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Lawrence L Rudel – One of the best experts on this subject based on the ideXlab platform.

  • Intestine-specific MTP and global ACAT2 deficiency lowers acute cholesterol absorption with chylomicrons and HDLs
    Journal of lipid research, 2014
    Co-Authors: Jahangir Iqbal, Lawrence L Rudel, Mohamed Boutjdir, M. Mahmood Hussain
    Abstract:

    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.

  • 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, 2013
    Co-Authors: Masaki Ohtawa, Lawrence L Rudel, Hiroshi Tomoda, Satoshi Ohte, Taichi Ohshiro, Daisuke Matsuda, Satoshi Ōmura, Hiroyuki Yamazaki, Tohru Nagamitsu
    Abstract:

    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.

  • ACAT2 and abcg5 g8 are both required for efficient cholesterol absorption in mice evidence from thoracic lymph duct cannulation
    Journal of Lipid Research, 2012
    Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L Rudel
    Abstract:

    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?

Matthew A. Davis – One of the best experts on this subject based on the ideXlab platform.

  • ACAT2 and abcg5 g8 are both required for efficient cholesterol absorption in mice evidence from thoracic lymph duct cannulation
    Journal of Lipid Research, 2012
    Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L Rudel
    Abstract:

    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?

  • ACAT2 and ABCG5/G8 are both required for efficient cholesterol absorption in mice: evidence from thoracic lymph duct cannulation.
    Journal of lipid research, 2012
    Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L Rudel
    Abstract:

    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?

  • 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, 2012
    Co-Authors: Jun Zhang, Robert V. Farese, Matthew A. Davis, Janet K. Sawyer, Stephanie M. Marshall, Martha D. Wilson, J. Mark Brown, Kathryn L. Kelley, Lawrence L Rudel
    Abstract:

    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.

Janet K. Sawyer – One of the best experts on this subject based on the ideXlab platform.

  • ACAT2 and abcg5 g8 are both required for efficient cholesterol absorption in mice evidence from thoracic lymph duct cannulation
    Journal of Lipid Research, 2012
    Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L Rudel
    Abstract:

    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?

  • ACAT2 and ABCG5/G8 are both required for efficient cholesterol absorption in mice: evidence from thoracic lymph duct cannulation.
    Journal of lipid research, 2012
    Co-Authors: Tam Nguyen, Carol R. Kent, Matthew A. Davis, Janet K. Sawyer, Kathryn L. Kelley, Lawrence L Rudel
    Abstract:

    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?

  • Abstract 129: Liver-Specific and Intestine-Specific ACAT2 Knockout Mice Are Equally Protected from Diet-Induced Hepatic Cholesterol Accumulation
    Arteriosclerosis Thrombosis and Vascular Biology, 2012
    Co-Authors: Jun Zhang, Matthew L. Davis, Janet K. Sawyer, Kathryn Kelley, Stephanie M. Marshall, Martha D. Wilson, Jonathan Mark Brown, Lawrence L Rudel
    Abstract:

    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.