly, diet-induced accumulation of hepatic CE was similarly decreased in both ACAT2L-/L- and ACAT2SI-/SI- mice, and free cholesterol did not build up in the liver. Compared with control mice, both ACAT2L-/L- and ACAT2SI-/SI- mice had lower levels of plasma VLDL-cholesterol but higher plasma triglycerides. ACAT2SI-/SI- but not ACAT2L-/L- mice had blunted cholesterol absorption. Collectively, both ACAT2L-/L- and ACAT2SI-/SI- mice were equally protected from diet-induced hepatic CE accumulation and hypercholesterolemia. These results suggest that inhibition of either intestinal or hepatic ACAT2 improves atherogenic hyperlipidemia and limits hepatic CE accumulation in mice, indicating that inhibition of ACAT2 expression in either tissue likely would be beneficial for atheroprotection.
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Tissue-specific knockouts of ACAT2 reveal that intestinal depletion is sufficient to prevent diet-induced cholesterol accumulation in the liver and blood
Journal of lipid research, 2012Co-Authors: Jun Zhang, Matthew A. Davis, Janet K. Sawyer, Stephanie M. Marshall, Martha D. Wilson, J. Mark Brown, Robert V. Farese, Kathryn L. Kelley, Lawrence L RudelAbstract:Acyl-CoA:cholesterol acyltransferase 2 (ACAT2) generates cholesterol esters (CE) for packaging into newly synthesized lipoproteins and thus is a major determinant of blood cholesterol levels. ACAT2 is expressed exclusively in the small intestine and liver, but the relative contributions of ACAT2 expression in these tissues to systemic cholesterol metabolism is unknown. We investigated whether CE derived from the intestine or liver would differentially affect hepatic and plasma cholesterol homeostasis. We generated liver-specific (ACAT2(L-/L-)) and intestine-specific (ACAT2(SI-/SI-)) ACAT2 knockout mice and studied dietary cholesterol-induced hepatic lipid accumulation and hypercholesterolemia. ACAT2(SI-/SI-) mice, in contrast to ACAT2(L-/L-) mice, had blunted cholesterol absorption. However, specific deletion of ACAT2 in the intestine generated essentially a phenocopy of the conditional knockout of ACAT2 in the liver, with reduced levels of plasma very low-density lipoprotein and hepatic CE, yet hepatic-free cholesterol does not build up after high cholesterol intake. ACAT2(L-/L-) and ACAT2(SI-/SI-) mice were equally protected from diet-induced hepatic CE accumulation and hypercholesterolemia. These results suggest that inhibition of intestinal or hepatic ACAT2 improves atherogenic hyperlipidemia and limits hepatic CE accumulation in mice and that depletion of intestinal ACAT2 is sufficient for most of the beneficial effects on cholesterol metabolism. Inhibitors of ACAT2 targeting either tissue likely would be beneficial for atheroprotection.
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Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation.
Journal of lipid research, 2011Co-Authors: Tam Nguyen, Janet K. Sawyer, Matthew A. Davis, Kathryn L. Kelley, Lawrence L RudelAbstract: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.
Kathryn L. Kelley - One of the best experts on this subject based on the ideXlab platform.
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ACAT2 and abcg5 g8 are both required for efficient cholesterol absorption in mice evidence from thoracic lymph duct cannulation
Journal of Lipid Research, 2012Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L RudelAbstract:Beginning in 1974, the identification of sitosterolemia (1), a rare recessive disorder characterized by elevated plasma and tissue concentrations of phytosterols, has focused attention on the basic molecular processes that govern how the body normally absorbs animal-derived dietary cholesterol while excluding all other similarly structured plant-derived sterols, generally called phytosterols. Phytosterols, including campesterol, stigmasterol, and sitosterol, differ from cholesterol mainly in possessing one or two additional carbons in side chains at C24. The average North American diet contains about equal amounts of cholesterol and phytosterols (∼150–400 mg/day) (2–5), yet <5% of phytosterols are absorbed compared with ∼50% of cholesterol (4, 6). Thus in healthy individuals, sensitive mechanisms exist that allow the body to distinguish among modestly different sterol structures. In general, different species of sterols have different absorption efficiencies; the closer the structural similarity to the cholesterol molecule, the higher the percentage absorption (4, 7). Even mildly hypercholesterolemic patients have serum concentrations of phytosterols that are 500 (campesterol) to 20,000 (sitosterol) times lower than that of cholesterol. Patients with sitosterolemia have increased fractional sterol absorption rates and impaired biliary secretion of neutral sterols, resulting in accumulations of these sterols in the blood and tissues (1, 8), premature atherosclerosis, and tendon/skin xanthomatosis (8, 9). Sitosterolemia has been linked to mutations in either adenosine triphosphate-binding cassette transporter G5 or G8 (ABCG5 or ABCG8), which are expressed almost exclusively in hepatocytes and enterocytes forming a heterodimer (G5G8) typically localized to the plasma membrane (10, 11). Another protein that is expressed exclusively in the same two cell types is acyl CoA:cholesterol acyltransferase type 2 (ACAT2), a cholesterol-esterifying enzyme residing in the endoplasmic reticulum (ER) membrane (12). Mice lacking G5G8 have phenotypes resembling patients with sitosterolemia, including increased fractional absorption of noncholesterol sterols, marked accumulation of sitosterol and campesterol in the blood and liver, reduced levels of biliary cholesterol, and various hemolytic disorders (7, 13). These findings suggested that the physiological role of G5G8 is to limit phytosterol accumulation in the body by limiting its absorption in the intestine and by promoting its secretion from the liver. Because sitosterolemic patients and G5G8 knockout (G5G8−/−) mice can still maintain the same rank order of absorption efficiency (cholesterol > campesterol > sitosterol), it has been suggested that other proteins independent of G5G8 are responsible for the selectivity of cholesterol over phytosterol absorption (13, 14). In the enterocyte, the esterification of a free cholesterol (FC) molecule with a fatty acid from acyl-CoA to synthesize a cholesterol ester (CE) molecule changes the physicochemical state of cholesterol from a relatively membrane-soluble lipid into a more insoluble CE molecule that must be packaged into the neutral lipid core of chylomicron particles for transport (15). Chylomicrons are secreted directly into the lymphatic system where they pool in the cisternae chyli, travel up the thoracic lymph duct, and enter the circulatory system at the subclavian vein (15, 16). Although the cross-talk between G5G8 and ACAT2 is unknown, it is possible that, together, the relative functions of G5G8 and ACAT2 in the enterocyte dictate the fate of newly absorbed cholesterol and phytosterols as they traverse the enterocyte from the gut lumen. The majority (70–92%) of the sterols exported into thoracic duct lymph of various animals, including rats, rabbits, monkeys, and humans, are esterified (15–19). More particularly, the percentage esterification of absorbed cholesterol is constant regardless of the extent of absorption (19). Lee et al. (12) showed that ACAT2, but not ACAT1, is exclusively expressed in hepatocytes and enterocytes. Buhman et al. (20) reported that the loss of ACAT2 activity in the intestine leads to a decrease in cholesterol absorption efficiency despite unchanged cholesterol synthesis. To date, the degree to which ACAT2 handles cholesterol differently than phytosterols remains unclear. In vitro studies using CaCo-2 cells and rabbit intestinal microsomes have concluded that membrane sitosterol does not interfere or compete with cholesterol for esterification, and it does not alter intracellular cholesterol trafficking or CE secretion (21, 22). On the contrary, experiments performed using microsomes isolated from Chinese hamster ovary cells overexpressing either ACAT1 or ACAT2 showed that ACAT2 displayed a greater capacity to differentiate cholesterol from sitosterol, with a preference for esterifying cholesterol over sitosterol (23). These conflicting reports led us to question what actually happens in animal models in vivo. We hypothesized that sterols enter the enterocyte through the brush border membrane via a Niemann-Pick C1-like 1 (NPC1L1)-mediated process but that G5G8 can limit the substrate availability for ACAT2 esterification by subsequently excreting newly absorbed phytosterols and cholesterol out of the cell, in the process possibly decreasing the overall absorption efficiency of both sterols. In addition, sterol esterification by ACAT2 may enhance absorption efficiency by generating sterol esters that get packaged into chylomicron particles for secretion into lymph and, in the process, become unavailable to G5G8. In this study, we investigated the relative contributions of G5G8 and ACAT2 in intestinal cholesterol absorption compared and contrasted with phytosterol absorption. Using the thoracic lymph duct cannulation (TLDC) technique, which allows for quantitative collection of newly absorbed sterols after a meal and characterization of chylomicron particles, this study directly addressed the following question: How does the absence of G5G8 affect cholesterol esterification and absorption efficiency during sterol metabolism in enterocytes of physiologically intact mice?
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ACAT2 and ABCG5/G8 are both required for efficient cholesterol absorption in mice: evidence from thoracic lymph duct cannulation.
Journal of lipid research, 2012Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L RudelAbstract:Beginning in 1974, the identification of sitosterolemia (1), a rare recessive disorder characterized by elevated plasma and tissue concentrations of phytosterols, has focused attention on the basic molecular processes that govern how the body normally absorbs animal-derived dietary cholesterol while excluding all other similarly structured plant-derived sterols, generally called phytosterols. Phytosterols, including campesterol, stigmasterol, and sitosterol, differ from cholesterol mainly in possessing one or two additional carbons in side chains at C24. The average North American diet contains about equal amounts of cholesterol and phytosterols (∼150–400 mg/day) (2–5), yet campesterol > sitosterol), it has been suggested that other proteins independent of G5G8 are responsible for the selectivity of cholesterol over phytosterol absorption (13, 14). In the enterocyte, the esterification of a free cholesterol (FC) molecule with a fatty acid from acyl-CoA to synthesize a cholesterol ester (CE) molecule changes the physicochemical state of cholesterol from a relatively membrane-soluble lipid into a more insoluble CE molecule that must be packaged into the neutral lipid core of chylomicron particles for transport (15). Chylomicrons are secreted directly into the lymphatic system where they pool in the cisternae chyli, travel up the thoracic lymph duct, and enter the circulatory system at the subclavian vein (15, 16). Although the cross-talk between G5G8 and ACAT2 is unknown, it is possible that, together, the relative functions of G5G8 and ACAT2 in the enterocyte dictate the fate of newly absorbed cholesterol and phytosterols as they traverse the enterocyte from the gut lumen. The majority (70–92%) of the sterols exported into thoracic duct lymph of various animals, including rats, rabbits, monkeys, and humans, are esterified (15–19). More particularly, the percentage esterification of absorbed cholesterol is constant regardless of the extent of absorption (19). Lee et al. (12) showed that ACAT2, but not ACAT1, is exclusively expressed in hepatocytes and enterocytes. Buhman et al. (20) reported that the loss of ACAT2 activity in the intestine leads to a decrease in cholesterol absorption efficiency despite unchanged cholesterol synthesis. To date, the degree to which ACAT2 handles cholesterol differently than phytosterols remains unclear. In vitro studies using CaCo-2 cells and rabbit intestinal microsomes have concluded that membrane sitosterol does not interfere or compete with cholesterol for esterification, and it does not alter intracellular cholesterol trafficking or CE secretion (21, 22). On the contrary, experiments performed using microsomes isolated from Chinese hamster ovary cells overexpressing either ACAT1 or ACAT2 showed that ACAT2 displayed a greater capacity to differentiate cholesterol from sitosterol, with a preference for esterifying cholesterol over sitosterol (23). These conflicting reports led us to question what actually happens in animal models in vivo. We hypothesized that sterols enter the enterocyte through the brush border membrane via a Niemann-Pick C1-like 1 (NPC1L1)-mediated process but that G5G8 can limit the substrate availability for ACAT2 esterification by subsequently excreting newly absorbed phytosterols and cholesterol out of the cell, in the process possibly decreasing the overall absorption efficiency of both sterols. In addition, sterol esterification by ACAT2 may enhance absorption efficiency by generating sterol esters that get packaged into chylomicron particles for secretion into lymph and, in the process, become unavailable to G5G8. In this study, we investigated the relative contributions of G5G8 and ACAT2 in intestinal cholesterol absorption compared and contrasted with phytosterol absorption. Using the thoracic lymph duct cannulation (TLDC) technique, which allows for quantitative collection of newly absorbed sterols after a meal and characterization of chylomicron particles, this study directly addressed the following question: How does the absence of G5G8 affect cholesterol esterification and absorption efficiency during sterol metabolism in enterocytes of physiologically intact mice?
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Tissue-specific knockouts of ACAT2 reveal that intestinal depletion is sufficient to prevent diet-induced cholesterol accumulation in the liver and blood
Journal of lipid research, 2012Co-Authors: Jun Zhang, Matthew A. Davis, Janet K. Sawyer, Stephanie M. Marshall, Martha D. Wilson, J. Mark Brown, Robert V. Farese, Kathryn L. Kelley, Lawrence L RudelAbstract:Acyl-CoA:cholesterol acyltransferase 2 (ACAT2) generates cholesterol esters (CE) for packaging into newly synthesized lipoproteins and thus is a major determinant of blood cholesterol levels. ACAT2 is expressed exclusively in the small intestine and liver, but the relative contributions of ACAT2 expression in these tissues to systemic cholesterol metabolism is unknown. We investigated whether CE derived from the intestine or liver would differentially affect hepatic and plasma cholesterol homeostasis. We generated liver-specific (ACAT2(L-/L-)) and intestine-specific (ACAT2(SI-/SI-)) ACAT2 knockout mice and studied dietary cholesterol-induced hepatic lipid accumulation and hypercholesterolemia. ACAT2(SI-/SI-) mice, in contrast to ACAT2(L-/L-) mice, had blunted cholesterol absorption. However, specific deletion of ACAT2 in the intestine generated essentially a phenocopy of the conditional knockout of ACAT2 in the liver, with reduced levels of plasma very low-density lipoprotein and hepatic CE, yet hepatic-free cholesterol does not build up after high cholesterol intake. ACAT2(L-/L-) and ACAT2(SI-/SI-) mice were equally protected from diet-induced hepatic CE accumulation and hypercholesterolemia. These results suggest that inhibition of intestinal or hepatic ACAT2 improves atherogenic hyperlipidemia and limits hepatic CE accumulation in mice and that depletion of intestinal ACAT2 is sufficient for most of the beneficial effects on cholesterol metabolism. Inhibitors of ACAT2 targeting either tissue likely would be beneficial for atheroprotection.
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Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation.
Journal of lipid research, 2011Co-Authors: Tam Nguyen, Janet K. Sawyer, Matthew A. Davis, Kathryn L. Kelley, Lawrence L RudelAbstract: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.
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Inhibition of Acyl-Coenzyme A:Cholesterol Acyltransferase 2 (ACAT2) Prevents Dietary Cholesterol-associated Steatosis by Enhancing Hepatic Triglyceride Mobilization
The Journal of biological chemistry, 2010Co-Authors: Heather M. Alger, Janet K. Sawyer, Ramesh Shah, Mark C. Willingham, Martha D. Wilson, J. Mark Brown, Kathryn L. Kelley, Lawrence L RudelAbstract: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.
Ta-yuan Chang - One of the best experts on this subject based on the ideXlab platform.
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Myeloid Acat1/Soat1 KO attenuates pro-inflammatory responses in macrophages and protects against atherosclerosis in a model of advanced lesions
The Journal of biological chemistry, 2019Co-Authors: Elaina M. Melton, Bao-liang Song, Catherine C. Y. Chang, Jalen Benson, Paul Sohn, Li-hao Huang, Ta-yuan ChangAbstract:Cholesterol esters are a key ingredient of foamy cells in atherosclerotic lesions; their formation is catalyzed by two enzymes: acyl-CoA:cholesterol acyltransferases (ACATs; also called sterol O-acyltransferases, or SOATs) ACAT1 and ACAT2. ACAT1 is present in all body cells and is the major isoenzyme in macrophages. Whether blocking ACAT1 benefits atherosclerosis has been under debate for more than a decade. Previously, our laboratory developed a myeloid-specific Acat1 knockout (KO) mouse (Acat1 -M/-M), devoid of ACAT1 only in macrophages, microglia, and neutrophils. In previous work using the ApoE KO (ApoE -/-) mouse model for early lesions, Acat1 -M/-M significantly reduced lesion macrophage content and suppressed atherosclerosis progression. In advanced lesions, cholesterol crystals become a prominent feature. Here we evaluated the effects of Acat1 -M/-M in the ApoE KO mouse model for more advanced lesions and found that mice lacking myeloid Acat1 had significantly reduced lesion cholesterol crystal contents. Acat1 -M/-M also significantly reduced lesion size and macrophage content without increasing apoptotic cell death. Cell culture studies showed that inhibiting ACAT1 in macrophages caused cells to produce less proinflammatory responses upon cholesterol loading by acetyl low-density lipoprotein. In advanced lesions, Acat1 -M/-M reduced but did not eliminate foamy cells. In advanced plaques isolated from ApoE -/- mice, immunostainings showed that both ACAT1 and ACAT2 are present. In cell culture, both enzymes are present in macrophages and smooth muscle cells and contribute to cholesterol ester biosynthesis. Overall, our results support the notion that targeting ACAT1 or targeting both ACAT1 and ACAT2 in macrophages is a novel strategy to treat advanced lesions.
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the ACAT2 expression of human leukocytes is responsible for the excretion of lipoproteins containing cholesteryl steryl esters
Acta Biochimica et Biophysica Sinica, 2016Co-Authors: Dongqing Guo, Bao-liang Song, Catherine C. Y. Chang, Ta-yuan Chang, Xiao-wei Zhang, Ming Zhu, Lei Qian, Ying XiongAbstract:Acyl-coenzymeA:cholesterol acyltransferase 2 (ACAT2) is abundantly expressed in intestine and fetal liver of healthy human. Our previous studies have shown that in monocytic cells the low-level expression of human ACAT2 gene with specific CpG-hypomethylated promoter is regulated by the CCAAT/enhancer binding protein (C/EBP) transcription factors. In this study, we further report that the ACAT2 gene expression is attributable to the C/EBPs in the human leukocytes and correlated with the excretion of fluorescent lipoproteins containing the ACAT2-catalyzed NBD22-steryl esters. Moreover, this lipoprotein excretion can be inhibited by the ACAT2 isoform-selective inhibitor pyripyropene A (PPPA) in a dose-dependent manner, and employed to determine the half maximum inhibitory concentration (IC50) values of PPPA. Significantly, it is found that the differentiation-inducing factor all-trans retinoic acid, but not the proinflammatory cytokine tumor necrosis factor-α, enhances this ACAT2-dependent lipoprotein excretion. These data demonstrate that the ACAT2 expression of human leukocytes is responsible for the excretion of lipoproteins containing cholesteryl/steryl esters (CE/SE), and suggest that the excretion of lipoproteins containing the ACAT2-catalyzed CS/SE may avoid cytotoxicity through decreasing the excess intracellular cholesterols/sterols (especially various oxysterols), which is essential for the action of the human leukocytes.
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Low-level expression of human ACAT2 gene in monocytic cells is regulated by the C/EBP transcription factors
Acta biochimica et biophysica Sinica, 2016Co-Authors: Dongqing Guo, Catherine C. Y. Chang, Xiao-wei Zhang, Ming Zhu, Ta-yuan ChangAbstract:Acyl-coenzyme A:cholesterol acyltransferases (ACATs) are the exclusive intracellular enzymes that catalyze the formation of cholesteryl/steryl esters (CE/SE). In our previous work, we found that the high-level expression of human ACAT2 gene with the CpG hypomethylation of its whole promoter was synergistically regulated by two transcription factors Cdx2 and HNF1α in the intestine and fetal liver. Here, we first observed that the specific CpG-hypomethylated promoter was correlated with the low expression of human ACAT2 gene in monocytic cell line THP-1. Then, two CCAAT/enhancer binding protein (C/EBP) elements within the activation domain in the specific CpG-hypomethylation promoter region were identified, and the expression of ACAT2 in THP-1 cells was evidently decreased when the C/EBP transcription factors were knock-downed using RNAi technology. Furthermore, ChIP assay confirmed that C/EBPs directly bind to their elements for low-level expression of human ACAT2 gene in THP-1 cells. Significantly, the increased expressions of ACAT2 and C/EBPs were also found in macrophages differentiated from both ATRA-treated THP-1 cells and cultured human blood monocytes. These results demonstrate that the low-level expression of human ACAT2 gene with specific CpG-hypomethylated promoter is regulated by the C/EBP transcription factors in monocytic cells, and imply that the lowly expressed ACAT2 catalyzes the synthesis of certain CE/SE that are assembled into lipoproteins for the secretion.
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acyl coa cholesterol acyltransferases acats soats enzymes with multiple sterols as substrates and as activators
The Journal of Steroid Biochemistry and Molecular Biology, 2015Co-Authors: Maximillian A. Rogers, Bao-liang Song, Catherine C. Y. Chang, Boliang Li, Ta-yuan ChangAbstract:Cholesterol is essential to the growth and viability of cells. The metabolites of cholesterol include: steroids, oxysterols, and bile acids, all of which play important physiological functions. Cholesterol and its metabolites have been implicated in the pathogenesis of multiple human diseases, including: atherosclerosis, cancer, neurodegenerative diseases, and diabetes. Thus, understanding how cells maintain the homeostasis of cholesterol and its metabolites is an important area of study. Acyl-coenzyme A:cholesterol acyltransferases (ACATs, also abbreviated as SOATs) converts cholesterol to cholesteryl esters and play key roles in the regulation of cellular cholesterol homeostasis. ACATs are most unusual enzymes because (i) they metabolize diverse substrates including both sterols and certain steroids; (ii) they contain two different binding sites for steroidal molecules. In mammals, there are two ACAT genes that encode two different enzymes, ACAT1 and ACAT2. Both are allosteric enzymes that can be activated by a variety of sterols. In addition to cholesterol, other sterols that possess the 3-beta OH at C-3, including PREG, oxysterols (such as 24(S)-hydroxycholesterol and 27-hydroxycholesterol, etc.), and various plant sterols, could all be ACAT substrates. All sterols that possess the iso-octyl side chain including cholesterol, oxysterols, various plant sterols could all be activators of ACAT. PREG can only be an ACAT substrate because it lacks the iso-octyl side chain required to be an ACAT activator. The unnatural cholesterol analogs epi-cholesterol (with 3-alpha OH in steroid ring B) and ent-cholesterol (the mirror image of cholesterol) contain the iso-octyl side chain but do not have the 3-beta OH at C-3. Thus, they can only serve as activators and cannot serve as substrates. Thus, within the ACAT holoenzyme, there are site(s) that bind sterol as substrate and site(s) that bind sterol as activator; these sites are distinct from each other. These features form the basis to further pursue ACAT structure-function analysis, and can be explored to develop novel allosteric ACAT inhibitors for therapeutic purposes. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'.
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acat1 knockdown gene therapy decreases amyloid β in a mouse model of alzheimer s disease
Molecular Therapy, 2013Co-Authors: Stephanie R Murphy, Catherine C. Y. Chang, Elena Y. Bryleva, Godwin Dogbevia, Zachary D Bowen, Mazahir T Hasan, Ta-yuan ChangAbstract:Both genetic inactivation and pharmacological inhibition of the cholesteryl ester synthetic enzyme acyl-CoA:cholesterol acyltransferase 1 (ACAT1) have shown benefit in mouse models of Alzheimer's disease (AD). In this study, we aimed to test the potential therapeutic applications of adeno-associated virus (AAV)-mediated Acat1 gene knockdown in AD mice. We constructed recombinant AAVs expressing artificial microRNA (miRNA) sequences, which targeted Acat1 for knockdown. We demonstrated that our AAVs could infect cultured mouse neurons and glia and effectively knockdown ACAT activity in vitro. We next delivered the AAVs to mouse brains neurosurgically, and demonstrated that Acat1-targeting AAVs could express viral proteins and effectively diminish ACAT activity in vivo, without inducing appreciable inflammation. We delivered the AAVs to the brains of 10-month-old AD mice and analyzed the effects on the AD phenotype at 12 months of age. Acat1-targeting AAV delivered to the brains of AD mice decreased the levels of brain amyloid-β and full-length human amyloid precursor protein (hAPP), to levels similar to complete genetic ablation of Acat1. This study provides support for the potential therapeutic use of Acat1 knockdown gene therapy in AD.
