Impaired Cholesterol Metabolism and Enhanced Atherosclerosis in Clock Mutant MiceClinical Perspective
Background—Clock is a key transcription factor that positively controls circadian regulation. However, its role in plasma cholesterol homeostasis and atherosclerosis has not been studied.
Methods and Results—We show for the first time that dominant-negative Clock mutant protein (ClockΔ19/Δ19) enhances plasma cholesterol and atherosclerosis in 3 different mouse models. Detailed analyses revealed that ClkΔ19/Δ19Apoe−/− mice display hypercholesterolemia resulting from the accumulation of apolipoprotein B48–containing cholesteryl ester–rich lipoproteins. Physiological studies showed that enhanced cholesterol absorption by the intestine contributes to hypercholesterolemia. Molecular studies indicated that the expression of Niemann Pick C1 Like 1, Acyl-CoA:Cholesterol acyltransferase 1, and microsomal triglyceride transfer protein in the intestines of ClkΔ19/Δ19Apoe−/− mice was high and that enterocytes assembled and secreted more chylomicrons. Furthermore, we identified macrophage dysfunction as another potential cause of increased atherosclerosis in ClkΔ19/Δ19Apoe−/− mice. Macrophages from ClkΔ19/Δ19Apoe−/− mice expressed higher levels of scavenger receptors and took up more modified lipoproteins compared with Apoe−/− mice, but they expressed low levels of ATP binding casette protein family A member 1 and were defective in cholesterol efflux. Molecular studies revealed that Clock regulates ATP binding casette protein family A member 1 expression in macrophages by modulating upstream transcription factor 2 expression.
Conclusions—ClockΔ19/Δ19 protein enhances atherosclerosis by increasing intestinal cholesterol absorption, augmenting uptake of modified lipoproteins by macrophages, and reducing cholesterol efflux from macrophages. These studies establish that circadian Clock activity is crucial in maintaining low plasma cholesterol levels and in reducing atherogenesis in mice.
- ATP binding cassette transporter 1
- circadian rhythm
- CLOCK proteins
- lipid metabolism
- upstream transcription factor 2
Circadian regulatory mechanisms synchronize biological functions to environmental stimuli such as light. Changes in light are transmitted from the eye by the retinal ganglion cells to the suprachiasmatic nuclei in the brain, where this information is translated into transcriptional regulation of certain transcription factors. Clock and Bmal1 are 2 transcription factors that increase the expression of other transcription factors to control the rhythmicity of different biological functions.1,2 Ablation of Clock has no significant effect on circadian rhythms because neuronal PAS domain containing protein 2 (NPAS2) can substitute for Clock deficiency by interacting with Bmal1.3,4 However, deletion of exon 19 in the Clock (Clk) gene results in the synthesis of ClockΔ19/Δ19 mutant protein, which acts as a dominant-negative regulator and disrupts clock function.5,6 Mice expressing ClockΔ19/Δ19 protein exhibit modest hypertriglyceridemia, hypercholesterolemia, hyperglycemia, and hyperleptinemia.7 We have previously shown that plasma triglycerides in ClkΔ19/Δ19 mutant mice do not exhibit circadian rhythms; instead, they show modest hypertriglyceridemia.8,9 Molecular studies showed that ClockΔ19/Δ19 protein disrupts plasma triglyceride homoeostasis by deregulating diurnal transcriptional regulation of short heterodimer partner (SHP) and microsomal triglyceride transfer protein (MTP).8 In this study, we examined the effects of ClockΔ19/Δ19 protein on the regulation of plasma cholesterol and atherosclerosis. Here, we show that ClockΔ19/Δ19 protein enhances atherosclerosis and identify different physiological pathways and molecular targets affected by the expression of ClockΔ19/Δ19 protein that contribute to atherosclerosis.
Clinical Perspective on p 1769
ClkΔ19/wt, ClkΔ19/wtLdlr−/−, and ClkΔ19/wtApoe−/− mice were bred to obtain ClkΔ19/Δ19, Clkwt/wt, ClkΔ19/Δ19Ldlr−/−, Ldlr−/−, ClkΔ19/Δ19Apoe−/−, and Apoe−/− mice. All mice on C57/Bl6 background were housed with a 12-hour light schedule (7 am–7 pm). Male 2- to 3-month-old mice were fed different diets (Table I in the online-only Data Supplement) for atherosclerosis studies. Animal experiments were approved by the Animal Care and Use Committee of the SUNY Downstate Medical Center and were performed in accordance with institutional guidelines.
Bone marrow–derived macrophages obtained from ClkΔ19/Δ19Apoe−/− mice and Apoe−/− mice10 were treated with or without oxidized low-density lipoprotein (ox-LDL) for 8 hours. For cholesterol efflux assays, macrophages were labeled with [3H]cholesterol for 24 hours, washed with PBS, and incubated in Dulbecco modified Eagle medium containing 0.2% BSA for 1 hour and then in the same media in the absence or presence of apolipoprotein (Apo) AI (15 μg/mL) or high-density lipoprotein (HDL; 50 μg/mL) for 8 hours. The human monocytic cell line THP-1 was maintained in RPMI 1640 media and differentiated by treatment with phorbol myristic acid.
After a 4-hour fast, plasma was obtained to measure lipids with the use of kits. Plasma ApoA-I, ApoB, and ApoE were quantified by Western blotting.11 Mice were not fasted when daily changes in plasma and tissue lipids were studied.
In Vivo Absorption of Lipids
Mice were injected intraperitoneally with 0.5 mL Poloxamer P407 in PBS (1:6 vol/vol) and gavaged with [3H]cholesterol at noon.
Uptake and Secretion of Lipids by Enterocytes
To study uptake, enterocytes from ClkΔ19/Δ19Apoe−/− mice and Apoe−/− mice were incubated in triplicate with [3H]cholesterol (1 μCi/mL) for different times. To measure secretion, enterocytes were incubated in triplicate with [3H]cholesterol for 1 hour, washed, and then incubated in fresh media containing oleic acid and taurocholate for different times.12 To study the distribution of cholesterol in chylomicrons and HDL, conditioned media was adjusted to a density of 1.10 g/mL by the addition of KBr, overlaid with solution of a different density, and centrifuged.12
Evaluation of Atherosclerosis
The proximal aorta was collected after saline perfusion. The aortic root and ascending aorta were sectioned at a thickness of 10 µm, and alternate sections were saved on slides for staining.13
J774A.1 cells were grown in suspension in DMEM supplemented with 10% FBS. Cells were radiolabeled with 5 μCi/mL 3H-cholesterol and 50 μg/mL acetylated LDL for 48 hours. The labeled foam cells were injected intraperitoneally into ClkΔ19/Δ19Apoe−/− and Apoe−/− mice. Blood, feces, and liver were collected for counting. Bone marrow–derived macrophages from ClockΔ19/Δ19Apoe−/− mice and Apoe−/− mice were incubated with acetylated LDL and [3H]cholesterol and injected into wild-type (WT) mice.
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation was used to study the binding of different transcription factors to the ABCA1 promoter using goat polyclonal antibodies against hypoxia-inducible factor (HIF)-1α, HIF1β, USF1, and USF2 with the use of kits as reported previously.8 ABCA1 promoter sequences were amplified with primers (Table II in the online-only Data Supplement).
Bone Marrow Transplantation
Apoe−/− mice (age 8 weeks) were lethally irradiated and transplanted with bone marrow cells derived from ClkΔ19/Δ19Apoe−/− or Apoe−/− mice.
Data are presented as mean±SD (n=6–12 animals). Mice were euthanized at each time point, and plasma and tissue samples were collected. Statistical testing was performed by the paired Student t test. Temporal comparisons between 2 groups were performed with 2-way ANOVA followed by Bonferroni posttest (GraphPad Prism). Differences were considered statistically significant when P<0.05.
ClkΔ19/Δ19 Mice Develop Atherosclerosis on an Atherogenic Diet
To test the hypothesis that ClkΔ19/Δ19 mice might be susceptible to atherosclerosis, WT (Clkwt/wt) and ClkΔ19/Δ19 siblings were fed either chow or an atherogenic diet15 ad libitum. Chow-fed ClkΔ19/Δ19 mice had higher lipids compared with their WT siblings (Table) but did not show any atherosclerotic lesions. However, after being fed an atherogenic diet for 2 months, ClkΔ19/Δ19 mice had 2- to 3-fold higher plasma cholesterol and triglyceride levels (Table), mainly very low-density lipoprotein/intermediate-density lipoprotein/LDL (Figure IA and IB in the online-only Data Supplement), increased amounts of ApoB100 and ApoB48 but reduced levels of ApoA1 and ApoE (Figure IC in the online-only Data Supplement), and higher ApoB/ApoAI ratios (Figure ID in the online-only Data Supplement) compared with WT siblings. These mice had 2.3- and 1.6- fold more lesions at the aortic arches (Figure 1A) and aortic root (Figure 1B), respectively, and 3- to 4-fold elevated amounts of lipid lesions in the abdominal aorta (Figure 1C). Thus, ClkΔ19/Δ19 mice show hyperlipidemia and develop more lesions on an atherogenic diet.
Clock Mutant Protein Increases Atherosclerosis in Ldlr−/− Mice
Besides mice fed an atherogenic diet, atherosclerosis is commonly studied in mouse models deficient in LDL receptors and ApoE.16 ClkΔ19/Δ19Ldlr−/− mice had higher plasma lipids when fed chow and Western diets (Table) and developed more atherosclerotic lesions than Ldlr−/− mice on both chow and Western diets, respectively (Figure 1D–1F).
Clock Mutant Protein Increases Atherosclerosis in Chow-Fed Apoe−/− Mice
Next, we studied the effect of ClockΔ19/Δ19 protein on atherosclerosis in Apoe−/− mice. ClkΔ19/Δ19Apoe−/− mice on a chow diet showed more extensive atherosclerotic lesions in the aortic arch than Apoe−/− mice (Figure 2A). The whole aorta showed 34-fold increased lipid staining (Figure 2B), whereas the cardiac/aortic junctions of ClkΔ19/Δ19Apoe−/− mice had 22-fold more lipid lesions. The lesions at the cardiac/aortic junction contained 4-fold more necrotic core (Figure 2C) and macrophages (Figure 2D). Furthermore, smooth muscle cells (Figure 2E) and collagen content (Figure 2F) were increased by 5-fold. These observations indicate the presence of advanced, stable plaques in ClkΔ19/Δ19Apoe−/− mice. These lesions were seen more frequently in male than in female mice, and their size increased with age (Figure II in the online-only Data Supplement). The brachiocephalic arteries of these mice had higher amounts of cholesterol/cholesteryl esters/triglycerides (Figure IIIA in the online-only Data Supplement), lipids (Figure IIIB in the online-only Data Supplement), necrotic area (Figure IIIC in the online-only Data Supplement), macrophages (Figure IIID in the online-only Data Supplement), smooth muscle cells (Figure IIIE in the online-only Data Supplement), and collagen (Figure IIIF in the online-only Data Supplement). ClkΔ19/Δ19Apoe−/− mice developed more atherosclerotic lesions on a Western diet (Figure IV in the online-only Data Supplement). These studies indicate that ClkΔ19/Δ19Apoe−/− mice develop extensive lesions throughout the aorta that are rich in lipids, macrophages, smooth muscle cells, and collagen compared with Apoe−/− mice, most likely representing stable plaques.
Plasma Lipids Are Higher in ClkΔ19/Δ19Apoe−/− Mice
Plasma of chow-fed ClkΔ19/Δ19Apoe−/− mice was more turbid (Figure 3A) and had 2-fold more total and esterified cholesterol (Table). Plasma total cholesterol levels were significantly higher and triglyceride levels were lower in these mice at all time points (Figure V in the online-only Data Supplement). Cholesterol levels were higher in non-HDL lipoproteins but were lower in HDL (Table). Plasma ApoB100 and ApoAI levels were lower (Figure 3C, protein blot) but ApoB48 and ApoB/ApoAI ratios were higher in these mice (Figure 3C). These studies indicate that ClkΔ19/Δ19Apoe−/− mice accumulate more ApoB48-containing cholesteryl ester–rich lipoproteins.
ClkΔ19/Δ19Apoe−/− Mice Absorb More Cholesterol
Because lipoprotein catabolism is impaired in Apoe−/− mice, we hypothesized that higher plasma ApoB48-containing cholesteryl ester–rich lipoproteins are attributable to increased cholesterol absorption by the intestine. To study absorption, mice were gavaged with radiolabeled cholesterol. Radiolabeled cholesterol–derived lipids were higher at 2 to 4 hours in the plasma of ClkΔ19/Δ19Apoe−/− mice (Figure 3D). Increased absorption could be attributable to increased uptake or secretion by enterocytes. Isolated primary enterocytes from ClkΔ19/Δ19Apoe−/− mice took up more cholesterol in a time-dependent manner (Figure 3E). Furthermore, pulse-chase studies showed that enterocytes secrete more cholesterol (Figure 3F). These studies revealed that uptake and secretion of cholesterol are higher in ClkΔ19/Δ19Apoe−/− enterocytes.
Cholesterol uptake in enterocytes is a balance between import by NPC1L1 and export by ABCG5/ABCG8.17 Measurement of mRNA levels revealed no change in ABCG5/ABCG8 (Figure 3G); instead, we found significant increases in NPC1L1 mRNA (Figure 3G) and protein (Figure 3H) levels. Therefore, increased expression of NPC1L1 might contribute to increased uptake of cholesterol in ClkΔ19/Δ19Apoe−/− enterocytes.
After uptake, cholesterol is secreted by enterocytes via HDL and chylomicrons.18 The HDL pathway transports free cholesterol involving ABCA1 and ApoAI. We found that ABCA1 was reduced but ApoA1 mRNA was similar in the enterocytes of ClkΔ19/Δ19Apoe−/− and in Apoe−/− mice (Figure 3G). The transport of cholesterol via chylomicrons depends on ACAT enzymes and MTP because dietary cholesterol is esterified by ACAT1/ACAT2, packaged in chylomicrons by MTP, and secreted. We observed significant increases in ACAT2, but not ACAT1, mRNA levels in ClkΔ19/Δ19Apoe−/− mice (Figure 3G). Moreover, MTP mRNA, protein (Figure 3G and 3H), and activity (Figure 3I) were significantly higher in ClkΔ19/Δ19Apoe−/− mice. Increases in MTP and ACAT2 suggested that chylomicron assembly and secretion pathway might be augmented in ClkΔ19/Δ19Apoe−/− mice. To test this, we incubated enterocytes with radiolabeled cholesterol and subjected conditioned media to ultracentrifugation. Cholesterol counts were higher in chylomicrons but not in HDL fractions (Figure 3J), indicating that ClkΔ19/Δ19Apoe−/− mice absorb more cholesterol by enhancing assembly and secretion of chylomicrons.
Plasma Cytokines Are Higher in ClkΔ19/Δ19Apoe−/− Mice
Inflammation is a hallmark of atherosclerosis. Therefore, we measured cytokines in ClkΔ19/Δ19Apoe−/− and Apoe−/− mice. Plasma of ClkΔ19/Δ19Apoe−/− mice contained ≈2- to 4-fold higher levels of interleukin (IL)-12, IL17A, and granulocyte colony-stimulating factor (Figure VIA in the online-only Data Supplement). It is known that macrophages contribute to plasma cytokines. Therefore, we looked at the expression of several of these cytokines in bone marrow–derived macrophages. ClkΔ19/Δ19Apoe−/− macrophages had higher mRNA levels of IL12, IL6, tumor necrosis factor-α, and granulocyte colony-stimulating factor but not IL17A (Figure VIB in the online-only Data Supplement); IL17 is produced mainly by lymphocytes.19 To determine whether Clock plays a role in the regulation of cytokine expression, we reduced Clock levels using siRNA in WT macrophages. siClock reduced Clock mRNA by 80% (Figure V in the online-only Data Supplement) and increased granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. These studies suggest that Clock suppresses the expression of different cytokines in macrophages.
ClkΔ19/Δ19Apoe−/− Macrophages Take up More Modified Lipoproteins as a Result of Increased Expression of CD36 and SR-A1
The studies described above showed that lesions in ClkΔ19/Δ19Apoe−/− mice were lipid and macrophage rich (Figure 2B and 2D and Figure IIIB and IIID in the online-only Data Supplement). To understand the mechanisms that might contribute to the accumulation of lipids in the aorta, we injected DiI-labeled acetylated LDL into Apoe−/− and ClkΔ19/Δ19Apoe−/− mice. There was 2-fold higher DiI labeling in the aorta of ClkΔ19/Δ19Apoe−/− mice than Apoe−/− mice (Figure 4A). It is known that macrophages are the principal cells that take up modified lipoproteins in the subintima; therefore, we studied the uptake of modified lipoproteins by bone marrow–derived macrophages from ClkΔ19/Δ19Apoe−/− and Apoe−/− mice. Compared with Apoe−/− mice, ClkΔ19/Δ19Apoe−/− macrophages took up 2-fold more DiI-labeled acetylated LDL and contained 2- to 3-fold more lipids, lipid peroxides, and total and esterified cholesterol (Figure 4B–4E). To explore reasons for lipid accumulation, we measured mRNA and protein levels of scavenger receptors involved in the uptake of modified lipoproteins. ClkΔ19/Δ19Apoe−/− macrophages expressed higher protein and mRNA levels of CD36 and SR-A1 (Figure 4F), suggesting that their increased expression could contribute to fat accumulation.
ClkΔ19/Δ19 reduces Clock activity by acting as a dominant-negative mutant.5 Therefore, to understand the mechanisms for increased expression of scavenger receptors, we reduced Clock expression using siRNA in Clkwt/wt macrophages. siClock reduced Clock mRNA levels by ≈80% in WT macrophages, and these levels were unaffected by ox-LDL treatment (Figure 4G). Reductions in Clock had no effect on the mRNA (Figure 4G) and protein (Figure 4H) levels of CD36 and SR-A1 in normal macrophages. However, incubation of these macrophages with ox-LDL increased the expression of scavenger receptors in both siControl- and siClock-treated cells, but increases in the protein and mRNA levels of these scavenger receptors were higher in siClock-treated cells (Figure 4G and 4H). Furthermore, siClock-treated macrophages took up 2-fold more amounts of DiI-labeled acetylated LDL (Figure 4I). Similarly, siClock-treated human THP-1 macrophages took up more DiI-labeled acetylated LDL (Figure VIIA in the online-only Data Supplement). These studies show that increases in scavenger receptors are higher when macrophages have reduced Clock expression and are exposed to ox-LDL. Thus, Clock reduces the expression of scavenger receptors when macrophages are exposed to modified lipoproteins.
ClkΔ19/Δ19Apoe−/− Macrophages Are Defective in Cholesterol Efflux as a Result of Reduced ABCA1 Expression
In addition to increased uptake, reduced efflux contributes to cholesterol accumulation in macrophages. Therefore, we studied in vivo reverse cholesterol transport from 3H-cholesterol–loaded J774 macrophages in Apoe−/− and ClkΔ19/Δ19Apoe−/− mice. The appearance of cholesterol into plasma, feces, and liver was significantly less in ClkΔ19/Δ19Apoe−/− mice compared with Apoe−/− mice (Figure 5A), indicating that ClkΔ19/Δ19Apoe−/− plasma is less efficient in reverse cholesterol transport from J774 macrophages, most likely secondary to low plasma HDL (Table) and ApoAI (Figure 3C) in these mice. Additionally, we studied the ability of ClkΔ19/Δ19Apoe−/− macrophages to give up cholesterol to plasma acceptors in WT mice. Injection of 3H-cholesterol–loaded ClkΔ19/Δ19Apoe−/− or Apoe−/− macrophages into WT mice revealed that ClkΔ19/Δ19Apoe−/− macrophages are defective in giving off cholesterol, as evidenced by lower amounts of cholesterol in plasma, feces, and liver (Figure 5B). Furthermore, isolated ClkΔ19/Δ19Apoe−/− macrophages gave up less cholesterol to extracellular ApoAI and HDL in culture (Figure 5C). Thus, ClkΔ19/Δ19Apoe−/− macrophages are defective in cholesterol efflux.
Clock Regulates ABCA1 Expression
To understand reasons for reduced cholesterol efflux, we measured mRNA and protein levels of transporters involved in cholesterol efflux and found lower amounts of ABCA1 and ABCG1 mRNA and protein levels in ClkΔ19/Δ19Apoe−/− macrophages but no change in SR-B1 and ABCG4 expression (Figure 5D). To determine whether low expression of ABCA1 was contributing to reduced cholesterol efflux, we expressed ABCA1 under the control of cytomegalovirus promoter. Overexpression of ABCA1 increased cholesterol efflux from ClkΔ19/Δ19Apoe−/− macrophages (Figure 5E).Next, we asked whether Clock regulates ABCA1. First, we asked whether ApoE deficiency is required for ClockΔ19/Δ19 to reduce ABCA1. This was not the case because ABCA1 levels were low in ClkΔ19/Δ19 macrophages compared with their WT littermates (Figure 5F). Second, knockdown of Clock in Clkwt/wt macrophages reduced ABCA1 mRNA (Figure 5G) and protein (Figure 5H, inset) levels, as well as efflux to ApoAI (Figure 5H). Similarly, Clock knockdown in human THP-1 macrophages reduced cholesterol efflux to HDL and ApoAI (Figure VIIB and VIIC in the online-only Data Supplement). In contrast, knockdown of PER1, CRY1, or BMAL1 in Clkwt/wt macrophages had no effect on ABCA1 mRNA (Figure VIIIA in the online-only Data Supplement) and cholesterol efflux (Figure VIIIB in the online-only Data Supplement). These data suggest that Clock regulates ABCA1 expression and cholesterol efflux.
Clock Modulates ABCA1 Expression Involving USF2
To determine whether Clock regulates ABCA1 at the transcriptional level, we expressed luciferase under the control of 1.3 kb ABCA1 promoter,20 along with a Clock expression plasmid or lentiviruses expressing shClock in WT macrophages. Overexpression of Clock increased whereas its knockdown significantly reduced promoter activity (Figure 6A), suggesting that Clock increases ABCA1 transcription. To identify transcription factors regulated by Clock and those involved in ABCA1 expression, we measured mRNA levels of various activators and repressors known to regulate ABCA1 gene expression.21 Activators of ABCA1 either were reduced or did not change in ClkΔ19/Δ19Apoe−/− macrophages compared with Apoe−/− macrophages (Figure IXA in the online-only Data Supplement). Quantification of various repressors showed that USF1, USF2, and TRβ were significantly increased (Figure IXB in the online-only Data Supplement). Furthermore, knockdown of Clock in WT macrophages either reduced or had no effect on activators (Figure IXC in the online-only Data Supplement). Although siClock had no significant effect on various repressors, it significantly increased mRNA (Figure IXD in the online-only Data Supplement) and protein (Figure 6B) levels of USF1 and USF2. These studies suggested that Clock may modulate USF1 and USF2 expression to regulate ABCA1.
Subsequently, we determined the role of USF1 and USF2 in the regulation of ABCA1 by Clock. USF1/USF2 and HIF1α/HIF1β bind to an E-box in the ABCA1 promoter to decrease and increase ABCA1 expression, respectively.21,22 Knockdown of USF1, USF2, HIF1α, and HIF1β had no effect on Clock mRNA, suggesting that Clock is not regulated by them (Figure X in the online-only Data Supplement). siUSF1 and siUSF2 increased ABCA1 expression, but siHIF1α and siHIF1β had no effect (Figure 6C), indicating that USF1 and USF2 suppress ABCA1 expression. Therefore, we asked whether Clock needs these transcription factors to regulate ABCA1. siClock reduced ABCA1 expression in siHIF1α-, siHIF1β-, and siUSF1-treated cells but not in siUSF2-treated cells (Figure 6C), indicating that siClock needs USF2 to reduce ABCA1 expression. To confirm the role of USF2 in ABCA1 regulation, we performed chromatin immunoprecipitation in Apoe−/− and ClkΔ19/Δ19Apoe−/− macrophages. In Apoe−/− macrophages, ABCA1 promoter was occupied by HIF1β, USF1, and USF2 (Figure 6D). However, in ClkΔ19/Δ19Apoe−/− macrophages, only USF1 and USF2 were found to be associated with the promoter. The amounts of USF2 associated with the promoter were higher in ClkΔ19/Δ19Apoe−/− macrophages. Therefore, increased binding of USF2 to the ABCA1 promoter in ClkΔ19/Δ19Apoe−/− macrophages might reduce expression.
To garner the in vivo significance of USF2 in cholesterol efflux, we hypothesized that a reduction of USF2 in ClkΔ19/Δ19Apoe−/− macrophages might enhance reverse cholesterol transport. To test this, bone marrow–derived ClkΔ19/Δ19Apoe−/− macrophages were treated with siControl or siUSF2, loaded with 3H-cholesterol, and injected in WT mice. After 48 hours, mice receiving siUSF2-treated macrophages contained higher 3H-cholesterol levels in the plasma, liver, and feces (Figure 6E). These studies indicate that siUSF2 increases reverse cholesterol transport from macrophages.
Cyclic Expression of ABCA1 and USF2 in Macrophages
The above studies indicated that Clock regulates macrophage ABCA1 expression and cholesterol efflux by regulating USF2. Nothing is known about the circadian regulation of ABCA1 and USF2 in macrophages or in other cells. To determine whether ABCA1 and USF2 expression shows diurnal changes, WT bone marrow macrophage cultures treated with siClock or not treated were synchronized by incubating them in 50% serum for 2 hours. Subsequently, changes in macrophage ABCA1 and USF2 were measured at different times. ABCA1 and USF2 expression showed cyclic expression in synchronized WT macrophages. ABCA1 and USF2 levels increased and decreased, respectively, in siClock-treated macrophages (Figure 6F). Furthermore, ABCA1 mRNA levels were low when USF2 levels were high. These studies indicate that ABCA1 and USF2 expression in macrophages shows cyclic change and that Clock plays an important role in these changes.
Effect of ClkΔ19/Δ19Apoe−/− Bone Marrow Cell Transplantation on Atherosclerosis in Apoe−/− Mice
To determine whether macrophage dysfunction contributes to increased atherosclerosis independently of hyperlipidemia, we transplanted bone marrow cells obtained from ClkΔ19/Δ19Apoe−/− or Apoe−/− mice into lethally irradiated Apoe−/− mice. Bone marrow transplantation slightly reduced total plasma cholesterol in Apoe−/− mice (Figure 7A). However, Apoe−/− mice that received bone marrow cells from ClkΔ19/Δ19Apoe−/− mice had 2- to 2.7-fold more atherosclerotic plaques in the ascending aortas and 3 main branching arteries (Figure 7B) and at the cardiac/aortic junctions (Figure 7C). Furthermore, there was 3-fold more lipid staining in the aorta (Figure 7D). Moreover, macrophages obtained from mice transplanted with bone marrow cells from ClkΔ19/Δ19Apoe−/− were defective in cholesterol efflux to ApoAI and HDL (Figure 7E). Gene expression analysis showed that macrophages isolated from Apoe−/− mice transplanted with ClkΔ19/Δ19Apoe−/− bone marrow cells had low mRNA levels of ABCA1/ABCG1 and higher levels of CD36/SR-A1 (Figure 7F). Further analysis of transcription factors that regulate ABCA1 revealed that these macrophages had higher levels of USF2 (Figure 7G). Thus, macrophage dysfunction caused by the expression of ClockΔ19/Δ19 protein contributes to atherosclerosis in Apoe−/− mice.
Using 3 different mouse models and 3 different diets, we show for the first time that Clock dysfunction resulting from the expression of a dominant-negative ClockΔ19/Δ19 protein increases atherosclerosis in mice. Different mouse models carrying ClockΔ19/Δ19 protein had higher cholesterol in ApoB-containing non-HDL lipoproteins. Mechanistic studies revealed that ClockΔ19/Δ19 protein enhances cholesterol absorption by enterocytes and uptake of modified lipoproteins by macrophages in Apoe−/− mice. In contrast, it reduces cholesterol efflux from macrophages. Thus, Clock plays an important and novel role in the regulation of cholesterol metabolism in enterocytes and macrophages to prevent hypercholesterolemia and atherosclerosis.
Biochemical analysis showed that hypercholesterolemia in ClkΔ19/Δ19Apoe−/− mice was attributable to accumulation of cholesteryl ester–rich ApoB48-containing lipoproteins. Physiological studies showed that enterocytes expressing ClockΔ19/Δ19 protein take up more cholesterol from the intestinal lumen and secrete more cholesterol with chylomicrons. Molecular studies demonstrated that increased cholesterol uptake was associated with enhanced expression of NPC1L1 with no significant changes in the cholesterol exporters ABCG1/ABCG8. After uptake, cholesterol is transported to plasma involving HDL and chylomicrons. The HDL pathway was not affected, but the chylomicron pathway was upregulated in ClkΔ19/Δ19Apoe−/− mice. Two proteins, ACAT2 and MTP, that are involved in the assembly of chylomicrons were increased in ClkΔ19/Δ19Apoe−/− mice. Thus, Clock regulates cholesterol absorption by modulating cholesterol uptake, cholesterol esterification, and chylomicron assembly.
This study shows that ClockΔ19/Δ19 protein disrupts several macrophage functions: secretion of cytokines, uptake of oxidized lipoproteins, and cholesterol efflux. ClkΔ19/Δ19Apoe−/− macrophages secrete more IL12, IL17, and granulocyte colony-stimulating factor. They take up more modified lipoproteins and retain more oxidized lipids. Additionally, we showed that ClkΔ19/Δ19Apoe−/− mice were defective in reverse cholesterol transport as a result of a combination of lower plasma cholesterol acceptors, ApoAI/HDL, and reduced macrophage expression of ABCA1 and ABCG1 transporters. Molecular studies revealed that Clock regulates ABCA1 and that USF2 might be an intermediary repressor that is regulated by Clock to modulate ABCA1 expression in macrophages. During Clock deficiency, USF2 levels are increased. Further binding of this repressor to the ABCA1 promoter is enhanced.
Consideration was given to the possibility that the effect observed in ClkΔ19/Δ19Apoe−/− mice might not be specific to Clock dysfunction. Instead, it could be a general effect resulting from deficiencies in other Clock genes or from off-target effects of the mutation. To address this, we performed several Clock knockdown experiments in WT macrophages. ABCA1 levels were reduced after siClock treatment. Furthermore, there were no significant differences, except for Per3 and Cry2 mRNA levels, in macrophages obtained from ClkΔ19/Δ19Apoe−/− and Apoe−/− mice (Figure XI in the online-only Data Supplement). Thus, Clock has a specific effect on lipid metabolism. It is known that Clock affects cholesterol synthesis and degradation by the liver. In our studies, we observed no significant increase in lipoprotein production by the liver. Therefore, we did not address whether hepatic cholesterol metabolism was affected in these mice.
Myocardial infarctions occur predominantly in the morning. It is known that circadian clocks regulate arrhythmogenesis, myocardial contractility, and oxidative metabolism. Using an isograft model, Cheng et al23 have shown that transplantation of arteries from Bmal1- and Per-deficient mice into WT mice elicits a pathological response resulting in intimal hyperplasia and wall thickening. This response was attributable to infiltration of WT cells and hyperplastic response by the Clock-deficient arteries. Here, we provide evidence that Clock deficiency alters lipid metabolism and macrophage function to enhance atherosclerosis. Thus, circadian Clock might play an important protective role against hyperlipidemia and atherosclerosis.
These studies show that Clock regulates cholesterol metabolism in the intestine and macrophages and acts as an antiatherogenic gene (Figure XII in the online-only Data Supplement). In the intestine, Clock deficiency increases lipid absorption. In macrophages, it augments uptake of modified lipoproteins and diminishes cholesterol efflux. These changes could contribute to enhanced atherosclerosis in ClkΔ19/Δ19Apoe−/− mice.
We are grateful to Drs Lita Freeman and Alan Remaley of the National Institutes of Health for plasmids expressing luciferase under the control of various ABCA1 promoter regions; Drs Roman Kondratov and Antoch Marino for plasmids expressing Clock; Yan Li and Joyce Queiroz for technical assistance in the early analysis of atherosclerotic plaques; and Wei Quan for technical assistance in fluorescence microscope.
Sources of Funding
This work was supported in part by National Institutes of Health grant DK-81879 to Dr Hussain and American Heart Association Scientist Development Grant 2300158 to Dr Pan.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.113.002885/-/DC1.
- Received March 27, 2013.
- Accepted August 22, 2013.
- © 2013 American Heart Association, Inc.
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- Fulton DJ,
- Rudic RD
Heart attacks happen mainly in the early hours of the day, suggesting that their occurrence might be related to circadian rhythms seen in various behavioral, physiological, and biochemical activities. Here, we show that disruption of circadian Clock activity as a result of a dominant-negative mutation (ClockΔ19/Δ19) increases susceptibility to atherosclerosis in various mouse models. ClockΔ19/Δ19 mice fed an atherogenic diet had increased plasma cholesterol, triglycerides, and atherosclerotic lesions compared with their wild-type siblings. Similarly, ClockΔ19/Δ19 protein increased cholesterolemia and atherosclerosis in Ldlr−/− and Apoe−/− mice fed a chow or Western diets. Physiological studies revealed that high plasma cholesterol in ClockΔ19/Δ19Apoe−/− mice was due in part to increased cholesterol uptake by enterocytes. In addition, macrophages in ClockΔ19/Δ19Apoe−/− mice displayed higher lipid uptake and reduced cholesterol efflux compared with Apoe−/− siblings. Molecular studies demonstrated that knockdown of Clock gene expression in wild-type macrophages reduces ABCA1 expression and cholesterol efflux. Furthermore, Clock overexpression increases ABCA1 transcription. Evidence is presented to suggest that USF2 could participate in the modulatory effect of Clock on ABCA1 expression. These studies provide significant evidence for the importance of Clock in the proper physiological functioning of enterocytes and macrophages. Hence, disruptions in Clock function as a result of either mutations or other environmental factors such as a high-fat diet, transcontinental flights, and night shift work might deregulate enterocyte and macrophage function, increasing the risk for atherosclerosis.