Peroxisome Proliferator-Activated Receptor α Reduces Cholesterol Esterification in Macrophages
Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor activated by fatty acid derivatives and hypolipidemic drugs of the fibrate class. PPARα is expressed in monocytes, macrophages, and foam cells, suggesting a role for this receptor in macrophage lipid homeostasis with consequences for atherosclerosis development. Recently, it was shown that PPARα activation promotes cholesterol efflux from macrophages via induction of the ABCA1 pathway. In the present study, the influence of PPARα activators on intracellular cholesterol homeostasis was investigated. In human macrophages and foam cells, treatment with fibrates, synthetic PPARα activators, led to a decrease in the cholesteryl ester (CE):free cholesterol (FC) ratio. In these cells, PPARα activation reduced cholesterol esterification rates and Acyl-CoA:cholesterol acyltransferase-1 (ACAT1) activity. However, PPARα activation did not alter ACAT1 gene expression, whereas mRNA levels of carnitine palmitoyltransferase type 1 (CPT-1), a key enzyme in mitochondrial fatty acid catabolism, were induced. Finally, PPARα activation blocked CE formation induced by TNF-α, possibly due to the inhibition of neutral sphingomyelinase activation by TNF-α. In conclusion, our results identify a role for PPARα in the control of cholesterol esterification in macrophages, resulting in an enhanced availability of FC for efflux through the ABCA1 pathway.
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate the expression of genes controlling lipid and glucose metabolism.1 PPARα, which is activated by fibrates, fatty acids, and eicosanoids,2 is highly expressed in liver, heart, muscle, and kidney, but also present in cells of the arterial wall including monocytes, macrophages, macrophage foam cells, smooth muscle, and endothelial cells.2 Recent observations suggest a role for PPARα in macrophage lipid homeostasis and cholesterol efflux, the first step of the reverse cholesterol transport pathway. In these cells, PPARα activators induce the expression of the scavenger receptor CLA-1/SR-BI, which binds HDL with high affinity.3 In addition, in differentiated macrophages and macrophage-derived foam cells PPARα activators induce ABCA1 gene expression and promote apoAI-mediated cholesterol efflux.4
In cholesterol-loaded macrophages, cholesterol efflux is closely related to the rate of cholesteryl ester (CE) turnover.5 Within cells, modified LDL-derived cholesteryl esters are hydrolyzed in lysosomes to free cholesterol (FC). This FC is probably initially transported to the plasma membrane where it integrates in the cell membrane.5 Excess membrane cholesterol is transported back to the endoplasmic reticulum where it is re-esterified by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) with fatty acids (FAs) and stored as lipid droplets.6 Thus, the cholesterol esterification rate also controlled by FA availability depends partially on their catabolism by enzymes such as carnitine palmitoyltransferase 1 CPT-1, a key enzyme regulating mitochondrial FA entry.7 On the other hand, FC can be effluxed from cells to extracellular acceptors such as apoAI through the ABCA1 pathway and as such, enter into the reverse cholesterol transport pathway. This mechanism limits CE accumulation in vascular macrophages, thus preventing foam cell formation.5 ACAT1 enzyme activity as well as cholesterol esterification rates are controlled by different factors.8,9 For instance, TNF-α has been reported to stimulate cholesteryl ester formation in human fibroblasts by activating the neutral sphingomyelinase (N-SMase) pathway.10 As such, TNF-α treatment probably leads to a decrease in plasma membrane levels of sphingomyelin, which functions as a trap for FC. In this way, FC could move from the plasma membrane, thus reaching the esterification site and providing more substrate for ACAT1 in the endoplasmic reticulum.5 Because PPARα is expressed in differentiated macrophages and macrophage-foam cells where it controls free cholesterol removal through activation of ABCA1,4 we decided to study the role of PPARα in processes upstream of cholesterol efflux. Our results demonstrate that, without influencing total cholesterol accumulation in macrophages,4 PPARα activation reduces the CE:FC ratio in macrophages and macrophage-derived foam cells by inhibiting cellular cholesteryl ester formation activity. Moreover, PPARα activation blocks TNF-α–stimulated cholesteryl ester formation activity possibly by negatively interfering with the N-SMase pathway. Our results further expand the role for PPARα in the control of cholesterol homeostasis in macrophages. Along with the induction of ABCA1 expression, a decrease in CE:FC ratio may contribute to an enhanced liberation and efflux of free cholesterol to extracellular acceptors, the first step of the reverse cholesterol transport pathway, after PPARα activation.
Materials and Methods
Mononuclear cells isolated from blood of healthy normolipidemic donors by Ficoll gradient centrifugation were suspended in RPMI 1640 medium containing gentamycin (40 mg/mL), glutamine (0.05%), and 10% pooled human serum.11 Differentiation of monocytes into macrophages occurs spontaneously by adhesion of the cells to the culture dishes. Mature monocyte-derived macrophages were used for experiments after 10 days of culture. For experiments, medium was changed to medium without serum but supplemented with 1% Nutridoma HU (Boehringer Mannheim).
RNA Extraction and Analysis
Total cellular RNA was extracted from 10-day–cultured primary human macrophages or macrophage-derived foam cells treated or not with the PPARα ligands Wy14643 (25, 50, and 100 μmol/L), ciprofibrate (50 μmol/L), bezafibrate (50 μmol/L), fenofibric acid (50 μmol/L), and GW647 (600 nmol/L) for 24 hours using Trizol (Life Technologies, France). For Northern blot analysis, membranes containing 10 μg of total RNA were hybridized with radiolabeled ACAT112 or 36B4 cDNA probes.
For quantitative PCR, total RNA were reverse transcribed using random hexameric primers and Superscript reverse transcriptase (Life Technologies, France). cDNAs were quantified by real-time PCR on a MX 4000 (Stratagene), using specific primers CPT-1: 5′-ACAGTCGGTGAGGCCTCTTATGAA-3′ and 5′-TCTTGCTG-CCTGAATGTGAGTTGG-3′ and cyclophilin: 5′-GCATACGGGT-CCTGGCATCTTGTCC-3′ and 5′-ATGGTGATCTTCTTGCTGG-TCTTGC-3′. PCR amplification was performed in a volume of 25 μL containing 100 nmol/L of each primer, 4 mmol/L MgCl2, the Brilliant Quantitative PCR Core Reagent Kit mix as recommended by the manufacturer (Stratagene) and SYBR Green 0.33X (Sigma-Aldrich). The conditions were 95°C for 10 minutes, followed by 40 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C. CPT-1 mRNA levels were subsequently normalized to cyclophilin mRNA.
Cellular Cholesterol Measurement
Ten-day–cultured human macrophages were pretreated for 24 hours and thereafter every 24 hours with the PPARα activators Wy14643 (25, 50 and 100 μmol/L), ciprofibrate (50 μmol/L), bezafibrate (50 μmol/L), fenofibric acid (50 μmol/L), and GW647 (600 nmol/L) and cholesterol loaded by incubation with AcLDL (50 μg/mL, containing or not [3H]cholesterol)13 in RPMI 1640 medium supplemented with 1% Nutridoma for 48 hours. Intracellular lipids were extracted by hexane/isopropanol, and total cholesterol (TC) and free cholesterol (FC) were measured by enzymatic assay (Boehringer). Cholesteryl esters (CEs) were calculated as the difference between TC and FC. In other experiments, intracellular lipids were separated by thin layer chromatography (TLC) in Petroleum ether/Diethyl ether/Acetic acid (180:20:10, vol:vol:vol). Spots corresponding to CE and FC were scraped and radioactivity measured by scintillation counting.
Measurement of Cholesteryl Ester Formation
Cholesteryl ester formation within the cells was assessed by measuring the incorporation of [14C]oleate into cholesteryl esters. Ten-day–cultured human macrophages were cholesterol loaded by incubation with AcLDL (50 μg/mL) in RPMI 1640 medium supplemented with 1% Nutridoma for 48 hours. The PPARα activators Wy14643 (25, 50, and 100 μmol/L), bezafibrate (50 μmol/L), fenofibric acid (50 μmol/L), and GW647 (600 nmol/L) were added to the culture medium 24 hours before cholesterol loading and thereafter every 24 hours. After the cholesterol-loading period, cells were incubated for 2 hours at 37°C with 1 μCi [14C]oleic acid per mL of medium. Oleic acid was presented to the cells as a BSA-sodium oleate complex, as described.14 In the experiments with TNF-α, cells were washed and subsequently incubated for 1 hour in fresh RPMI 1640 medium containing or not TNF-α (60 ng/mL) and Wy14643 (50 μmol/L). At the end of the assay, cells were washed and lipids extracted with hexane/isopropanol. [14C]oleic acid incorporation into cholesteryl esters was measured on lipid extracts after separation by TLC in hexane/diethyl ether/acetic acid (90:10:1, vol:vol:vol). Spots corresponding to cholesteryl oleate and oleic acid were scraped and radioactivity measured by scintillation counting.
Measurement of N-SMase Activity
To determine N-SMase activity, 10-day–cultured human macrophages were treated for 24 hours and thereafter every 24 hours with Wy14643 (50 μmol/L) and labeled with [3H]choline (2.5 μCi/mL) for 48 hours.10 After this period, cells were washed and subsequently incubated for 1 hour in fresh RPMI 1640 medium containing or not TNF-α (60 ng/mL) and Wy14643 (50 μmol/L). Medium was subsequently removed, cells were washed in PBS and intracellular lipids were extracted by hexane:isopropanol, as described above. The different phospholipids present were separated by TLC using chloroform/methanol/formic acid (65:25:4, vol:vol:vol) as solvent.10 After chromatography, the gel area corresponding to sphingomyelin was scraped and the radioactivity measured. Results are expressed as the difference of [3H] radioactivity associated to sphingomyelin per mg of cellular protein, between values obtained in the presence or in the absence of TNF-α.
PPARα Activation Decreases the Cholesteryl Ester:Free Cholesterol Ratio in Cholesterol-Loaded Macrophages
To determine the influence of PPARα activation on the cholesterol distribution between FC and CE, primary human monocyte-derived differentiated macrophages were loaded with AcLDL (50 μg/mL) for 48 hours and treated with the specific synthetic PPARα ligand Wy14643 (50 μmol/L) added 24 hours before cholesterol loading and thereafter every 24 hours. As previously shown,4 PPARα activation by Wy14643 did not modify macrophage AcLDL-induced total cholesterol accumulation. However, the cholesterol distribution changed with a significant decrease of the CE fraction (*P<0.05) (Figure 1A). A similar effect was also observed in unloaded macrophages (Figure 1A). In order to demonstrate that the variation of intracellular cholesterol distribution was not due to the action of the PPARα ligand on de novo cholesterol synthesis, the distribution of radiolabeled [3H]cholesterol between FC and CE was analyzed by TLC analysis after [3H]cholesterol-AcLDL loading (Figure 1B). Treatment with Wy14643 at different concentrations (25, 50, and 100 μmol/L) significantly decreased the amount of [3H]cholesterol present in the cholesteryl ester fraction (Figure 1B), indicating that this effect is not due to an action on endogenous cholesterol synthesis and is dependent on the concentration of activator used.
Similar effects were observed with other specific PPARα ligands, such as ciprofibrate (50 μmol/L), bezafibrate (50 μmol/L), fenofibric acid (50 μmol/L), and GW647 (600 nmol/L) at concentrations specifically activating human PPARα15,16 (Figure 2). Under these conditions, no net increase in total cholesterol accumulation was observed with any of the drugs tested (data not shown). These data suggest a role for PPARα in the control of macrophage cholesterol esterification.
PPARα Activation Decreases Cholesteryl Ester Formation in Human Macrophages
Because PPARα activation reduces the amount of CE in macrophages, the influence of PPARα activators on cholesterol esterification was assessed in macrophage foam cells. Treatment of AcLDL-loaded macrophages with different PPARα activators, including Wy14643 (50 μmol/L), bezafibrate (50 μmol/L), fenofibric acid (50 μmol/L), and GW647 (600 nmol/L), resulted in a reduction of cholesteryl ester formation as measured by a decreased incorporation of [14C]oleic acid into cholesteryl esters (Figure 3). To determine whether these compounds act as direct ACAT inhibitor, their effects on in vitro ACAT activity in microsomal preparation were tested. Our results demonstrate that none of these PPARα activators are competitive ACAT inhibitors (data not shown).
ACAT1 Gene Expression Is Not Decreased by PPARα Activation in Human Macrophages and Macrophage-Foam Cells
To determine whether PPARα decreases cholesteryl ester formation through regulation of ACAT1 gene expression, Northern blot analysis was performed using RNA from human macrophages and foam cells incubated with Wy14643. As previously reported,12 4 different ACAT1 transcripts were observed in differentiated macrophages as well as in macrophage-derived foam cells (Figure 4A). PPARα activation did not affect ACAT1 mRNA levels in either cell types (Figure 4B), thus excluding a role for PPARα action via changes in ACAT1 gene expression.
PPARα Activation Increases CPT-1 Expression in Primary Human Macrophages
In order to evaluate whether PPARα may control FA availability for cholesterol esterification by ACAT1, the effect of PPARα ligands was studied on the expression of CPT-1α (the CPT-1 isoform that is highly expressed in liver), a key enzyme in mitochondrial fatty acid catabolism. Treatment with different PPARα activators significantly induced the expression of CPT-1 mRNA levels in primary human monocyte-derived macrophages (Figure 5A). This induction occurred in a dose-dependent manner, as shown for Wy14643 (Figure 5B). The effects on CPT-1 induction by Wy14643 correlated with the observed reduction of cholesteryl ester levels (Figure 1B). These observations provide one potential mechanism by which PPARα controls cholesteryl ester accumulation.
PPARα Decreases TNF-α–Induced Cholesteryl Ester Formation in Human Macrophage-Foam Cells
To identify other potential biochemical mechanisms contributing to the effects of PPARα activation on cholesteryl ester formation, it was analyzed whether PPARα activation interferes with cholesteryl ester formation stimulated by TNF-α.10 Incubation of cholesterol-loaded macrophages with TNF-α (60 ng/mL) increased cholesteryl ester formation as demonstrated by enhanced incorporation of [14C]oleic acid into cholesterol (Figure 6A). The presence of Wy14643 blocked the inductive effect of TNF-α on cholesterol esterification (Figure 6A). These data suggest the existence of a negative cross-talk between the TNF-α pathway and PPARα in the control of cholesterol esterification.
PPARα Decreases TNF-α–Induced N-SMase Activity in Primary Human Macrophages
The stimulatory effect of TNF-α on cholesteryl ester formation may be mediated via the activation of cell membrane–associated neutral sphingomyelinase (N-SMase).10 Treatment with TNF-α resulted in a stimulation of N-SMase activity, as determined by a decrease in [3H]choline incorporation into sphingomyelin (Figure 6B). TNF-α–induced N-SMase activity was blocked in the presence of Wy14643 (Figure 6B). However, basal macrophage N-SMase gene expression was not affected by PPARα activation (data not shown). These findings indicating that PPARα activation interferes negatively with the N-SMase pathway may thus constitute another mechanism contributing to the decreased cholesteryl ester formation activity due to a reduced supply of free cholesterol substrate for the ACAT1 enzyme.
PPARα is a lipid-activated transcription factor that regulates genes involved in lipid and glucose metabolism and inflammation control.1 PPARα is expressed in human endothelial cells, smooth muscle cells, and macrophages in vitro as well as in resident atherosclerotic lesion macrophages where it plays an important role in the control of vascular cholesterol homeostasis and inflammation.2 In macrophages, PPARα ligands enhance ABCA1 expression as well as apoAI-mediated cholesterol efflux,4 indicating a role of PPARα in the control of macrophage cholesterol homeostasis. In this study, we show that PPARα also regulates the balance between free cholesterol and cholesteryl esters, a process upstream of and closely related to cholesterol removal, in human macrophages and foam cells.
PPARα activation did not affect lipid accumulation after AcLDL loading, thus confirming our previous observations4 that PPARα activation does not promote foam cell transformation of primary human macrophages. Moreover, in the present study, we demonstrate that, in human macrophages as well as in macrophage-derived foam cells, PPARα activation reduces cholesteryl ester levels. These effects are not due to a PPARα effect on endogenous cholesterol synthesis, because esterification of exogenously added radiolabeled cholesterol was also reduced. Instead, PPARα activation led to a decrease of cholesteryl ester formation. These actions of PPARα are not due to a decreased expression of the ACAT1 gene, the enzyme responsible for cholesteryl ester formation in macrophages, because ACAT1 mRNA levels do not change. By contrast, PPARα agonists increase the expression of CPT-1,17,18 an enzyme located in the mitochondrial outer membrane catalyzes the entry of long chain FAs into the mitochondria, thus determining the flux of FAs into mitochondria for β-oxidation.7 Increased expression of CPT-1 mRNA may thus result in a reduced availability of long chain FAs as substrate for ACAT1, thus leading to decreased cholesterol esterification. The potential role of CPT-1 in the control of CE levels in macrophages is supported by previous studies showing that CPT-1 inhibition by etomoxir results in a 3.5-fold induction of cholesterol incorporation into CE.19 Our data add CPT-1 to the list of genes that are upregulated by PPARα through a PPRE-dependent mechanism in macrophages, along with LPL20 and the nuclear receptor LXRα.4 Moreover, to determine if the decrease in cholesteryl ester content on PPARα activation could lead to an increased triglyceride synthesis in human macrophages, the triglyceride cellular content was determined. Our data demonstrated that Wy14643 did not affect the triglyceride levels in macrophages and AcLDL-loaded macrophages (triglyceride content; untreated cells, 73.7±5.4 μg/mg protein; Wy14643, 69.7±6.4; AcLDL, 89.1±4.1; AcLDL+Wy14643, 92.9±5.6). Interestingly, PPARα also inhibits cholesterol esterification in human macrophage-foam cells induced by TNF-α, a factor known to stimulate cholesteryl ester formation in other cell types, such as human fibroblasts.10 Although, TNF-α may also induce cellular death by apoptosis,21 these experiments were performed using TNF-α at lower concentrations and shorter incubation times than those reported to induce macrophage apoptosis.11 This negative regulation of TNF-α activity is another example of the antiinflammatory action of PPARα in macrophages. A defect in the delivery of cholesterol substrate to ACAT1, by trapping it within phospholipid-containing pools in cell membranes, could indeed result in the inhibition of CE formation. Thus, PPARα may inhibit cholesteryl ester formation activity via two complementary mechanisms both leading to decreased substrate availability: on the one hand, by inhibiting membrane cholesterol mobilization due to inhibition of TNF-α activity and, on the other hand, by increasing FA oxidation due to CPT-1 gene induction. PPARα activation could also influence the cholesteryl ester hydrolysis by affecting neutral cholesteryl ester hydrolase (NCEH) activity. However, PPARα activators rather reduced cholesteryl ester hydrolysis activity in the macrophages (data not shown), thus indicating that the effects of PPARα on CE:FC ratio occurs rather via inhibition cholesterol esterification than by stimulation of cholesteryl ester hydrolysis. Further studies are required to determine whether PPARα also controls other steps of intracellular cholesterol homeostasis, such as cholesterol trafficking.
The inhibition of cholesteryl ester formation by PPARα agonists could have broader implications than to regulate cholesterol efflux. First, macrophage cholesteryl esters appear to be an important stimulus for the secretion of matrix metalloproteinases (MMPs) in advanced lesions.22 PPARα activators reduce the secretion of MMP9 in human monocytic THP1 cells.23 The reduced secretion of MMPs after PPARα activation could be mediated, in part, via the control of cholesteryl ester formation by PPARα in advanced lesion macrophages. Second, biochemical analysis has shown that large pools of cholesteryl ester derivatives can predispose plaques to rupture.24 PPARα activators could thus influence plaque instability by controlling the amounts of cholesteryl esters in plaques. In vivo studies performed using the apoE−/− mouse showed that treatment with the specific PPARα activator fenofibrate, results in a reduction of the cholesterol content of the plaque, an effect that is predominantly due to a decrease in cholesteryl ester accumulation.25 These in vivo observations extend our results obtained in human macrophages in vitro.
In conclusion, our results demonstrate a novel role for PPARα in the control of the balance between free cholesterol and cholesteryl esters, an effect which, associated with the induction of ABCA1, may contribute to enhanced liberation and efflux of free cholesterol and stimulation of the initial step of the reverse cholesterol transport pathway.
This work was supported by grants from Fondation Leducq, the European Community (grant QLRT-1999-01007), and from the FEDER and Conseil Régional Région Nord/Pas-de-Calais (Genopole project No. 01360124). We thank D. Junquero and A. Delhon (Pierre Fabre, Castres) for the measurement of in vitro ACAT activity and P. Brown (Glaxo Smith Kline) for providing us the GW647 compound. B. Noel, R. Barbeau, and L. Thumerel are acknowledged for the technical help.
Original received July 26, 2002; revision received November 22, 2002; accepted December 11, 2002.
Chinetti G, Gbaguidi GF, Griglio S, Mallat Z, Antonucci M, Poulain P, Chapman J, Fruchart JC, Tedgui A, Najib-Fruchart J, Staels B. CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferator-activated receptors. Circulation. 2000; 101: 2411–2417.
Chinetti G, Lestavel S, Bocher V, Remaley AT, Neve B, Pineda Torra I, Teissier E, Minnich A, Jaye M, Duverger N, Brewer BH, Fruchart JC, Clavey V, Staels B. PPARα and PPARγ activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway. Nat Med. 2001; 7: 53–58.
Panousis CG, Zuckerman SH. Regulation of cholesterol distribution in macrophage-derived foam cells by interferon-gamma. J Lipid Res. 2000; 41: 75–83.
Maung K, Miyazaki A, Nomiyama H, Chang CC, Chang TY, Horiuchi S. Induction of acyl-coenzyme A: cholesterol acyltransferase-1 by 1,25-dihydroxyvitamin D(3) or 9-cis-retinoic acid in undifferentiated THP-1 cells. J Lipid Res. 2001; 42: 181–187.
Chatterjee S. Neutral sphingomyelinase action stimulates signal transduction of tumor necrosis factor-alpha in the synthesis of cholesteryl esters in human fibroblasts. J Biol Chem. 1994; 269: 879–882.
Chinetti G, Griglio S, Antonucci M, Pineda Torra I, Delerive P, Majd Z, Fruchart JC, Chapman J, Najib J, Staels B. Activation of peroxisome proliferator-activated receptors α and γ induces apoptosis of human monocyte-derived macrophages. J Biol Chem. 1998; 273: 25573–25580.
Wang H, Germain SJ, Benfield PP, Gillies PJ. Gene expression of acyl-coenzyme-A: cholesterol-acyltransferase is upregulated in human monocytes during differentiation and foam cell formation. Arterioscler Throm Vasc Biol. 1996; 16: 809–814.
Basu SK, Goldstein JL, Anderson GW, Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976; 73: 3178–3182.
Mascaro C, Acosta E, Ortiz J, Marrero P, Hegardt F, Haro D. Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor. J Biol Chem. 1998; 273: 8560–8563.
Yu GS, Lu YC, Gulick T. Co-regulation of tissue-specific alternative human carnitine palmitoyltransferase Iβ gene promoters by fatty acid enzyme substrate. J Biol Chem. 1998; 273: 32901–32909.
Gbaguidi GF, Chinetti G, Milosavljevic D, Teissier E, Chapman J, Olivecrona G, Fruchart JC, Griglio S, Fruchart-Najib J, Staels B. Peroxisome proliferator-activated receptor (PPAR) agonists decrease lipoprotein lipase secretion and glycated LDL uptake by human macrophages. FEBS Lett. 2002; 512: 85–90.
Galis ZS, Sukhova GK, Kranzhofer R, Clark S, Libby P. Macrophage foam cells from experimental atheroma constitutively produce matrix-degrading proteinases. Proc Natl Acad Sci U S A. 1995; 92: 402–406.
Duez H, Chao YS, Hernandez M, Torpier G, Poulain P, Mundt S, Mallat Z, Teissier E, Burton CA, Tedgui A, Fruchart JC, Fievet C, Wright SD, Staels B. Reduction of atherosclerosis by the PPARα agonist fenofibrate in mice. J Biol Chem. 2002; 277: 48051–48057.