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• W2801233784 abstract "Intracellular cholesterol transport proteins move cholesterol to different subcellular compartments and thereby regulate its final metabolic fate. In hepatocytes, for example, delivery of high-density lipoprotein (HDL)–associated cholesterol for bile acid synthesis or secretion into bile facilitates cholesterol elimination from the body (anti-atherogenic effect), whereas delivery for esterification and subsequent incorporation into apolipoprotein B–containing atherogenic lipoproteins (e.g. very-low-density lipoprotein (VLDL)) enhances cholesterol secretion into the systemic circulation (pro-atherogenic effect). Intracellular cholesterol transport proteins such as sterol carrier protein-2 (SCP2) should, therefore, play a role in regulating these pro- or anti-atherosclerotic processes. Here, we sought to evaluate the effects of SCP2 deficiency on the development of diet-induced atherosclerosis. We generated LDLR−/− mice deficient in SCP2/SCPx (LS) and examined the effects of this deficiency on Western diet–induced atherosclerosis. SCP2/SCPx deficiency attenuated atherosclerosis in LS mice by >80% and significantly reduced plasma cholesterol and triglyceride levels. Investigation of the likely underlying mechanisms revealed a significant reduction in intestinal cholesterol absorption (given as an oral gavage) in SCP2/SCPx-deficient mice. Consistently, siRNA-mediated knockdown of SCP2 in intestinal cells significantly reduced cholesterol uptake. Furthermore, hepatic triglyceride/VLDL secretion from the liver or hepatocytes isolated from SCP2/SCPx-deficient mice was significantly reduced. These results indicate an important regulatory role for SCP2 deficiency in attenuating diet-induced atherosclerosis by limiting intestinal cholesterol absorption and decreasing hepatic triglyceride/VLDL secretion. These findings suggest targeted inhibition of SCP2 as a potential therapeutic strategy to reduce Western diet–induced dyslipidemia and atherosclerosis. Intracellular cholesterol transport proteins move cholesterol to different subcellular compartments and thereby regulate its final metabolic fate. In hepatocytes, for example, delivery of high-density lipoprotein (HDL)–associated cholesterol for bile acid synthesis or secretion into bile facilitates cholesterol elimination from the body (anti-atherogenic effect), whereas delivery for esterification and subsequent incorporation into apolipoprotein B–containing atherogenic lipoproteins (e.g. very-low-density lipoprotein (VLDL)) enhances cholesterol secretion into the systemic circulation (pro-atherogenic effect). Intracellular cholesterol transport proteins such as sterol carrier protein-2 (SCP2) should, therefore, play a role in regulating these pro- or anti-atherosclerotic processes. Here, we sought to evaluate the effects of SCP2 deficiency on the development of diet-induced atherosclerosis. We generated LDLR−/− mice deficient in SCP2/SCPx (LS) and examined the effects of this deficiency on Western diet–induced atherosclerosis. SCP2/SCPx deficiency attenuated atherosclerosis in LS mice by >80% and significantly reduced plasma cholesterol and triglyceride levels. Investigation of the likely underlying mechanisms revealed a significant reduction in intestinal cholesterol absorption (given as an oral gavage) in SCP2/SCPx-deficient mice. Consistently, siRNA-mediated knockdown of SCP2 in intestinal cells significantly reduced cholesterol uptake. Furthermore, hepatic triglyceride/VLDL secretion from the liver or hepatocytes isolated from SCP2/SCPx-deficient mice was significantly reduced. These results indicate an important regulatory role for SCP2 deficiency in attenuating diet-induced atherosclerosis by limiting intestinal cholesterol absorption and decreasing hepatic triglyceride/VLDL secretion. These findings suggest targeted inhibition of SCP2 as a potential therapeutic strategy to reduce Western diet–induced dyslipidemia and atherosclerosis. Hydrophobicity of nonesterified or free cholesterol (FC) 2The abbreviations used are: FCfree or unesterified cholesterolHDLhigh-density lipoproteinCEcholesteryl esterDPMdisintegrations per minuteH&Ehematoxylin and eosinVLDLvery-low-density lipoproteinWDWestern dietAcLDLacetylated low-density lipoproteinLDLRlow density lipoprotein receptorFBSfetal bovine serum7α-OOH7α-hydroperoxycholesterol. precludes its free movement within the cell and between different cellular membranes, a process that is essential to multiple cellular functions, including FC esterification, conversion to bile acids in hepatocytes, or synthesis of steroid hormones in steroidogenic tissues. Although release of FC from the donor membrane is typically rate-limiting, and physical proximity with the acceptor membrane is not required (1Phillips M.C. Johnson W.J. Rothblat G.H. Mechanisms and consequences of cellular cholesterol exchange and transfer.Biochim. Biophys. Acta. 1987; 906 (3297153): 223-27610.1016/0304-4157(87)90013-XGoogle Scholar, 2Zilversmit D.B. Lipid transfer proteins.J. Lipid Res. 1984; 25 (6397561): 1563-1569Google Scholar), transfer of FC between membranes is enhanced by intracellular transfer/carrier proteins, such as members of the steroidogenic acute regulatory (StAR) family (3Soccio R.E. Breslow J.L. StAR-related lipid transfer (START) proteins: mediators of intracellular lipid metabolism.J. Biol. Chem. 2003; 278 (12724317): 22183-2218610.1074/jbc.R300003200Google Scholar), which are highly specific for sterols, or nonspecific transporters including sterol carrier protein 2 (SCP2) and fatty acid binding protein 1 (FABP1) (4Scallen T.J. Pastuszyn A. Noland B.J. Chanderbhan R. Kharroubi A. Vahouny G.V. Sterol carrier and lipid transfer proteins.Chem. Phys. Lipids. 1985; 38 (3910286): 239-26110.1016/0009-3084(85)90019-2Google Scholar, 5Gallegos A.M. Atshaves B.P. Storey S.M. Starodub O. Petrescu A.D. Huang H. McIntosh A.L. Martin G.G. Chao H. Kier A.B. Schroeder F. Gene structure, intracellular localization, and functional roles of sterol carrier protein-2.Prog. Lipid Res. 2001; 40 (11591437): 498-56310.1016/S0163-7827(01)00015-7Google Scholar). Consequently, these transfer proteins not only regulate the distribution of cholesterol in cell organelles but also play an important role in intracellular cholesterol metabolism and tissue-specific distribution. Furthermore, expression of these proteins is closely related to the rate of cholesterol metabolism in each tissue (6Chanderbhan R.F. Kharroubi A.T. Noland B.J. Scallen T.J. Vahouny G.V. Sterol carrier protein 2: further evidence for its role in adrenal steroidogenesis.Endocr. Res. 1986; 12 (3030719): 351-37010.3109/07435808609035445Google Scholar). Therefore, the role of these proteins is extensively studied in steroidogenic tissues such as adrenals (where these proteins regulate steroid hormone synthesis by modulating the delivery of FC to appropriate subcellular organelles) and the liver (the principal organ responsible for maintaining whole-body cholesterol homeostasis). free or unesterified cholesterol high-density lipoprotein cholesteryl ester disintegrations per minute hematoxylin and eosin very-low-density lipoprotein Western diet acetylated low-density lipoprotein low density lipoprotein receptor fetal bovine serum 7α-hydroperoxycholesterol. The Scp2 gene encodes the 58-kDa sterol carrier protein-x (SCPx) and 15-kDa pro-SCP2 proteins, both of which contain a 13-kDa SCP2 domain in their C termini. SCPx is localized primarily to peroxisomes and functions as a thiolase. Pro-SCP2 can either be obtained by proteolytic cleavage of SCPx, or it is independently transcribed using an internal/alternate transcription start site (7Ohba T. Rennert H. Pfeifer S.M. He Z. Yamamoto R. Holt J.A. Billheimer J.T. Strauss 3rd., J.F. The structure of the human sterol carrier protein X/sterol carrier protein 2 gene (SCP2).Genomics. 1994; 24 (7698762): 370-37410.1006/geno.1994.1630Google Scholar). Mature SCP2 (13 kDa), representing the C-terminal end of SCPx, as well as pro-SCP2 is, however, obtained by proteolytic cleavage. Mechanisms that regulate transcription initiation from the internal start site to directly generate pro-SCP2 or proteolytic cleavage of SCPx or pro-SCP2 to generate mature SCP2 are not completely defined. In addition to being involved in maintaining differential cholesterol content of the plasma membrane leaflets (8Schroeder F. Frolov A.A. Murphy E.J. Atshaves B.P. Jefferson J.R. Pu L. Wood W.G. Foxworth W.B. Kier A.B. Recent advances in membrane cholesterol domain dynamics and intracellular cholesterol trafficking.Proc. Soc. Exp. Biol. Med. 1996; 213 (8931661): 150-17710.3181/00379727-213-44047Google Scholar), replenishing mitochondrial membrane cholesterol (9Gallegos A.M. Schoer J.K. Starodub O. Kier A.B. Billheimer J.T. Schroeder F. A potential role for sterol carrier protein-2 in cholesterol transfer to mitochondria.Chem. Phys. Lipids. 2000; 105 (10727111): 9-2910.1016/S0009-3084(99)00128-0Google Scholar), and regulating lipid rafts and signaling (10Schroeder F. Atshaves B.P. McIntosh A.L. Gallegos A.M. Storey S.M. Parr R.D. Jefferson J.R. Ball J.M. Kier A.B. Sterol carrier protein-2: new roles in regulating lipid rafts and signaling.Biochim. Biophys. Acta. 2007; 1771 (17543577): 700-71810.1016/j.bbalip.2007.04.005Google Scholar), SCP-2 is also involved in regulating hepatic cholesterol homeostasis. Niemann–Pick C disease, characterized by hepatic cholesterol accumulation in lysosomes and the Golgi, is associated with reduced levels of hepatic 13-kDa SCP2 (11Roff C.F. Pastuszyn A. Strauss 3rd, J.F. Billheimer J.T. Vanier M.T. Brady R.O. Scallen T.J. Pentchev P.G. Deficiencies in sex-regulated expression and levels of two hepatic sterol carrier proteins in a murine model of Niemann-Pick type C disease.J. Biol. Chem. 1992; 267: 15902-15908Google Scholar). SCP2 affects hepatic cholesterol accumulation (12Atshaves B.P. McIntosh A.L. Martin G.G. Landrock D. Payne H.R. Bhuvanendran S. Landrock K.K. Lyuksyutova O.I. Johnson J.D. Macfarlane R.D. Kier A.B. Schroeder F. Overexpression of sterol carrier protein-2 differentially alters hepatic cholesterol accumulation in cholesterol-fed mice.J. Lipid Res. 2009; 50 (19289417): 1429-144710.1194/jlr.M900020-JLR200Google Scholar) and enhances HDL-mediated cholesterol uptake in primary hepatocytes (13Storey S.M. McIntosh A.L. Huang H. Landrock K.K. Martin G.G. Landrock D. Payne H.R. Atshaves B.P. Kier A.B. Schroeder F. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.Am. J. Physiol. Gastrointest. Liver Physiol. 2012; 302: G824-G839Google Scholar). Earlier studies from our laboratory have demonstrated an increase in flux of cholesterol from HDL-associated cholesteryl esters (HDL-CE) to bile acids by adenovirus-mediated transient overexpression of SCP2 as well as FABP1 (14Wang J. Bie J. Ghosh S. Intracellular cholesterol transport proteins enhance hydrolysis of HDL-CEs and facilitate elimination of cholesterol into bile.J. Lipid Res. 2016; 57 (27381048): 1712-171910.1194/jlr.M069682Google Scholar). Studies with mice lacking SCP-2/SCPx or FABP1 or both have provided evidence for the role of these two proteins in modulating biliary lipid levels (15Martin G.G. Atshaves B.P. Landrock K.K. Landrock D. Storey S.M. Howles P.N. Kier A.B. Schroeder F. Ablating L-FABP in SCP-2/SCP-x null mice impairs bile acid metabolism and biliary HDL-cholesterol secretion.Am. J. Physiol. Gastrointest. Liver Physiol. 2014; 307 (25277800): G1130-G114310.1152/ajpgi.00209.2014Google Scholar, 16Martin G.G. Landrock D. Landrock K.K. Howles P.N. Atshaves B.P. Kier A.B. Schroeder F. Relative contributions of L-FABP, SCP-2/SCP-x, or both to hepatic biliary phenotype of female mice.Arch. Biochem. Biophys. 2015; 588 (26541319): 25-32Google Scholar). Given the inability of mammalian systems to metabolize the steroid nucleus, conversion of hydrophobic cholesterol to more water-soluble bile acids and solubilization of cholesterol by these detergents, followed by excretion into bile, represents a major mechanism for cholesterol elimination from the body. A homeostatic balance between cholesterol intake/de novo synthesis and cholesterol elimination is central to the development of atherosclerosis. Despite the significant role of intracellular cholesterol transfer proteins such as SCP-2 in regulating the expression of genes involved in hepatic cholesterol homeostasis (17Martin G.G. Atshaves B.P. Landrock K.K. Landrock D. Schroeder F. Kier A.B. Loss of L-FABP, SCP-2/SCP-x, or both induces hepatic lipid accumulation in female mice.Arch. Biochem. Biophys. 2015; 580 (26541319): 41-4910.1016/j.abb.2015.10.018Google Scholar), the direct role of these proteins in modulating atherogenesis has not been examined to date. Based on our earlier studies, where transient overexpression of SCP2 led to increased flux of cholesterol from HDL to bile, we speculated an anti-atherogenic role for SCP2 (14Wang J. Bie J. Ghosh S. Intracellular cholesterol transport proteins enhance hydrolysis of HDL-CEs and facilitate elimination of cholesterol into bile.J. Lipid Res. 2016; 57 (27381048): 1712-171910.1194/jlr.M069682Google Scholar). To directly address this hypothesis, in this study we examined the effects of SCP2/SCPx deficiency on the development of Western diet–induced atherosclerosis in the LDLR−/− background. Contrary to the expected increase in diet-induced atherosclerosis, a dramatic attenuation of lesion development was noted in mice lacking SCP2/SCPx. Data are presented to indicate that a significant decrease in dietary cholesterol absorption in the intestine as well as in hepatic VLDL secretion by SCP2/SCPx deficiency collectively represent the mechanism underlying the observed attenuation of plasma lipid levels and, consequently, the reduction in Western diet–induced atherosclerosis in LDLR−/− mice. Intracellular cholesterol-binding proteins facilitate delivery of cholesterol to appropriate organelles for subsequent metabolism, and we have demonstrated earlier that adenovirus-mediated overexpression of SCP2 increases the flux of cholesterol from HDL to bile and feces, indicative of a potential anti-atherogenic role of this protein (14Wang J. Bie J. Ghosh S. Intracellular cholesterol transport proteins enhance hydrolysis of HDL-CEs and facilitate elimination of cholesterol into bile.J. Lipid Res. 2016; 57 (27381048): 1712-171910.1194/jlr.M069682Google Scholar). However, as shown in Fig. 1A, deficiency of SCP2/SCPx (LS) dramatically reduced diet-induced atherosclerotic lesions in LDLR−/− mice. Compared with LDLR−/− mice, the lesion area in the aortic arch in LS mice was significantly reduced in both males (37.31 ± 7.79 versus 6.85 ± 5.47, p < 0.0001) and females (29.77 ± 4.43 versus 14.09 ± 6.62, p < 0.011). Similar trends were seen when total aortic lesion areas in LS mice were compared (Fig. 1C) (males 19.15 ± 2.87 versus 3.31 ± 2.16, p < 0.0001; females 13.35 ± 2.68 versus 6.41 ± 2.36, p < 0.0001). The aortic root represents another anatomical site of plaque development, and changes in plaque area at this site were also examined using H&E-stained aortic root sections. Consistent with the en face analyses shown in Fig. 1, a significant reduction in the area occupied by the plaque in the aortic root was noted in LS mice (Fig. 2, A and B). To evaluate changes in plaque characteristics, if any, Masson’s trichrome–stained aortic root sections were analyzed to determine the percentage of necrotic area. A significant reduction in plaque necrosis was seen in LS mice (Fig. 2, C and D). Plasma cholesterol and triglyceride levels are considered a reliable marker of whole-body lipid homeostasis, and elevated levels associated with development of atherosclerosis are either the result of increased uptake of dietary lipids or enhanced hepatic secretion of pro-atherogenic ApoB–containing VLDL. SCP2 deficiency significantly reduced plasma cholesterol levels in LS mice (1747.38 ± 670.62 versus 543.00 ± 144.57, p < 0.0001 in males and 1424.33 ± 205.1 versus 966.83 ± 214.97, p = 0.0118 in females; Fig. 3A). Furthermore, comparison of changes in percent total cholesterol associated with non-HDL or HDL fractions showed that the observed reduction in plasma cholesterol was primarily determined by reduction in non-HDL cholesterol or cholesterol associated with LDL and VLDL fractions (86.15 ± 11.93 versus 71.55 ± 5.85, p = 0.0077 in males and 91.42 ± 1.44 versus 85.95 ± 2.04, p = 0.0003 in females, Fig. 3B). In contrast, there was a small but statistically significant increase in the percentage of cholesterol associated with the HDL fraction (13.85 ± 11.93 versus 28.45 ± 5.85, p = 0.0077 in males and 8.58 ± 1.44 versus 14.05 ± 2.04, p = 0.0003 in females; Fig. 3B). To facilitate a direct demonstration of the effects of SCP2 deficiency on cholesterol distribution between plasma lipoprotein fractions, the data are also represented after normalization to total cholesterol levels observed in LDLR−/− mice of the same sex. Significant reduction in non-HDL cholesterol is apparent in LS mice of both sexes (87.31 ± 32.41 versus 39.27 ± 11.04, p = 0.0014 in males and 91.39 ± 11.45 versus 60.78 ± 6.31, p = 0.0002 in females; Fig. 3C). It is noteworthy that, in WD-fed LDLR−/− mice, >90% plasma cholesterol is associated with the non-HDL fraction (18Huszar D. Varban M.L. Rinninger F. Feeley R. Arai T. Fairchild-Huntress V. Donovan M.J. Tall A.R. Increased LDL cholesterol and atherosclerosis in LDL receptor-deficient mice with attenuated expression of scavenger receptor B1.Arterioscler. Thromb. Vasc. Biol. 2000; 20 (10764675): 1068-107310.1161/01.ATV.20.4.1068Google Scholar). A strong positive correlation was obtained between the total lesion area and plasma cholesterol levels (Fig. 3D). Plasma triglyceride levels were also significantly reduced in male LS mice (471.88 ± 292.22 versus 121.75 ± 48.20, p = 0.0016; Fig. 3E), but this decrease did not reach statistical significance in female mice (411.33 ± 268.1 versus 225.67 ± 139.25, p = 0.1631). Although total lesions increased with increasing plasma triglyceride levels, this correlation did not reach statistical significance (Fig. 3F). These data suggest that global SCP2 deficiency–mediated attenuation of plasma lipids likely underlies the observed decrease in atherosclerotic lesions in LS mice. Diet-induced hypercholesterolemia underlies the development of atherosclerosis in this model of Western diet–induced atherosclerosis in LDLR−/− mice. Therefore, any reduction in absorption of dietary cholesterol is likely to affect the development of atherosclerosis. To examine whether SCP2 deficiency–mediated changes in intestinal cholesterol absorption represent the mechanism underlying the observed reduction in plasma lipids as well as atherosclerosis, the appearance of orally administered [3H]cholesterol in plasma was monitored under conditions where tissue uptake of absorbed cholesterol is prevented by tyloxapol-mediated inhibition of lipoprotein lipase. A significant reduction in plasma DPM was seen in SCP2−/− mice (286.23 ± 60.58 versus 180.81 ± 11.43, p = 0.007 in males and 236.11 ± 71.06 versus 169.68 ± 35.09, p = 0.036 in females; Fig. 4A). These data suggest that SCP2/SCPx deficiency leads to reduced intestinal absorption of dietary cholesterol, resulting in attenuation of diet-induced hypercholesterolemia. Although absorption of cholesterol occurs via NPC1L1, intestinal cells secrete cholesterol along with phytosterols back into the lumen via ABCG5/G8. Furthermore, cholesterol absorption is not uniform along the length of the intestine. Expression of NPC1L1 or ABCG5 and ABCG8 along the intestine was evaluated in WT and SCP2−/− mice to determine the effects SCP2 deficiency. Although no significant difference was noted in the expression of NPC1L1, there was a significant reduction in the expression of ABCG5 and ABCG8 in the distal P4 segment of the intestine of SCP2−/− mice (Fig. 4B). Consistent with global deficiency of SCP2 in these mice, no expression of SCP2 protein was noted along the length of the intestine, i.e. segments P1 through P4 (Fig. 4C). Because the mice used in this study have a global deficiency of SCP2/SCPx, to directly assess the effects of SCP2 deficiency in intestinal cells on cholesterol absorption/uptake, SCP2 was knocked down in the human intestinal epithelial cell line HT-29 using SCP2-specific siRNA. A concentration-dependent decrease in SCP2 mRNA was noted, along with maximum reduction in SCP2 protein seen at the highest siRNA concentration tested (Fig. 4D). Cholesterol uptake was monitored in HT-29 cells transfected with either scrambled or SCP2 siRNA. A time-dependent increase in cholesterol uptake was seen, and this increase was significantly attenuated when SCP2 was knocked down with siRNA (Fig. 4E). These data indicate that deficiency of SCP2 in intestinal epithelial cells limits cholesterol absorption and likely represents the mechanism underlying the observed reduction in plasma lipids and attenuated atherosclerosis in LS mice. Upon WD feeding, increased absorbed lipids are repackaged in the liver and secreted as pro-atherogenic lipoprotein VLDL. Therefore, in addition to the effects of altered lipid absorption, changes in secretion of pro-atherogenic lipoproteins from the liver are also likely to modify plasma lipids and the downstream development of atherosclerosis. Time-dependent accumulation of VLDL-associated triglycerides in the plasma compartment following inhibition of lipoprotein lipase is routinely used to evaluate hepatic VLDL secretion. To examine whether SCP2 deficiency affects hepatic VLDL secretion, we monitored plasma triglyceride levels in WT and SCP2−/− mice following tyloxapol-mediated inhibition of lipoprotein lipase. As shown in Fig. 5A, triglyceride secretion rates calculated from the plasma triglyceride levels were significantly reduced in SCP2−/− mice (12.05 ± 0.73 versus 7.46 ± 1.03, p = 0.0021). These data suggest that SCP2 deficiency attenuates pro-atherogenic VLDL secretion from the liver. Because the mice used in these studies lack SCP2 in all tissues, to confirm the effects of SCP2 deficiency on VLDL/triglyceride secretion from hepatocytes, direct secretion of triglycerides as well as cholesteryl ester into the incubation medium from primary hepatocytes isolated from WT or SCP2−/− mice was assessed. Secretion of [3H]oleic acid–labeled triglycerides (Fig. 5B) as well as cholesteryl esters (Fig. 5C) by SCP2/SCPx-deficient hepatocytes was significantly reduced, confirming the reduction in hepatic secretion rates observed in vivo. Reduced secretion of VLDL could potentially lead to hepatic accumulation of lipids, and therefore morphological changes in the livers of WD-fed LDLR−/− and LS mice were compared. Significant lipid accumulation was noted in livers from WD-fed LDLR−/− mice compared with LS mice, indicating that SCP2 deficiency does not lead to increased accumulation of lipids in the liver (Fig. 6A). Lack of SCP2 expression in livers form LS mice was confirmed by almost undetectable levels of Scp2 mRNA in LS mice (Fig. 6B). Consistent with lack of lipid accumulation in the liver, no significant difference in the expression of lipogenic genes (namely, ApoB, Fas, Srebp-1c, and Srebp-2) was noted between livers from WT and SCP2−/− mice (Fig. 6C). These data indicate that SCP2/SCPx deficiency leads to reduced VLDL secretion from liver/hepatocytes and that SCP2 deficiency likely leads to reduced plasma lipids by not only limiting intestinal absorption but also by decreasing hepatic VLDL secretion. Furthermore, SCP2 deficiency also reduces WD-induced lipid accumulation in the liver without any significant change in expression of genes involved in lipogenesis. An imbalance between forward transport of cholesterol to peripheral tissues and its return to the liver for final elimination underlies accumulation of lipid-laden macrophage foam cells within the artery wall. Although the data presented above provide strong evidence for SCP2 deficiency–mediated reduction in plasma lipids because of intestinal lipid absorption as well as hepatic lipid secretion, the effects of SCP2 deficiency on reverse flux of cholesterol from macrophages to the liver and final elimination in bile were also monitored to examine any direct effects of SCP2 deficiency on these processes. Changes, if any, in the uptake of modified LDL by macrophages from WT or SCP2−/− were examined, and, as shown in Fig. 7A, no apparent difference in Oil Red O–stained neutral lipid was seen in WT control and SCP2−/− macrophages before or after incubation with acetylated LDL (AcLDL). Consistently, although there was an increase in total cellular cholesterol upon loading with AcLDL in both genotypes, there was no significant difference in total cellular cholesterol mass between WT and SCP2−/− macrophages before or after loading (Fig. 7B). The observed lack of difference in the expression of SR-A (Fig. 7C) further suggests that SCP2 deficiency does not affect the uptake of modified LDL. When efflux of cholesterol from AcLDL-loaded macrophages was measured, a small but significant reduction in cholesterol efflux was observed in LS macrophages. An increase in cholesterol efflux capacity is associated with reduction in the development of atherosclerosis, and this observed reduction in cholesterol efflux from SCP2−/− macrophages suggests that SCP2 deficiency in macrophages may not be the major underlying mechanism for the observed decrease in atherosclerosis in LS mice. Final elimination of cholesterol from the body, required to maintain whole-body cholesterol homeostasis and limit development of atherosclerosis, occurs via biliary or nonbiliary routes. To evaluate the effects of SCP2 deficiency on modulating cholesterol elimination from the body, secreted bile acids and cholesterol were monitored in gall bladder bile and normalized to total biliary phospholipid content. Deficiency of SCP2/SCPx did not affect biliary bile acid (Fig. 8A) or free cholesterol (Fig. 8B) secretion. Although increased biliary elimination of cholesterol from the body as bile acids or free cholesterol is generally viewed as anti-atherogenic, a dramatic reduction in atherosclerosis with no change in biliary bile acids or cholesterol in LS mice points to a possible limited contribution of the cholesterol elimination pathway to the observed attenuation of atherosclerosis by deficiency of SCP2/SCPx in these global knockout mice. While recognizing the role of extracellular trafficking of cholesterol in the progression of atherosclerosis, Billheimer and Reinhart (19Billheimer J.T. Reinhart M.P. Intracellular trafficking of sterols.Subcell. Biochem. 1990; 16 (2238007): 301-33110.1007/978-1-4899-1621-1_10Google Scholar) argued more than 3 decades ago for a major role of intracellular cholesterol trafficking, possibly by regulating transport of cholesterol back to the liver from the peripheral tissues and secretion into the bile. Although ample evidence exists for defining the role of SCP2/SCPx in modulating hepatic cholesterol/bile acid metabolism, the data presented here describe, for the first time, the dramatic anti-atherogenic (>80% decrease) effects of SCP2/SCPx deficiency in athero-susceptible LDLR−/− mice. Furthermore, SCP2/SCPx deficiency led to a significant reduction in plasma cholesterol and triglyceride levels despite consumption of a high-fat, high-cholesterol–containing Western diet. Consistently, we demonstrate that SCP2/SCPx deficiency reduces absorption of cholesterol in the intestine and attenuates VLDL secretion from the liver, identifying SCP2 as a potential therapeutic target to modulate dyslipidemia and its downstream adverse effects. Dyslipidemia is central to the development of atherosclerotic plaques, and LDLR−/− mice develop atherosclerosis only after a dietary challenge with a high-fat, high-cholesterol–containing Western diet. Therefore, the contribution of dietary lipids to overall dyslipidemia is paramount in this model of atherogenesis. Sterol carrier protein–like activity was described in the rat intestine by Kharroubi et al. (20Kharroubi A. Wadsworth J.A. Chanderbhan R. Wiesenfeld P. Noland B. Scallen T. Vahouny G.V. Gallo L.L. Sterol carrier protein2-like activity in rat intestine.J. Lipid Res. 1988; 29 (3379341): 287-292Google Scholar), and the widespread distribution of SCP2-like protein in the intestine was thought to be related to the potential transfer functions in all phases of cholesterol processing. Wouters et al. (21Wouters F.S. Markman M. de Graaf P. Hauser H. Tabak H.F. Wirtz K.W. Moorman A.F. The immunohistochemical localization of the non-specific lipid transfer protein (sterol carrier protein-2) in rat small intestine enterocytes.Biochim. Biophys. Acta. 1995; 1259 (7488641): 192-19610.1016/0005-2760(95)00163-7Google Scholar) confirmed the expression of SCP-2 in rat small intestine enterocytes and, based on the intracellular distribution, suggested that this protein may play a role in the intracellular processing of absorbed lipids. The data presented here show that SCP2/SCPx deficiency significantly reduces intestinal cholesterol absorption and that siRNA-dependent knockdown of SCP2 in human intestinal epithelial cells leads to a significant reduction in cholesterol accumulation, indicating that intestinal SCP2 regulates the uptake of dietary cholesterol and is likely responsible for the reduced plasma lipids in LS mice. Future development of an intestine-specific SCP2 knockout will facilitate the direct confirmation of this hypothesis. Reduction in cholesterol absorption is used as a clinical intervention, and supplementation with ezetimibe reduced plasma cholesterol by 32%, leading to a reduction in atherosclerosis by 52–59% in LDLR−/− mice (22Basso F. Freeman L.A. Ko C. Joyce C. Amar M.J. Shamburek R.D. Tan" @default.
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• W2801233784 date "2018-06-01" @default.
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• W2801233784 title "Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice" @default.
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• W2801233784 doi "https://doi.org/10.1074/jbc.ra118.002290" @default.
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